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Chemistry of
Protein and Nucleic Acid
Cross-Linking and
Conjugation
Second Edition
Chemistry of
Protein and Nucleic Acid
Cross-Linking and
Conjugation
Second Edition
Shan S. Wong
David M. Jameson
Boca Raton London New York
CRC Press is an imprint of the

Taylor & Francis Group, an informa business
CRC Press
Taylor & Francis Group
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Contents
Preface�� xv
Authors��xvii
Chapter 1 Overview of Protein Conjugation.................................................................................. 1

References..................................................................................................................... 6
Chapter 2 Review of Protein and Nucleic Acid Chemistry........................................................... 9

2.1 Introduction........................................................................................................ 9
2.2 Protein Composition......................................................................................... 10
2.2.1 Amino Acids....................................................................................... 10
2.2.2 Prosthetic Groups................................................................................ 10
2.3 Protein Functional Groups............................................................................... 13
2.3.1 Reactive Amino Acid Side Chains...................................................... 13
2.3.1.1 Relationship between Nucleophilicity and Reactivity......... 14
2.3.1.2 Effects of pH........................................................................ 15
2.3.1.3 Effects of Microenvironment............................................... 16
2.3.2 Chemically Introduced Reactive Groups............................................ 17
2.3.2.1 Reduction of Disulfide Bonds.............................................. 18
2.3.2.2 Interconversion of Functional Groups................................. 19
2.3.2.3 Introduction of Carbohydrate Prosthetic Groups................25
2.3.2.4 Activation of Carbohydrates by Periodate........................... 27
2.4 Nucleic Acid Chemistry................................................................................... 27
2.4.1 Photochemical Reactivities of Nucleic Acids...................................... 27
2.4.2 Chemical Reactivities of Nucleic Acids.............................................. 29
References................................................................................................................... 31
Chapter 3 Reagents Targeted to Specific Functional Groups...................................................... 35

3.1 Introduction...................................................................................................... 35
3.2 Sulfhydryl Reagents......................................................................................... 35
3.2.1 α-Haloacetyl Compounds.................................................................... 35
3.2.2 N-Maleimide Derivatives.................................................................... 37
3.2.3 Mercurial Compounds......................................................................... 38
3.2.4 Disulfide Reagents............................................................................... 38
3.3 Amino Group–Specific Reagents..................................................................... 38
3.3.1 Alkylating Agents............................................................................... 39
3.3.1.1 α-Haloacetyl Compounds.................................................... 39
3.3.1.2 N-Maleimide Derivatives..................................................... 39
3.3.1.3 Aryl Halides.........................................................................40
3.3.1.4 Aldehydes and Ketones....................................................... 41
3.3.2 Acylating Agents................................................................................. 42
v
vi Contents
3.4 Reagents Directed toward Carboxyl Groups.................................................... 43

3.4.1 Diazoacetate Esters and Diazoacetamides.......................................... 43
3.4.2 Carbodiimides.....................................................................................44
3.5 Tyrosine Selective Reagents.............................................................................44
3.5.1 Acylating Agents.................................................................................44
3.5.2 Electrophilic Reagents........................................................................ 45
3.6 Arginine-Specific Reagents.............................................................................. 45
3.7 Histidine-Selective Reagents............................................................................46
3.8 Methionine-Alkylating Reagents..................................................................... 47
3.9 Tryptophan-Specific Reagents......................................................................... 47
3.10 Serine-Modifying Reagents............................................................................. 48
References................................................................................................................... 48
Chapter 4 How to Design and Choose Cross-Linking Reagents................................................. 53

4.1 Introduction...................................................................................................... 53
4.2 Use of Nucleophilic Reactions......................................................................... 55
4.2.1 The Basic Reaction.............................................................................. 55
4.2.1.1 Electrophilicity of the Substrate.......................................... 55
4.2.1.2 Leaving Group Reactivity................................................... 56
4.2.2 Alkylation............................................................................................ 56
4.2.3 Acylation............................................................................................. 58
4.3 Use of Electrophilic Reactions.........................................................................60
4.4 Incorporating Group-Directed Reagents.......................................................... 61
4.4.1 Disulfide Reagents............................................................................... 62
4.4.2 Mercurial Reagents............................................................................. 62
4.4.3 Reductive Alkylation........................................................................... 62
4.4.4 Vicinal Dicarbonyl Reagents............................................................... 63
4.5 Incorporating Photoactivatable Nonspecific Groups........................................ 63
4.6 Changing the Water Solubility of Cross-Linkers............................................. 65
4.7 Incorporating Special Characteristics in the Bridge Spacer............................66
4.7.1 Incorporation of Cleavable Bonds.......................................................66
4.7.1.1 Disulfide Bond.....................................................................66
4.7.1.2 Mercurial Group..................................................................66
4.7.1.3 Vicinal Glycol Bond............................................................66
4.7.1.4 Azo Linkage........................................................................66
4.7.1.5 Sulfone Linkage................................................................... 69
4.7.1.6 Selenoethylene Group.......................................................... 69
4.7.1.7 Ester Bond........................................................................... 69
4.7.1.8 Thioester Bond.................................................................... 69
4.7.1.9 Maleylamide Linkage.......................................................... 69
4.7.1.10 Acetals, Ketals, and Ortho Esters........................................ 69
4.7.2 Incorporating Molecular Distance Rulers........................................... 70
4.7.3 Incorporating Reporter Groups........................................................... 72
4.7.3.1 UV–VIS Absorption Chromophores................................... 72
4.7.3.2 Infrared-Absorbing Chromophores..................................... 73
4.7.3.3 Fluorescent Probes............................................................... 73
4.7.3.4 Spin Labels.......................................................................... 74
4.7.3.5 Radioactive and Nonradioactive Isotopes........................... 75
References................................................................................................................... 76
Contents vii
Chapter 5 Homobifunctional Cross-Linking Reagents................................................................ 81

5.1 Introduction...................................................................................................... 81
5.2 Amino Group–Directed Cross-Linkers........................................................... 82
5.2.1 Bisimidoesters (Bisimidates)............................................................... 82
5.2.2 Bis-Succinimidyl Derivatives (N-Hydroxysuccinimidyl
Esters, NHS Esters)............................................................................. 85
5.2.3 Bifunctional Aryl Halides................................................................... 86
5.2.4 DiIsocyanates and DiIsothiocyanates................................................. 87
5.2.5 Bifunctional Sulfonyl Halides............................................................. 87
5.2.6 Bis-Nitrophenyl Esters........................................................................ 88
5.2.7 Bifunctional Acylazides...................................................................... 88
5.2.8 Dicarbonyl Compounds....................................................................... 88
5.2.9 Other Amino Group–Reacting Cross-Linking Reagents.................... 91
5.3 Sulfhydryl Group–Directed Cross-Linkers..................................................... 93
5.3.1 Mercurial Reagents............................................................................. 95
5.3.2 Disulfide-Forming Reagents............................................................... 95
5.3.3 Bismaleimides.....................................................................................97
5.3.4 Bis-Haloacetyl Derivatives.................................................................. 98
5.3.5 Di-Alkyl Halides................................................................................. 98
5.3.6 Chloro-s-Triazines...............................................................................99
5.3.7 Aziridines (Ethyleneimines)...............................................................99
5.3.8 Bis-Epoxides (Bisoxiranes).................................................................99
5.3.9 Sulfone Derivatives........................................................................... 100
5.4 Carboxyl Group–Directed Cross-Linking Agents......................................... 101
5.5 Phenolate and Imidazolyl Group–Directed Cross-Linking Reagents............ 102
5.6 Arginine Residue–Directed Cross-Linkers.................................................... 102
5.7 Methionine Residue Cross-Linking Agent..................................................... 103
5.8 Carbohydrate Moiety–Specific Reagents....................................................... 103
5.9 Nondiscriminatory Photoactivatable Cross-Linkers...................................... 104
5.10 Noncovalent Homobifunctional Cross-Linking Reagents.............................. 104
5.11 Nucleic Acid Cross-Linking Reagents........................................................... 105
5.11.1 Metal Compounds............................................................................. 105
5.11.2 Azinomycin Bis-Epoxides................................................................. 140
5.11.3 Bis-Pyrrolobenzodiazepines............................................................. 141
5.11.4 Bis-Cyclopropylpyrroloindole (CPI)-Based Reagents....................... 143
5.11.5 Bis-Cyclopropanebenz[e]indoline (CBI)-Based Reagents................ 145
5.11.6 Diaziridinyl Benzoquinones.............................................................. 146
5.11.7 Mitomycin C Dimers......................................................................... 147
5.11.8 Bis-Chloroethylamine Derivatives.................................................... 147
5.11.9 Bis-Carbamate Derivatives............................................................... 158
5.11.10 Pyrrolizidine Alkaloids (PAs)........................................................... 160
5.11.11 Bis-Catechol Derivatives................................................................... 161
5.11.12 Quinone Methides............................................................................. 162
5.11.13 Nitrosourea Derivatives..................................................................... 164
References................................................................................................................. 165
Chapter 6 Heterobifunctional Cross-Linkers............................................................................. 191

6.1 Introduction.................................................................................................... 191
6.2 Group-Selective Heterobifunctional Reagents
for Protein Cross-Linking....................................................................... 191
viii Contents
6.2.1 Amino- and Sulfhydryl-Group–Directed Cross-Linkers................. 191

6.2.2 Cross-Linkers Directed toward Carboxyl and Either Sulfhydryl
or Amino Groups.............................................................................. 199
6.2.3 Carbonyl- and Amino- or Sulfhydryl-Group–Directed
Cross-Linkers....................................................................................200
6.2.4 Miscellaneous Heterobifunctional Cross-Linkers
with Undefined Specificity................................................................200
6.3 Protein-Photosensitive Heterobifunctional Cross-Linking Reagents.............202
6.3.1 Amino Group–Anchored Photosensitive Reagents........................... 203
6.3.2 Sulfhydryl Group–Anchored Photoactivatable Reagents.................204
6.3.3 Guanidinyl Group–Anchored Photoactivatable Reagents................205
6.3.4 Carboxyl-, Carboxamide-, and Carbonyl-Group–Anchored
Photoactivatable Reagents................................................................. 205
6.3.5 Photoaffinity-Labeling Reagents....................................................... 205
6.4 Noncovalent Immunoglobulin Cross-Linking System...................................206
6.5 Heterobifunctional Nucleic Acid Cross-Linking Reagents............................208
References................................................................................................................. 225
Chapter 7 Multifunctional Cross-Linking Reagents.................................................................. 239

7.1 Introduction.................................................................................................... 239
7.2 Trifunctional Cross-Linkers........................................................................... 239
7.3 Tetrafunctional Cross-Linkers........................................................................ 256
7.4 Multifunctional Cross-Linkers....................................................................... 257
7.5 Noncovalent Cross-Linkers............................................................................ 258
7.5.1 Avidin and Streptavidin.................................................................... 258
7.5.2 Lectins............................................................................................... 259
7.5.3 Multifunctional Antibodies...............................................................260
References................................................................................................................. 261
Chapter 8 Monofunctional and Zero -Length Cross -Linking Reagents.................................... 265

8.1 Introduction.................................................................................................... 265
8.2 Monofunctional Cross-Linking Reagents......................................................266
8.2.1 Imidoesters........................................................................................266
8.2.2 Formaldehyde....................................................................................266
8.2.3 Chloroformates.................................................................................. 268
8.2.4 Mercuric Ion...................................................................................... 269
8.2.5 Functional Group–Modifying Reagents........................................... 269
8.3 Zero-Length Cross-Linking Reagents............................................................ 270
8.3.1 Carboxyl Group–Activating Reagents.............................................. 270
8.3.1.1 Carbodiimides................................................................... 270
8.3.1.2 Isoxazolium Compounds................................................... 276
8.3.1.3 Ethylchloroformate............................................................ 276
8.3.1.4 Carbodiimidazole.............................................................. 277
8.3.1.5 N-Alkoxycarbonyl-2-Alkoxy-1,2-Dihydroquinolines........ 278
8.3.1.6 Diethylpyrocarbonate........................................................ 279
8.3.2 Reagents for Disulfide Formation..................................................... 279
8.3.3 Oxidation Cross-Linking Reagents...................................................280
8.3.4 Carbohydrate Activation Reagents.................................................... 281
8.3.5 Enzymes as Zero-Length Cross-Linkers.......................................... 282
Contents ix
8.3.5.1 Transglutaminase............................................................... 282

8.3.5.2 Tyrosinase.......................................................................... 282
8.3.5.3 Peroxidases........................................................................ 283
8.3.5.4 Xanthine Oxidase and Others........................................... 283
8.3.6 Radiation as Zero-Length Cross-Linker...........................................284
8.3.7 Miscellaneous Reagents.................................................................... 285
8.3.7.1 Tetranitromethane.............................................................. 285
8.3.7.2 Potassium Nitrosyl Disulfonate......................................... 286
8.3.7.3 Bisulfite.............................................................................. 286
References................................................................................................................. 286
Chapter 9 General Approaches for Chemical Cross-Linking.................................................... 297

9.1 Introduction.................................................................................................... 297
9.2 Classification of Cross-Linking Procedures................................................... 297
9.2.1 One-Step Cross-Linking Reactions................................................... 297
9.2.2 Two-Step Cross-Linking Reactions.................................................. 298
9.2.3 Three-Step Cross-Linking Reactions................................................300
9.2.4 Multistep Cross-Linking Reactions..................................................300
9.3 General Conditions for Cross-Linking........................................................... 303
9.3.1 Choice of Reaction Medium.............................................................. 303
9.3.2 Choice of Reaction Temperature and Time....................................... 303
9.3.3 Choice of Reactant Concentrations...................................................304
9.4 Cross-Linking Protocols for Commonly Used Reagents...............................304
9.4.1 Examples for Zero-Length Cross-Linker..........................................304
9.4.1.1 Cross-Linking a Peptide and a Protein Using EDC..........304
9.4.1.2 Cross-Linking of Porcine Luteinizing Hormone
with EDC to Study a and b Subunit Interactions............... 305
9.4.2 Examples for Homobifunctional Reagents........................................ 305
9.4.2.1 Bis-Imidoesters.................................................................. 305
9.4.2.2 Bis-N-Hydroxysuccinimide (NHS) Esters......................... 305
9.4.2.3 Bis-Maleimido Reagents....................................................306
9.4.2.4 Bis-α-Haloacetyl Reagents................................................307
9.4.3 Examples for Heterobifunctional Reagents.......................................307
9.4.3.1 Conjugation of Human Serum Albumin (HSA)
and Monoclonal Antibody (mAb) with SPDP...................308
9.4.3.2 Cross-Linking of Demineralized Bone Matrix (DBM)
and Monoclonal Antibody with Sulfo-SMCC...................308
9.4.4 Examples for Heterobifunctional Photosensitive Reagents...............309
9.4.4.1 Cross-Linking of Proteins with the Photoreagent
N-(4-Azido-2,3,5,6-Tetrafluorobenzyl)-3-
Maleimidylpropionamide (TFPAM-3)..............................309
9.4.4.2 Cross-Linking UvsY Hexamer Protein Complex
with the Photo-Reagent Ruthenium(II)
Tris-Bipyridyl Dichloride (Ru(II)bpy3Cl2)........................ 310
9.5 Cross-Linking Protocols Based on Biological Systems................................. 310
9.5.1 Soluble Macromolecules................................................................... 310
9.5.1.1 Cross-Linking Nonassociated Proteins............................. 310
9.5.1.2 Cross-Linking Multisubunit Complexes............................ 311
9.5.2 Membrane-Bound Proteins............................................................... 311
9.5.3 Nucleic Acids and Nucleic Acid–Protein Complexes....................... 312
x Contents
9.6 Conditions for Cleavage of Cross-Linked Complexes................................... 313

9.6.1 Disulfide Linkages............................................................................ 313
9.6.2 Glycol Bonds..................................................................................... 313
9.6.3 Azo Bonds......................................................................................... 313
9.6.4 Sulfone Linkages............................................................................... 313
9.6.5 Ester and Thioester Bonds................................................................ 314
9.6.6 Acetals, Ketals, and Orthoesters....................................................... 314
9.7 Reaction Complications.................................................................................. 314
9.7.1 General Considerations..................................................................... 314
9.7.2 Immunogenicity................................................................................ 315
9.7.3 Stability............................................................................................. 315
References................................................................................................................. 316
Chapter 10 Analysis of Cross-Linked Products........................................................................... 321

10.1 Introduction.................................................................................................... 321
10.2 Techniques...................................................................................................... 321
10.2.1 Size-Exclusion Chromatography....................................................... 321
10.2.2 Electrophoresis.................................................................................. 323
10.2.3 Light Scattering................................................................................. 323
10.2.4 Mass Spectrometry............................................................................ 325
References................................................................................................................. 326
Chapter 11 Applications of Chemical Cross-Linking to the Study of Biological

Macromolecules........................................................................................................ 327
11.1 Introduction.................................................................................................... 327
11.2 Determination of Tertiary Structures of Proteins.......................................... 328
11.2.1 Molecular Distances of Cross-Linking Reagents............................. 328
11.2.2 Examples of Interresidue Distance Measurements........................... 329
11.2.3 Examples of Applications to 3D Protein Structure Determination.......330
11.3 Determination of Quaternary Structures of Proteins..................................... 331
11.3.1 Nearest Neighbor Analysis................................................................ 332
11.3.2 Examples of Determination of Geometric Arrangements
of Subunits within a Multiprotein Complex...................................... 332
11.3.2.1 Subunit Arrangement in Hexameric Protein Oligomers......332
11.3.2.2 Three-Dimensional Arrangement of F1-Adedosine
Triphosphatase Subunits.................................................... 332
11.3.2.3 Three-Dimensional Structure of the RNA
Polymerase II–TFIIF Complex.......................................... 333
11.3.2.4 Three-Dimensional Structure of the Ribosome................ 334
11.3.2.5 Organization of Contractile Protein Systems.................... 336
11.4 Determination of Protein–Protein Interactions.............................................. 338
11.4.1 Examples of Determinations of Protein–Protein
Interactions of Soluble Proteins........................................................ 338
11.4.2 Examples of Protein–Protein Interactions of
Membrane-Bound Proteins: Ligand–Receptor Interactions............. 339
11.4.2.1 Interactions between Membrane-Bound Proteins.............340
11.4.2.2 Interactions between Membrane-Bound Proteins
and Soluble Proteins..........................................................340
11.5 Detection of Protein Conformational Changes.............................................. 341
Contents xi
11.6 Determination of Nucleic Acid Interaction and Nucleic

Acid– Protein Interaction................................................................................344
11.7 Effects of Cross-Linking on Structural Stability and Biological Activity..... 345
11.7.1 Increased Structural Stability and Activity....................................... 345
11.7.2 Conformation Lock........................................................................... 345
References.................................................................................................................346
Chapter 12 Applications of Chemical Conjugation in the Preparation

of Immunoconjugates and Immunogens................................................................... 353
12.1 Introduction.................................................................................................... 353
12.2 Preparation of Immunoconjugates................................................................. 353
12.2.1 Components of Enzyme Immunoconjugates.................................... 354
12.2.1.1 Enzymes............................................................................ 354
12.2.1.2 Antibodies and Their Fragments....................................... 356
12.2.2 Introduction of Thiol Groups into Immunoglobulins....................... 356
12.2.3 Preparation of Horseradish Peroxidase Immunoconjugates............. 358
12.2.3.1 Conjugation with Amino- and Thiol-Directed
Cross-Linkers.................................................................... 358
12.2.3.2 Conjugation through Disulfide Formation......................... 358
12.2.3.3 Conjugation with Glutaraldehyde...................................... 359
12.2.3.4 Conjugation Using Periodate Oxidation............................360
12.2.3.5 Zero-Length Conjugation In Vacuo...................................360
12.2.3.6 Conjugation with Miscellaneous Cross-Linkers................360
12.2.4 Preparation of Alkaline Phosphatase Immunoconjugates................ 361
12.2.4.1 Conjugation with Amino- and Thiol-Directed Reagents...... 361
12.2.4.2 Conjugation with Glutaraldehyde...................................... 361
12.2.4.3 Conjugation with Periodate Oxidation.............................. 362
12.2.4.4 Zero-Length Conjugation.................................................. 362
12.2.5 Preparation of α-d-Galactosidase Immunoconjugates...................... 362
12.2.5.1 Conjugation with Amino- and Thiol-Directed
Reagents........................................................................ 362
12.2.5.2 Conjugation with Thiol Group–Directed Dimaleimides......362
12.2.5.3 Conjugation with Phenolate and Thiol
Group–Directed Reagent................................................... 363
12.2.5.4 Conjugation with Glutaraldehyde......................................364
12.2.6 Preparation of Glucose-6-Phosphate Dehydrogenase
Immunoconjugates............................................................................364
12.2.7 Preparation of Glucose Oxidase Immunoconjugates........................364
12.2.7.1 Coupling with N-Ethoxycarbonyl-2-Ethoxy-
1,2-Dihydroquinoline.........................................................364
12.2.7.2 Coupling with Amino- and Thiol-Directed Reagents.......364
12.2.7.3 Coupling with Other Cross-Linkers..................................364
12.2.8 Preparation of Other Enzyme Immunoconjugates............................364
12.2.9 Preparation of Nonenzyme Protein Immunoconjugates.................... 365
12.2.10 Coupling Enzymes to Proteins Other than Antibodies..................... 365
12.2.10.1 Examples of Conjugations of Enzymes
and Biotin-Binding Proteins............................................ 365
12.2.10.2 Examples of Conjugation of Enzymes and Other
Proteins............................................................................ 365
12.2.10.3 Examples of Conjugation of Enzymes and Antigens....... 366
xii Contents
12.3 Preparation of Immunogens........................................................................... 366

12.3.1 Examples of Conjugation of Hapten to Albumin.............................. 366
12.3.2 Examples of Conjugation of Hapten to Keyhole Limpet
Hemocyanin...................................................................................... 368
12.3.3 Examples of Hapten Conjugation to Other Carriers......................... 368
12.4 Characterization of Conjugation Methods..................................................... 369
References................................................................................................................. 369
Chapter 13 Application of Chemical Conjugation for the Preparation of Immunotoxins

and Other Drug Conjugates for Targeting Therapeutics........................................... 377
13.1 Introduction.................................................................................................... 377
13.2 Targeting Agents and Toxins.......................................................................... 377
13.2.1 Choice of Targeting Agents............................................................... 377
13.2.1.1 Antibodies.......................................................................... 378
13.2.1.2 Other Naturally Occurring Molecules............................... 378
13.2.1.3 Synthetic Peptides and Nucleotides................................... 380
13.2.1.4 Synthetic Polymers............................................................ 381
13.2.2 Choice of Toxins................................................................................ 382
13.3 Preparation of Therapeutic Conjugates.......................................................... 383
13.3.1 Choice of Cross-Linking Reagents................................................... 383
13.3.2 Conjugation through Disulfide Bond................................................ 384
13.3.2.1 Coupling with N-Succinimidyl-3-(2-Pyridylodithio)
propionate.......................................................................... 384
13.3.2.2 Coupling with Other Disulfide Generating Agents........... 387
13.3.3 Conjugation through Thioether Linkage........................................... 390
13.3.3.1 Use of Iodoacetyl Compounds........................................... 390
13.3.3.2 Use of Amino and Thiol Directed Cross-Linkers............. 392
13.3.4 Conjugation with Activated Chlorambucil........................................ 392
13.3.5 Conjugation with Acid-Labile Cross-Linkers................................... 393
13.3.6 Conjugation with Photocleavable Cross-Linkers.............................. 396
13.3.7 Coupling through Carbohydrate Residues........................................ 396
13.3.7.1 Use of Intrinsic Carbohydrate Moieties............................. 396
13.3.7.2 Use of Polysaccharide Spacers.......................................... 398
13.3.8 Conjugation Using Avidin–Biotin Linkage....................................... 398
13.3.9 Conjugation Using Enzymes............................................................. 399
13.3.10 Conjugation Using Solid-Phase Procedures...................................... 399
13.3.11 Conjugation with Glutaraldehyde and Carbodiimides......................400
References.................................................................................................................400
Chapter 14 Application of Chemical Conjugation to Solid-State Chemistry..............................409

14.1 Introduction....................................................................................................409
14.2 Functionalities of Matrices.............................................................................409
14.3 Protein Immobilization by Matrix Activation................................................ 412
14.3.1 Activation of Hydroxyl Groups......................................................... 412
14.3.2 Activation of Carboxyl Groups......................................................... 413
14.3.3 Activation of Acyl Hydrazide............................................................ 413
Contents xiii
14.3.4 Activation of Amines........................................................................ 414

14.3.4.1 Use of Nitrous Acid........................................................... 414
14.3.4.2 Use of Phosgene and Thiophosgene.................................. 414
14.3.4.3 Use of Cyanogen Bromide................................................. 414
14.3.5 Activation of Polyacrylonitrile.......................................................... 415
14.4 Cross-Linking Reagents Commonly Used for Immobilization
of Biomolecules.............................................................................................. 415
14.4.1 Use of Zero-Length Cross-Linking Reagents................................... 415
14.4.2 Use of Mono- and Homobifunctional Cross-Linkers........................ 417
14.4.2.1 Glutaraldehyde................................................................... 417
14.4.2.2 Chloroformates and Carbonyldiimidazole........................ 417
14.4.2.3 Heterocyclic Halides.......................................................... 418
14.4.2.4 Bisoxiranes........................................................................ 418
14.4.2.5 Divinylsulfone................................................................... 419
14.4.2.6 Quinones............................................................................ 419
14.4.2.7 Transition Metal Ions......................................................... 419
14.4.2.8 Other Homobifunctional Cross-Linkers............................ 419
14.4.3 Use of Heterobifunctional Cross-Linkers......................................... 420
14.4.3.1 Monohalogenacetyl Halide................................................ 420
14.4.3.2 Epichlorohydrin................................................................. 420
14.4.3.3 Amino- and Thiol-Group–Directed Reagents.................. 421
14.5 Immobilization by Cross-Linking through Carbohydrate Chains................. 422
14.6 Examples of Applications of Solid-Phase Immobilization Chemistry.......... 422
14.6.1 Affinity Chromatography.................................................................. 422
14.6.2 Biosensors......................................................................................... 425
14.6.3 Microarrays....................................................................................... 426
14.6.3.1 DNA Microarrays.............................................................. 426
14.6.3.2 Protein/Peptide Microarrays.............................................. 431
14.6.3.3 Antibody Microarrays....................................................... 433
14.6.3.4 Carbohydrate Microarrays................................................. 434
14.6.4 Industrial Applications...................................................................... 435
References................................................................................................................. 436
Appendix A: Amino Group–Directed Homobifunctional Cross-Linkers..................................... 447

Appendix B: Sulfhydryl Group–Directed Homobifunctional Cross-Linkers............................... 483
Appendix C: Phenolate- and Imidazolyl-Group–Directed Reagents: Bisdiazonium Precursors........ 513
Appendix D: Group Selective Heterobifunctional Cross-Linkers................................................. 515
Appendix E: Photoactivatable Heterobifunctional Cross-Linking Reagents................................ 549
Preface
It has been 20 years since the publication of Chemistry of Protein Conjugation and Cross-Linking.
During this period, great advances have been made in the area of cross-linking of biological
molecules. New cross-linking reagents, including multifunctional cross-linkers, have been devel-
oped and synthesized. The completion of the human genome project has opened a new area for
studying nucleic acid and protein interactions using nucleic acid cross-linking reagents. Advances
have also been made in the area of biosensors and microarray biochips for the detection and analysis
of genes, proteins, and carbohydrates. In addition, physical techniques, especially novel mass spec-
trometry approaches with unprecedented sensitivity and resolution, have facilitated the analysis of
cross-linked products. All these advances warrant a new edition of the old text.
This book offers an overview of the chemical principles underlying the processes of cross-
linking and conjugation. Attempts have been made to list all, or at least most, cross-linking reagents
published in the literature up to now, covering monofunctional, homobifunctional, heterobifunc-
tional, and multifunctional as well as zero-length cross-linkers. A general methodology for experi-
mental applications of these cross-linkers is provided. This book also includes reviews on the use
of these reagents in studying protein tertiary structures, geometric arrangements of subunits within
complex proteins and nucleic acids, near neighbor analysis, protein-to-protein or ligand–receptor
interactions, and conformational changes of biomolecules. In addition, applications in the area of
immonoconjugation for immunoassays, immunotoxin for targeted therapy, microarray technology
for analysis of various biomolecules, and solid state chemistry for immobilizations are presented.
Therefore, this book is intended to be a valuable reference for multidisciplinary approaches.
It has taken a long time to prepare this book, and the authors thank the publishers for their
enormous patience. Shan Wong, as always, is indebted to Lee-Jun Wong for her patience and under-
standing during the entire book project (yet again!) as well as for her patience and understanding in
day-to-day life. David Jameson wishes to thank Marcin Bury and Nicholas James, in his laboratory,
for proofreading much of the book, and Dudley Williams and Don Laudicina from Allergan, Inc.
for helpful discussions on mass spectrometry. In addition, he wishes to thank Sandra Kopels for her
unwavering support in all aspects of his life!
xv
Authors
Shan S. Wong, PhD, has recently retired from the National Institutes of Health, where he served
as a scientific review administrator and a program officer. In his latter capacity, he oversaw sci-
entific programs in the area of alternative and complementary medicine. Previously, he served as
director of clinical chemistry at Hermann Hospital and Lyndon B. Johnson General Hospital in
Houston, Texas, and as a faculty at the University of Texas Health Science Center at Houston, Texas.
Before joining the University of Texas, Dr. Wong was a full professor of chemistry at the University
of Massachusetts at Lowell, Lowell, Massachusetts. In addition to teaching at the University of
Massachusetts at Lowell, he also taught chemistry courses at Denison University, Granville, Ohio,
and Ohio State University, Columbus, Ohio.
Dr. Wong graduated in 1970 from the Oregon State University, Corvallis, Oregon, with
a BS in chemistry and received his PhD in 1974 from the Department of Chemistry at Ohio
State University. After doing postdoctoral work at Temple University, Philadelphia, and Ohio State
University, he joined the University of Massachusetts at Lowell.
Dr. Wong has published extensively in various scientific journals in the area of enzymology and
clinical chemistry. He has received numerous honors and awards and was active in various profes-
sional societies.
David M. Jameson, PhD, joined the Department of Cell and Molecular Biology at the John A.
Burns School of Medicine at the University of Hawaii in 1989, where he is presently a full professor.
Before moving to Hawaii he was on the faculty of the Pharmacology Department at the University
of Texas Southwestern Medical School in Dallas.
Dr. Jameson received his BS in chemistry from Ohio State University in 1971 and his PhD in
biochemistry from the University of Illinois at Urbana-Champaign in 1978. His thesis advisor was
Gregorio Weber, who laid the foundations of modern fluorescence spectroscopy. He carried out post-
doctoral research at the Université Paris-Sud at Orsay before returning to the University of Illinois
for a postdoctoral period in Gregorio Weber’s laboratory. In 1983, he joined the Pharmacology
Department at the University of Texas Southwestern Medical Center at Dallas as an assistant
professor. In 1989, he moved to the University of Hawaii.
Dr. Jameson’s primary research focus has always been the development and application of flu-
orescence approaches for the study of biomolecular interactions, in particular protein–protein and
protein–ligand interactions. He has published extensively in this area (~130 publications to date)
and has received funding from the National Science Foundation, the American Heart Association,
and the National Institutes of Health. He has also received the Established Investigator Award
from the American Heart Association and the 2004 Gregorio Weber Award for Excellence in
Fluorescence Theory and Application. He lectures at numerous fluorescence workshops around
the world and is co-organizer of the International Weber Symposium on Innovative Fluorescence
Methodologies in Biochemistry and Medicine held every three years in Hawaii.
xvii
1 Overview of Protein
Conjugation
Completion of the human genome project has opened up tremendous opportunities for the study of
complex biological processes at the molecular level.1 We now know that only about 1%–2% of the
genome encodes for proteins.2 These gene products perform all cellular functions from metabolism
to developmental control to apoptosis and cell death. In order to comprehend how the cell works
and thus the whole organism, it is important to know the detailed functions of these proteins. From
the start, we need to elucidate their three-dimensional (3D) structures and their relationships and
interactions with other proteins. Some proteins, such as myoglobin, exist freely in the cytosol as
monomers. Others associate into protein complexes, the simplest of which are dimers, either with
another identical protein subunit (homodimer), for example, malate dehydrogenase, or a different
protein subunit (heterodimer), for example, creatine kinase.3 Still others may associate into higher
architectural organizations such as tetramers, pentamers, hexamers, and larger multicomponent
aggregates. Examples of these organizations are shown in Figure 1.1.4–8 As the number of compo-
nents increases, so do the complexities of the protein interactions. It then becomes more difficult to
elucidate the sites of protein contacts and the 3D dispositions of the individual subunits.
Some proteins associate to regulate or alter their activities. For example, bovine galactosyltrans-
ferase normally transfers galactose from UDP-galactose to N-acetylglucosamine, either free or as
the terminal sugar of glycoproteins.9 However, when it binds with bovine α-lactalbumin, glucose
becomes the preferred galactose acceptor leading to the formation of lactose.10 The protein–protein
interactions become an important aspect of the regulatory process.
Association of proteins as a regulatory process is seen practically in all signaling pathways. An
obvious example is that of the hedgehog (Hh) signaling pathway, which is depicted in Figure 1.2
for Drosophila.11–13 In Drosophila, the Hh signaling molecules associate with the Patched (Ptc)
receptor, a 12-pass membrane protein. This interaction activates the Smoothened (Smo) G-protein
leading to the release of active CI155 from a microtubule, Cos2, Fu, SuFu, and CI protein complex.14
The active CI155 ultimately controls the transcription of specific target genes. In the absence of
Hh, Ptc interacts with and inhibits Smo, a seven-pass membrane protein, and Fu, Cos2, and SuFu
bind to CI, preventing its activation and retaining it in the cytoplasm. CI in the complex is cleaved
to yield CI75 upon phosphorylation by Adenylate Cyclase (AC)-induced protein kinase A (PKA),
which involves Slimb and GSK3H. This culmination of protein binding events leads to inhibition of
transcription. It is obvious that Cos2, Fu, and SuFu play multiple and complex roles in CI control.15
In order to understand the details of the signal transduction pathway, it is necessary to reveal exactly
how the individual proteins in the assembly of complex protein networks interact with each other.
In this example, it would be of interest to understand how the association of Hh with Ptc alters its
protein structure such that Smo is activated. It would also be of interest to know the structural orga-
nization of the microtubule, Cos2, Fu, SuFu, and CI complex. Even the activation of PKA through
CA is an interesting regulatory process.
Cyclic AMP-dependent PKA consists of two regulatory and two catalytic subunits.16 In its tetra-
meric holoenzyme form, the catalytic subunits are inactive. However, binding of cAMP to the regu-
latory subunits results in the dissociation of the ternary complex into a regulatory dimer and two
active catalytic monomers as represented in Figure 1.3, demonstrating another level of regulation
through protein–protein interactions. Using the lysine-specific bifunctional cross-linking reagent
1
2 Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation
(A) (B) (C)
(D) (E)
FIGURE 1.1 (See color insert.) Examples of different molecular structures of proteins. (A) Myoglobin
molecule. (After Phillips, S. E. V. J. Mol. Biol., 142, 531, 1980.) (B) Dimeric creatine kinase. (After
Shen, Y. Q. et al., Acta Crystallogr. D Biol. Crystallogr., 57, 1196, 2010.) (C) Tetrameric hemoglobin.
(After Paoli, M. et al., J. Mol. Biol., 256, 775, 1996.) (D) Bovine cytochrome C oxidase with 2 copies
of 13 different components. (From Shinzawa-Itoh, K. et al., EMBO J., 26, 1713, 2007. With permission.)
(E) Yeast 80S ribosome of multicomponent proteins and RNA. (Reprinted from Cell, 107, Beckmann, R.
et al., Architecture of the protein-conducting channel associated with the translating 80S ribosome, 361,
Copyright 2001, with permission from Elsevier.)
dimethyl suberimidate, Charlton et al.17 have demonstrated the dynamics of dissociation of the tet-
ramer in the presence of cAMP and MgATP. Other information on the 3D architecture should be
available using the same technique.
There are numerous other ways in which proteins interact with each other in complex biological
processes. In addition, proteins also interact with nucleic acids. As we have seen above in the Hh
signaling pathway, CI regulates the cell cycle by binding to nuclear DNA to modulate gene expres-
sion. Also, in the structure of ribosomes, protein–RNA interactions are of paramount importance.18
Such protein–nucleic acid interactions are significant in diverse genetic networks and protein path-
ways. Determining the interactions of protein–protein and protein–nucleic acid systems is crucial to
understanding how biological systems function and how they contribute to cellular and organismal
phenotypes.
There are many methods to study protein structures and their interactions. X-ray crystallography
has been successfully used to elucidate tens of thousands of protein structures, from monomers to
multicomponents complexes. However, proteins in the biological environment are dynamic, and
x-ray structures, being restricted by crystal packing, are inherently static, although some measures
of the elasticity of these crystal structures are available.19 This powerful technique has even been
able to elucidate fairly high-resolution structures of ribosomes.18 Because it is based on crystallog-
raphy, the technique is limited in studying protein interactions that occur transiently as in signal
transduction pathways. In recent years, nuclear magnetic resonance (NMR) has become a powerful
method for elucidating protein structures in solution, but is limited to relatively small proteins,
for example, proteins less than about 30 kDa.20 The field of computationally based protein structure
Overview of Protein Conjugation 3
In presence of Hh In absence of Hh
Hh N
o
Sm AC
C
Ptc teins
C G-pro
Hh
N
Ptc C
Mi
Fu
cro
tub
SuFu PKA
Cos2
ule
N o N
Sm C
CI155 P
s
ein P P
r ot P
-p Fu
G
SuFu Slimb
Cos2
bule
crotu
Mi CI155 CIl75
GSK3H
Release of
microtubule
CI155
Target GRepression
of target genes
Target genes (Hh, Dpp)
(WNT, Dpp, Ptc) CIl75
CI155
FIGURE 1.2 Hedgehog signaling pathway in Drosophila.
C
C R R -cAMP
+ cAMP
C R R -cAMP
C
Inactive PKA Regulatory Free active

holoenzyme dimer catalytic subunit
FIGURE 1.3 Activation of PKA by cAMP. Cyclic-AMP binds to the regulatory subunits causing the release
of active free catalytic subunits.
prediction has made significant advances in recent years but is not likely to replace experimental
structure determinations in the near future.21 Other proteomic approaches include mass spectrometry,22
microarrays,23 the two-hybrid system,24,25 coprecipitation, and computational statistical analy-
sis.26 Modern molecular biology techniques of gene knockout,27 knock-down approaches,28 and use
of small molecular inhibitors29 have facilitated the determination of protein functions. Of these
methods, however, chemical cross-linking provides detailed characterization of protein–protein

interactions and enables non-covalent interactions to be captured in vivo.30
Chemical cross-linking of biological components is a special application of chemical modifica-
tion of proteins.31 The history of chemical modifications dates back to the 1920s when enzymes
were shown to be proteins.32 At this early stage, the technique was used mainly to identify the
particular amino acids responsible for their catalytic activity. Due to the lack of instrumentation
and analytical methods, progress in the development of new procedures and reagents was initially
slow. It was only during and immediately after World War II that significant advances started to
take place. The first reviews on the subject were published in 1947.33,34 Since then, the application
of chemical modification has grown exponentially hand-in-hand with the development of biochemi-
cal analysis techniques. For example, an effective procedure for the determination of amino acid
sequences was introduced by Edman and Begg in 1956.35 In 1960, automated amino acid analyz-
ers became available.36 Various new ion exchange and gel filtration chromatography media were
developed and different forms of gel electrophoresis also became commonplace during the same
time period. Numerous review articles and specialized monographs began to appear during the last
30 years.36–44 New directions such as photoaffinity labeling40,45,46 and active-site-directed affinity
reagents were developed.40,47–49 The very powerful method of site-directed mutagenesis is also pro-
viding results beyond those attainable by chemical modification, especially for the determination of
the function of individual residues in the structure and reactivity relationship of a molecule.50
Cross-linking as a special form of chemical modification has particular capabilities of its own,
unparalleled by in vitro mutagenesis.41,51,52 The process involves joining of two molecular compo-
nents by a covalent bond achieved through the use of cross-linking reagents. The components may
be proteins, peptides, drugs, nucleic acids, or solid particles. The chemical cross-linkers are multi-
functional reagents containing reactive functional groups derived from classical chemical modifica-
tion agents. Bifunctional cross-linkers are the most common. These reagents are capable of reacting
with the side chains of the amino acids of proteins. They may be classified into homobifunctional,
heterobifunctional, and zero-length cross-linkers. The zero-length cross-linkers are essentially
group-activating reagents, which cause the formation of a covalent bond between the components
without incorporation of any extrinsic atoms. Thus, dicyclohexyl carbodiimide, which has been
used extensively to bring about the formation of amide bonds between carboxyl and amino groups
in peptide synthesis, is an example of a zero-length cross-linker. The homobifunctional reagents
consist of two identical functional groups and the heterobifunctional reagents contain two different
types of reactive functional moieties. They therefore form bridges between the reactive amino acid
side chains in proteins. Homobifunctional reagents, such as dialkyl halides and bis-imidoesters,
were among the early cross-linkers developed,53–56 although formaldehyde and other reagents had
been used in the tanning industry many years prior without known chemical reactions. Since the
first application of a bifunctional reagent by Zahn in the 1950s,53–55 research in this area has flour-
ished, particularly during the last two decades. The introduction of photoactivatable aryl azides
marked the beginning of heterobifunctional reagents.46,57–59 Further advancement in the application
of these reagents has led to the design and synthesis of cleavable bifunctional compounds.60,61 Over
300 cross-linkers have now been synthesized, and more are forthcoming. The diversity of these
molecules is as complicated as organic chemistry itself, limited only by the creative imaginations of
the researchers. The presence of the Journal of Bioconjugate Chemistry will attest to the complex-
ity of these reagents and their usefulness in various applications.
The application of chemical cross-linking is multidisciplinary, ranging from basic protein bio-
chemistry to applied biotechnology and engineering, and from immunology to medicine. These
reagents have been used to stabilize tertiary structures of proteins,62,63 to study protein–protein
interactions of subunits in oligomeric proteins,64 and in complex structures such as ribosomes,65
to determine distances between reactive groups within or between protein subunits,56,66 to attach
ligands to solid supports,67 and to identify membrane receptors.68,69 Applications in the pharmaceu-
tical area have led to the coupling between target-specific proteins and metal-chelating agents for
diagnostics in in vivo imaging of human patients,70,71 as well as toxins and enzymes for therapeu-
tics.72–76 With the advent of enzyme-linked immunoassay and genetic probe technology, chemical
cross-linking provides a means for preparation of enzyme-immunoglobulin conjugates and DNA
probes.77–79 These applications will be reviewed and summarized in the second half of this book.
Although the terms cross-linking and conjugation are often used interchangeably, there is a fine
distinction in connotation between them. Cross-linking usually refers to the joining of two molecu-
lar species that have some sort of affinity between them, that is, they either exist as an aggregate or
can associate under certain conditions. Thus, the chemical bonding between a ligand and its recep-
tor is usually referred to as cross-linking. Similarly, cross-linking is used for the covalent bonding
between subunits of enzymes. Conjugation, on the other hand, denotes the coupling of two unre-
lated species. For example, the linking between an enzyme and an immunoglobulin is conjugation.
The product is referred to as a conjugate, and in this case, an immunoconjugate.
No matter whether it is conjugation or cross-linking, two types of products usually result from a
cross-linking reaction. One is derived from intramolecular cross-linking, the other as a consequence
of intermolecular joining of two or more species. The possible chemical reactions of a cross-linking
reagent with a protein dimer are illustrated in Figure 1.4. As an example, suppose a protein exists
as a monomer in dilute solutions and associates or interacts with another protein molecule at higher
concentrations. When the monomeric form reacts with the chemical reagent, intramolecular cross-
linking will take place since protein molecules are, on average, far apart. At high concentrations,
the molecules will be in closer proximity or will associate to form dimeric or oligomeric aggre-
gates. Under these conditions, the reagent will provide intermolecular cross-linking. Thus, at very
low concentrations, intramolecular bonding prevails, whereas intermolecular coupling is important
at high concentrations. Cross-linkers have been used to determine distances between two reac-
tive groups in a protein that is close in space, particularly those at the active sites of enzymes.80–83
Intermolecular cross-linking may conjugate molecules of the same kind or of different kinds to
form homopolymers or heteropolymers, respectively, thus providing a means for the preparation
of high-molecular-weight complexes. Intermolecular coupling of different kinds of proteins also
provides a tool for the study of antibody–antigen interactions, multienzyme complexes, membrane
X–X
X –X
FIGURE 1.4 Cross-linking reactions of a hypothetical protein dimer. Reaction of the cross-linker, X–X,
with the monomeric form usually yields intramolecular cross-linking such as in the case of dilute protein solu-
tions. At high protein concentrations or at conditions where the protein molecules associate, intermolecular
cross-linking will result.
protein structures, hormone and receptor recognitions, and other protein–protein interactions at the
quaternary structural level.
To help understand the basic principles of reactions of these cross-linking reagents, this book
begins with a review of the chemical reactivity of amino acid side chains and their reactions with
specific chemical reagents. With the background of chemical modification, bifunctional reagents
are introduced. All the existing cross-linking reagents are surveyed and classified into homo-, het-
ero-, and zero-length cross-linkers. Various specific applications of these reagents are mentioned.
Examples of conjugation are provided as well as the conditions for the reaction. The reader should
find this book useful not only as a reference for the basic information about cross-linking reagents
but also as a handbook for experimental application of these reagents.
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—that he even makes some rather serious mistakes from the mere
“point of view of the examiner”—is of course undeniable. He has a
good deal of the merely traditional mode of Renaissance respect for
classical—and for some modern—authority. That, for instance, there
is a good deal to be said, and that not only from the point of view of
Ben Jonson, against Spenser’s half-archaic half-rustic dialect in the
Calendar, few would refuse to grant. But Theocritus did use dialect:
it would not in the least matter whether either he or Virgil did not;
and if it did, what has the modern and partly vernacular name of
Sannazar to do with the matter? It can only be replied that Spenser,
by permitting “E. K.'s” annotation, did much to invite this sort of
criticism; and that Englishmen’s reluctance to rely on the inherent
powers of the English language was partly justified (for hardly any
dead poet but Chaucer and no dead prose-writers but Malory and
perhaps Berners deserved the title of “great”), partly came from very
pardonable ignorance.
It has been already observed that Sidney is by no means
peremptory about the “new versifying”; and in particular has
absolutely none of the craze against rhyme as rhyme which
animated persons of every degree of ability, from Milton to
Stanyhurst, during more than a century. His remarks on versification
are, however, too scanty to need much comment.
There remain his two major heresies, the declaration that verse is
not inseparable from poetry, and the denunciation of tragi-comedy.
and major In both the authority of the ancients must again
heresies. bear good part of the blame, but in both he has
additional excuses. As to the “pestilent heresy of prose poetry,” he is
at least not unwilling to argue on the hypothesis that verse were
necessary to poetry, though he does not think it is. He is quite sure
that verse is anyhow a nobler medium than prose. As for the plays,
there is still more excuse for him. His classical authorities were quite
clear on the point; and as yet there was nothing to be quoted on the
other side—at least in English. Spanish had indeed already made the
experiment of tragi-comic and anti-unitarian treatment; but I do not
think any of the best Spanish examples had yet appeared, and there
is great difference between the two theatres. In English itself not
one single great or even good play certainly existed on the model at
Sidney’s death; and, from what we have of what did exist, we can
judge how the rough verse, the clumsy construction, or rather
absence of construction, the entire absence of clear character-
projection, and the higgledy-piggledy of huddled horrors and
horseplay, must have shocked a taste delicate in itself and nursed
The excuses upon classical and Italian literature. And it is
of both, noteworthy that even Gorboduc, with all its
regularity and “Senecation,” does not bribe Sidney to overlook at
least some of its defects. He is here, as elsewhere,—as indeed
throughout,—neither blind nor bigoted. He is only in the position of
a man very imperfectly supplied with actual experiments and
observations, confronted with a stage of creative production but just
improving from a very bad state, and relying on old and approved
methods as against new ones which had as yet had no success.
And had his mistakes been thrice what they are, the tone and
temper of his tractate would make us forgive them three times over.
and their That “moving of his heart as with a trumpet”
ample communicates itself to his reader even now, and
compensatio shows us the motion in the heart of the nation at
n.
large that was giving us the Faerie Queene, that
was to give us Hamlet and As You Like It. What though the
illustrations sometimes make us smile? that the praise of the moral
and political effects of poetry may sometimes turn the smile into a
laugh or a sigh? Poetry after all, like all other human things, has a
body and a soul. The body must be fashioned by art—perhaps the
body is art; but the soul is something else. The best poetry will not
come without careful consideration of form and subject, of kind and
style; but it will not necessarily come with this consideration. There
must be the inspiration, the enthusiasm, the afflatus, the glow; and
they are here in Sidney’s tractate. Nor must we fail to draw
attention, once more, to the difference of the English critical spirit
here shown as regards both Italian and French.
In the decade which followed,[247] three notable books of English
criticism appeared, none of them exhibiting Sidney’s afflatus, but all
showing the interest felt in the subject, and one exceeding in
method, and at least attempted range, anything that English had
King James’s known, or was to know, for more than a century.
Reulis and These were King James the First’s (as yet only “the
Cautelis. Sixth’s”) Reulis and Cautelis to be observit and
eschewit in Scottis Poesie, 1585; William Webbe’s Discourse of
English Poesie, next year; and the anonymous Arte of English
Poesie, which appeared in 1589, and which (on rather weak
evidence, but with no counter-claimant) is usually attributed to
George Puttenham.[248]
The first is the slightest; but it is interesting for more than its
authorship. It was attached to James’s Essays of a Prentice in the
Divine Art, of which it gives some rules: it shows that Buchanan had
taken pains with his pupil; and it also exhibits that slightly scholastic
and “peddling,” but by no means unreal, shrewdness and acumen
which distinguished the British Solomon in his happier moments. It is
characteristic that James is not in the least afraid of the charge of
attending to mint, anise, and cumin. He plunges without any
rhetorical exordium into what he calls “just colours”—do not rhyme
on the same syllable, see that your rhyme is on accented syllables
only, do not let your first or last word exceed two or three syllables
at most. This dread of polysyllables, so curious to us, was very
common at the time: it was one of the things from which
Shakespeare’s silent sovereignty delivered us by such touches of
spell-dissolving mastery as
“The multitudinous seas incarnadine.”
Then he passes to feet, of which he practically allows only the iamb;

while he very oddly gives the word “foot” to the syllable, not the
combination of syllables; and lays down the entirely arbitrary rule
that the number of “feet”—i.e., syllables—must be even, not odd.
There is to be a sharp section (“cæsura”) in the middle of every line,
long or short; and the difference of long, short, and “indifferent”
(common) feet or syllables is dwelt upon, with its influence of
“flowing,” as the King calls rhythm. Cautions on diction follow, and
some against commonplaces, which look as if the royal prentice had
read Gascoigne, a suggestion confirmed elsewhere.[249] Invention is
briefly touched; and the tract finishes with a short account of the
kinds of verse: “rhyme”—i.e., the heroic couplet, “quhilk servis onely
for lang historieis”; a heroical stanza of nine lines, rhymed
aabaabbab; ottava rima, which he calls “ballat royal”; rhyme royal,
which he calls “Troilus verse”; “rouncifals,” or “tumbling verse”
(doggerel alliterative, with bob and wheel); sonnets; “common”
verse (octosyllable couplets); “all kinds of broken or cuttit verse,” &c.
The tract is, as has been said, interesting, because it is an honest,
and by no means unintelligent, attempt to make an English prosody,
with special reference to a dialect which had done great things in its
short day, but which had been specially affected—not to say
specially disorganised—by the revived and bastard alliteration of the
fifteenth century. Probably it was the study of French (where the
iamb had long been the only foot) which, quite as much as mere
following of Gascoigne, induced James to extend that crippling
limitation to English; and the same influence may be seen in his
insistence on the hard-and-fast section. These things (the latter of
which at least rather endeared him to Dr Guest)[250] are, of course,
quite wrong; but they express the genuine and creditable desire of
the time to impose some order on the shambling doggerel of the
generation or two immediately preceding. We find the same
tendency even in Spenser, as far as rigid dissyllabic feet and sections
are concerned; and it is certainly no shame for the Royal prentice to
follow, though unknowing, the master and king of English poetry at
the time when he wrote.
One would not, however, in any case have expected from James
Webbe’s evidence of the root of the matter in poetry. There
Discourse. is more of this root, though less scholarship and
also more “craze,” in the obscure William Webbe, of whom we know
practically nothing except that he was a Cambridge man, a friend of
Robert Wilmot (the author of Tancred and Gismund) and private
tutor to the sons of Edward Sulyard of Flemyngs, an Essex squire.
The young Sulyards must have received some rather dubious
instruction in the classics, for Webbe, in his inevitable classical
exordium, thinks that Pindar was older than Homer, and that Horace
came after—apparently a good while after—Ovid, and about the
same time as Juvenal and Persius. He was, however, really and
deeply interested in English verse; and his enthusiasm for Spenser
—“the new poet,” “our late famous poet,” “the mightiest English poet
that ever lived,” is, if not in every case quite according to knowledge,
absolutely right on the whole, and very pleasant and refreshing to
read. It is, indeed, the first thing of the kind that we meet with in
English; for the frequent earlier praises of Chaucer are almost always
long after date, always uncritical, and for the most part[251] much
rather expressions of a conventional tradition than of the writer’s
deliberate preference.
It was Webbe’s misfortune, rather than his fault, that, like his idol
(but without that idol’s resipiscence), and, like most loyal Cambridge
men, with the examples of Watson, Ascham, and Drant before them,
Slight in he was bitten with “the new versifying.” It was
knowledge, rather his fault than his misfortune that he seems to
have taken very little pains to acquaint himself with the actual
performance of English poetry. Even of Gower he speaks as though
he only knew him through the references of Chaucer and others:
though three editions of the Confessio—Caxton’s one and
Berthelette’s two—were in print in his time. His notice of Chaucer
himself is curiously vague, and almost limited to his powers as a
satirist, while he has, what must seem to most judges,[252] the
astonishing idea of discovering “good proportion of verse and meetly
current style” in Lydgate, though he reproves him for dealing with
“superstitious and odd matters.” That he thinks Piers Plowman later
than Lydgate is unlucky, but not quite criminal. He had evidently
read it—indeed the book, from its kinship in parts to the Protestant,
not to say the Puritan, spirit, appealed to Elizabethan tastes, and
Crowley had already printed two editions of it, Rogers a third. But he
makes upon it the extraordinary remark, “The first I have seen that
observed the quantity of our verse without the curiosity of rhyme.”
What Webbe here means by “quantity,” or whether he had any clear
deliberate meaning at all, it is impossible to see: it is needless to say
that Langland is absolutely non-quantitative in the ordinary sense,
that if “quantity” means number of syllables he observes none, and
that he can be scanned only on the alliterative-accentual system. For
Gascoigne Webbe relies on “E. K.”; brackets “the divers works of the
old Earl of Surrey” with a dozen others; is copious on Phaer, Golding,
&c., and mentions George Whetstone and Anthony Munday in words
which would be adequate for Sackville (who is not named), and
hardly too low for Spenser; while Gabriel Harvey is deliberately
ranked with Spenser himself. Yet these things, rightly valued, are not
great shame to Webbe. If he borrows from “E. K.” some scorn of the
“ragged rout of rakehell rhymers,” and adds more of his own, he
specifies nobody; and his depreciation is only the defect which
almost necessarily accompanies the quality of his enthusiasm.
His piece, though not long, is longer than those of Gascoigne,
but Sidney, and King James. After a dedication (not
enthusiastic, more than excusably laudatory) to his patron
Sulyard, there is a curious preface to “The Noble Poets of England,”
who, if they had been inclined to be censorious, might have replied
that Master Webbe, while complimenting them, went about to show
that the objects of his compliment did not exist. “It is,” he says, “to
be wondered of all, and is lamented of many, that, while all other
studies are used eagerly, only Poetry has found fewest friends to
amend it.” We have “as sharp and quick wits” in England as ever
were Greeks and Romans: our tongue is neither coarse nor harsh, as
she has already shown. All that is wanted is “some perfect platform
or Prosodia of versifying”: either in imitation of Greeks and Latins, or
with necessary alterations. So, if the Noble Poets would “look so low
from their divine cogitations”, and “run over the simple censure” of
Master Webbe’s “weak brain,” something might, perhaps, be done.
if uncritical, The treatise itself begins with the usual
etymological definition of poetry, as “making,” and
the usual comments on the word “Vates”; but almost immediately
digresses into praise of our late famous English poet who wrote the
Shepherd’s Calendar and a wish to see his “English Poet” (mentioned
by E. K.), which, alas! none of us have ever seen. This is succeeded,
first by the classical and then by the English historical sketches,
which have been commented upon. It ends with fresh laudation of
Spenser.
Webbe then turns to the general consideration of poetry
(especially from the allegoric-didactic point of view), subject, kinds,
&c.; and it is to be observed that, though he several times cites
Aristotle, he leans much more on Horace, and on Elyot’s translations
from him and other Latins. He then proceeds to a rather
unnecessarily elaborate study of the Æneid, with large citations both
from the original and from Phaer’s translation, after which he returns
once more to Spenser, and holds him up as at least the equal of
Virgil and Theocritus. Indeed the Calendar is practically his theme all
through, though he diverges from and embroiders upon it. Then,
after glancing amiably enough at Tusser, and mentioning a
translation of his own of the Georgics, which has got into the hands
of some piratical publisher, he attacks the great rhyme-question, to
which he has, from the Preface onwards, more than once alluded.
Much of what he says is borrowed, or a little advanced, from
Ascham; but Webbe is less certain about the matter than his master,
and again diverges into a consideration of divers English metres,
always illustrated, where possible, from the Calendar. Still harking
back again, he decides that “the true kind of versifying” might have
been effected in English: though (as Campion, with better wits, did
after him) he questions whether some alteration of the actual Greek
and Latin forms is not required. He gives a list of classical feet (fairly
correct, except that he makes the odd confusion of a trochee and a
tribrach), and discusses the liberties which must be taken with
English to adjust it to some of them. Elegiacs, he thinks, will not do:
Hexameters and Sapphics go best. And, to prove this, he is rash
enough to give versions of his own, in the former metre, of Virgil’s
first and second eclogues, in the latter of Spenser’s beautiful
“Ye dainty nymphs that in this blessed brook.”
It is enough to say that he succeeds in stripping all three of every

rag of poetry. A translation of Fabricius’[253] prose syllabus of
Horace’s rules, gathered not merely out of the Ep. ad Pisones but
elsewhere, and an “epilogue,” modest as to himself, sanguine as to
what will happen when “the rabble of bald rhymes is turned to
famous works,” concludes the piece.
in On the whole, to use the hackneyed old phrase
appreciation. once more, we could have better spared a better
critic than Webbe, who gives us—in a fashion invaluable to map-
makers of the early exploration of English criticism—the workings of
a mind furnished with no original genius for poetry, and not much
for literature, not very extensively or accurately erudite, but
intensely interested in matters literary, and especially in matters
poetical, generously enthusiastic for such good things as were
presented to it, not without some mother-wit even in its crazes, and
encouraged in those crazes not, as in Harvey’s case, by vanity,
pedantry, and bad taste, but by its very love of letters. Average
dispositions of this kind were, as a rule, diverted either into active
life—very much for the good of the nation—or—not at all for its good
—into the acrid disputes of hot-gospelling and Puritanism. Webbe, to
the best of his modest powers, was a devotee of literature: for which
let him have due honour.
Puttenham—or whosoever else it was, if it was not Puttenham[254]
—has some points of advantage, and one great one of disadvantage,
Puttenham’s in comparison with Webbe. In poetical faculty there
(?) Art of is very little to choose between them—the abundant
English specimens of his own powers, which the author or
Poesie.
The Art of English Poesie gives (and which are eked
out by a late copy of one of the works referred to, Partheniades),
deserve the gibes they receive in one of our scanty early notices of
the book, that by Sir John Harington (v. infra). On the other hand,
Puttenham has very little of that engaging enthusiasm which atones
for so much in his contemporary. But this very want of enthusiasm
somewhat prepares us for, though it need not necessarily
accompany, merits which we do not find in Webbe, considered as a
critic. The Art of English Poesy, which, as has been said, appeared in
1589, three years later than Webbe’s, but which, from some
allusions, may have been written, or at least begun, before it, and
which, from other allusions, must have been the work of a man well
advanced in middle life, is methodically composed, very capable in
range and plan, and supported with a by no means contemptible
erudition, and no inconsiderable supply of judgment and common-
sense. It was unfortunate for Puttenham that he was just a little too
old: that having been—as from a fairly precise statement of his he
must have been—born cir. 1530-35, he belonged to the early and
uncertain generation of Elizabethan men of letters, the Googes and
Turbervilles, and Gascoignes, not to the generation of Sidney and
Spenser, much less to that of Shakespeare and Jonson. But what he
had he gave: and it is far from valueless.
The book is “to-deled” (as the author of the Ancren Riwle would
say) into three books—“Of Poets and Poesie,” “Of Proportion,” and
Its erudition. “Of Ornament.” It begins, as usual, with
observations on the words poet and maker,
references to the ancients, &c.; but this exordium, which is fitly
written in a plain but useful and agreeable style, is commendably
short. The writer lays it down, with reasons, that there may be an
Art of English as of Latin and Greek poetry; but cannot refrain from
the same sort of “writing at large” as to poets being the first
philosophers, &c., which we have so often seen.[255] Indeed we must
lay our account with the almost certain fact that all writers of this
period had seen Sidney’s Defence at least in MS. or had heard of it.
He comes closer to business with his remarks on the irreption of
rhyme into Greek and Latin poetry; and shows a better knowledge of
leonine and other mediæval Latin verse, not merely than Webbe, but
even than Ascham. A very long section then deals with the question
—all-interesting to a man of the Renaissance—in what reputation
poets were with princes of old, and how they be now contemptible
(wherein Puttenham shows a rather remarkable acquaintance with
modern European literature), and then turns to the subject or matter
of poesy and the forms thereof, handling the latter at great length,
and with a fair sprinkle of literary anecdote. At last he comes to
English poetry; and though, as we might expect, he does not go
behind the late fourteenth century, he shows rather more knowledge
than Webbe and (not without slips here and elsewhere) far more
comparative judgment. It must, however, be admitted that,
engaging as is his description of Sir Walter Raleigh’s “vein most lofty,
insolent, and passionate,” he does not show to advantage in the
patronising glance in passing at “that other gentleman who wrote
the late Shepherd’s Calendar,” contrasted with the description of the
Queen our Sovereign lady, “whose Muse easily surmounteth all the
rest in any kind on which it may please her Majesty to employ her
pen.” But here the allowance comes in: the stoutest Tory of later
days can never wholly share, though he may remotely comprehend,
the curious mixture of religious, romantic, patriotic, amatory, and
interested feelings with which the men of the sixteenth century
wrote about Gloriana.
The second book deals with Proportion, in which word Puttenham
includes almost everything belonging to Prosody in its widest sense
—staff, stanza, measure, metres, and feet, “cæsure,” rhyme, accent,
cadence, situation (by which he means the arrangement of the
Systematic rhymes), and proportion in figure. On most of these
arrangement heads he speaks more or less in accordance with his
fellows (though he very noticeably abstains from extreme
commendation or condemnation of rhyme), save that, for the
moment, he seems to neglect the “new versifying.” It is, however,
but for a moment. After his chapters on “proportion” in figure (the
fanciful egg, wheel, lozenge, &c., which he himself argues for, and
which were to make critics of the Addisonian type half-angry and
half-sad), he deals with the subject.
About this “new versifying” he is evidently in two minds. He had
glanced at it before (and refers to the glance now)[256] as “a nice and
scholastic curiosity.” However, “for the information of our young
makers, and pleasure to those who be delighted in novelty, and to
the intent that we may not seem by ignorance or oversight to
omit,”[257] he “will now deal with it.” Which he does at great length;
and, at any rate sometimes, with a clearer perception of the
prosodic values than any other, even Spenser, had yet shown. But he
does not seem quite at home in the matter, and glides off to a
discussion of feet—classical feet—in the usual rhymed English verse.
The third book is longer than the first and second put together,
and is evidently that in which the author himself took most pleasure.
It is called “Of Ornament,” but practically deals with the whole
and question of lexis or style, so that it is at least
exuberant common to Rhetoric and Poetics. In one respect,
indulgence in too, it belongs more specially to the former, in that
Figures.
it contains the most elaborate treatment of
rhetorical figures to be found, up to its time, in English literature.
Full eighty pages are occupied with the catalogue of these “Figures
Auricular” wherein Puttenham (sometimes rather badly served by his
pen or his printer) ransacks the Greek rhetoricians, and compiles a
list (with explanations and examples) of over one hundred and
twenty. It is preceded and followed by more general remarks, of
which some account must be given.
Beginning with an exordial defence of ornament in general,
Puttenham proceeds to argue that set speeches, as in Parliament,
not merely may but ought to be couched in something more than a
conversational style. This added grace must be given by (1)
Language, (2) Style, (3) Figures. On diction he has remarks both
shrewd and interesting, strongly commending the language of the
Court and of the best citizens, not provincial speech, or that of
seaports, or of universities, or in other ways merely technical. “The
usual speech of the Court and that of London and the shires lying
about London, within ten miles and not much above” is his norm.
There is also a noteworthy and very early reference to English
dictionaries, and a cautious section on neologisms introduced from
other tongues to fill wants. Style he will have reached by “a constant
and continual tenor of writing,” and gives the usual subdivision of
high, low, and middle. And so to his Figures.
The details and illustrations of the long catalogue of these invite
comment, but we must abstain therefrom. When the list is finished,
Puttenham returns to his generalities with a discussion of the main
principle of ornament, which he calls Decorum or “Decency,” dividing
and illustrating the kinds of it into choice of subject, diction, delivery,
and other things, not without good craftsmanship, and with a
profusion of anecdotes chiefly of the Helotry kind. He then (rather
oddly, but not out of keeping with his classical models) has a chapter
of decorum in behaviour, turns to the necessity of concealing art,
and ends with a highly flattering conclusion to the Queen.
We have yet to mention some minorities; less briefly, the two
champions—Campion and Daniel, who brought the question of
“Rhyme v. 'Verse'” to final arbitrament of battle; the great name (not
so great here as elsewhere) of Francis Bacon; and lastly, one who, if
representative of a further stage, is far the greatest of Elizabethan
critics, and perhaps the only English critic who deserves the
adjective great before Dryden.
The earliest (1591) of these is Sir John Harington, in the prefatory
matter[258] of his translation of the Orlando, which contains the gibe
Minors: at Puttenham above referred to. It is otherwise
Harington, much indebted to Sidney, from whom, however,
Meres, Harington differs in allowing more scope to
Webster,
Bolton, &c. allegorical interpretation. Then comes Francis
Meres, whose Palladis Tamia[259] (1598) is to be
eternally mentioned with gratitude, because it gives us our one real
document about the order of Shakespeare’s plays, but is quite
childish in the critical characterisation which it not uninterestingly
attempts. Webster’s equally famous, and universally known,
epitheting of the work of Shakespeare and others in the Preface to
The White Devil (1612) adds yet another instance of the short sight
of contemporaries; but tempting as it may be to comment on these,
it would not become a Historian of Criticism to do so in this context.
Sir W. Vaughan in The Golden Grove (1600) had earlier dealt, and
Bolton[260] in his Hypercritica (1616), and Peacham in his Complete
Gentleman (1622), were later to deal, with Poetry, but in terms
adding nothing to, and probably borrowed from, the utterances of
Sidney, Webbe, and Puttenham. Their contributions are “sma’ sums,”
as Bailie Nicol Jarvie says, and we must neglect them.
The most interesting literary result of the “new versifying” craze is
to be found, without doubt, in the Observations in the Art of English
Poesy of Thomas Campion[261] and the subsequent Defence of
Campion and Rhyme of Samuel Daniel. The former was issued in
his 1602, and the latter still later:—that is to say more
Observations. than twenty years after Spenser’s and Harvey’s
letters, and more than thirty after the appearance—let alone the
writing—of Ascham’s Schoolmaster. In the interval the true system
of English prosody had put itself practically beyond all real danger;
but the critical craze had never received its quietus. Nay, it survived
to animate Milton: and there are persons whom we could only name
for the sake of honour, and who do not seem to see that it is dead
even yet. Both the writers mentioned were true poets: and the
curious thing is that the more exquisitely romantic poet of the two
was the partisan of classical prosody. But Campion—who dedicated
his book to Lord Buckhurst, the doyen (except poor old Churchyard)
of English poetry at the time, and one whose few but noble
exercises in it need hardly vail their crest to any contemporary
poetry but Spenser’s and Shakespeare’s—was far too wise a man, as
well as far too good a poet, to champion any longer the break-neck
and break-jaw hexameters of Harvey and Stanyhurst. We have seen
that almost from the first there had been questions of heart among
the partisans of the New Versifying. That English is not tolerant of
dactyls—that the dactyl, do what you will, in English, will tilt itself
into an anapæst with an anacrusis—is a truth which no impartial
student of metre with an ear, and with an eye to cover the history of
English poetry, can deny. Some even of these pioneers had seen
this: Campion has the boldness to declare it in the words, “It [the
dactylic hexameter] is an attempt altogether against the nature of
our language.” But though he was bold so far, he was not quite bold
enough. He could not surmount the queer Renaissance objection to
rhyme. That all the arguments against the “barbarism” of this tell
equally against Christianity, chivalry, the English constitution, the
existence of America, gunpowder, glass-windows, coal-fires, and a
very large number of other institutions of some usefulness, never
seems to have occurred to any of these good folk. But no man can
escape his time. Campion, not noticing, or not choosing to notice,
the intensely English quality of the anapæst, limits, or almost limits,
our verse to iambs and trochees. It was possible for him—though it
still appears to be difficult for some—to recognise the tribrach, the
mere suggestion of which in English verse threw Dr Guest into a
paroxysm of “!!!!’s,” but which exists as certainly as does the iamb
itself. On the contrary he shows himself in advance of Guest, and of
most behind Guest to his own time, by admitting tribrachs in the
third and fifth places. Nay, he even sees that a trochee may take the
place of an iamb (Milton’s probably borrowed secret) in the first
place, though his unerring ear (I think there is no verse of Campion’s
that is unmusical) insists on some other foot than an iamb following
—otherwise, he says, “it would too much drink up the verse.” But, on
the whole, he sets himself to work, a self-condemned drudge, to
make iambic and trochaic verses without rhyme. And on these two,
with certain licences, he arranges schemes of English elegiacs,
anacreontics, and the rest. Some of the examples of these are
charming poems, notably the famous “Rose-Cheeked Laura,” and the
beautiful “Constant to None,” while Campion’s subsequent remarks
on English quantity are among the acutest on the subject. But the
whole thing has on it the curse of “flying in the face of nature.” You
have only to take one of Campion’s own poems (written mostly after
the Observations) in natural rhyme, and the difference will be seen
at once. It simply comes to this—that the good rhymeless poems
would be infinitely better with rhyme, and that the bad ones, while
they might sometimes be absolutely saved by the despised invention
of Huns and Vandals, are always made worse by its absence.
In the preface of Daniel’s answering Defence of Rhyme to all the
worthy lovers and learned professors [thereof] within His Majesty’s
dominions,[262] he says that he wrote it “about a year since,” upon
the “great reproach” given by Campion, and some give it the date of
1603 or even 1602; but Dr Grosart’s reprint is dated five years later.
Daniel and The learned gentleman to whom it was specially
his Defence written was no less a person than William Herbert,
of Rhyme. Earl of Pembroke, whom some of us (acknowledging
that the matter is no matter) do not yet give up as “Mr W. H.” The
advocate affects, with fair rhetorical excuse (though of course he
must have known that the craze was nearly half a century old, and
had at least not been discouraged by his patron’s uncle nearly a
generation before), to regard the attack on rhyming as something
new, as merely concerned with the “measures” of Campion. Daniel,
always a gentleman, deals handsomely with his antagonist, whom
he does not name, but describes as “this detractor whose
commendable rhymes, albeit now an enemy to rhyme, have given
heretofore to the world the best notice of his worth,” and as a man
“of fair parts and good reputation.” And having put himself on the
best ground, in this way, from the point of view of morals and
courtesy, he does the same in matter of argument by refusing to
attack Campion’s “numbers” in themselves (“We could,” he says well,
“have allowed of his numbers, had he not graced his rhymes”), and
by seizing the unassailable position given by custom and nature
—“Custom that is before all law; Nature that is above all art.” In fact,
not Jonson himself, and certainly none else before Jonson, has
comprehended, or at least put, the truth of the matter as Daniel
puts it, that arbitrary laws imposed on the poetry of any nation are
absurd—that the verse of a language is such as best consorts with
the nature of that language. This seems a truism enough perhaps;
but it may be very much doubted whether all critics recognise it, and
its consequences, even at the present day. And it is certain that we
may search other early English critics in vain for a frank recognition
of it. With an equally bold and sure foot he strides over the silly stuff
about “invention of barbarous ages” and the like. Whatever its origin
(and about this he shows a wise carelessness), it is “an excellence
added to this work of measure and harmony, far happier than any
proportion quantity could ever show.” It “gives to the ear an echo of
a delightful report,” and to the memory “a deeper impression of
what is delivered.” He is less original (as well as, some may think,
less happy) in distinguishing the accent of English from the quantity
of the classical tongues; but the classicisers before Campion, if not
Campion himself, had made such a mess of quantity, and had played
such havoc with accent, that he may well be excused. The
universality of rhyme is urged, and once more says Daniel (with that
happy audacity in the contemning of vain things which belongs to
the born exploders of crazes), “If the barbarian likes it, it is because
it sways the affections of the barbarian; if civil nations practise it, it
works upon their hearts; if all, then it has a power in nature upon
all.” But it will be said, “Ill customs are to be left.” No doubt: but the
question is begged. Who made this custom “ill”? Rhyme aims at
pleasing—and it pleases. Suffer then the world to enjoy that which it
knows and what it likes, for all the tyrannical rules of rhetoric cannot
make it otherwise. Why are we to be a mere servum pecus, only to
imitate the Greeks and Latins? Their way was natural to them: let
ours be so to us. “Why should laboursome curiosity still lay affliction
on our best delights?” Moreover, to a spirit whom nature hath “fitted
for that mystery,” rhyme is no impediment, “but rather giveth wings
to mount.” The necessary historical survey follows, with a surprising
and very welcome justification of the Middle Ages against both
Classics and Renaissance. “Let us,” says this true Daniel come to
judgment, “go not further, but look upon the wonderful architecture
of the State of England, and see whether they were unlearned times
that could give it such a form?” And if politically, why not poetically?
Some acute and, in the other sense, rather sharp criticism of
Campion’s details follows, with a few apologetic remarks for mixture
of masculine and feminine rhymes on his own part: and the whole
concludes in an admirable peroration with a great end-note to it. Not
easily shall we find, either in Elizabethan times or in any other, a
happier combination of solid good sense with eager poetic
sentiment, of sound scholarship with wide-glancing intelligence, than
in this little tractate of some thirty or forty ordinary pages, which
dispelled the delusions of two generations, and made the poetical
fortune of England sure.
The contributions of “large-browed Verulam” to criticism have
sometimes been spoken of with reverence: and it is not uncommon
to find, amid the scanty classics of the subject, which until recently
have been recommended to the notice of inquirers, not merely a
place, but a place of very high honour, assigned to The
Bacon. Advancement of Learning. Actual, unprejudiced, and
to some extent expert, reference to the works,
however, will not find very much to justify this estimate: and,
indeed, a little thought, assisted by very moderate knowledge, would
suffice to make it rather surprising that Bacon should give us so
much, than disappointing that he should give us little or nothing. A
producer of literature who at his best has few superiors, and a user
of it for purposes of quotation, who would deserve the name of
genius for this use alone if he had no other title thereto—Bacon was
yet by no means inclined by his main interests and objects, or by his
temperament, either towards great exaltation of letters, or towards
accurate and painstaking examination of them. Indeed, it is in him—
almost first of all men, certainly first of all great modern men—that
we find that partisan opposition between literature and science
which has constantly developed since. It is true that his favourite
method of examination into “forms” might seem tempting as applied
to literature; and that it would, incidentally if not directly, have
yielded more solid results than his Will-o'-the-wisp chase of the Form
of Heat. But this very craze of his may suggest that if he had
undertaken literary criticism it would have been on the old road of
Kinds and Figures and Qualities, in which we could expect little but
glowing rhetorical generalisation from him.
Nor is the nature of such small critical matter as we actually have
The Essays. from him very different. The Essays practically give
us nothing but the contents of that Of Studies, a
piece too well known to need quotation; too much in the early
pregnant style of the author to bear compression or analysis; and
too general to repay it. For the critic and the man of letters generally
it is, in its own phrase, to be not merely tasted, nor even swallowed,
but chewed and digested; yet its teachings have nothing more to do
with the critical function of “study” than with all others.
The Advancement[263] at least excuses the greatness thrust upon it
in the estimates above referred to, not merely by the apparent
necessity that the author should deal with Criticism, but by a certain
The appearance of his actually doing so. Comparatively
Advancement early in the First Book he tackles the attention to
of Learning. style which sprang up at the Renaissance, opening
his discussion by the ingenious but slightly unhistorical attribution of
it to Martin Luther, who was forced to awake all antiquity, and call
former times to his succour, against the Bishop of Rome. Not a few
names, for the best part of two centuries before the great cause of
Martinus v. Papam was launched, from Petrarch and Boccaccio to
Erasmus and Reuchlin, will put in evidence before the tribunal of
chronology against this singular assertion; and though the Italian
Humanists of the fifteenth century might not (at least in thought)
care anything for the Pope except as a source of donatives and
benefices, it is certain that most of them were as constitutionally
disinclined to abet Luther as they were chronologically disabled from
in any way abetting him. Bacon’s argument and further survey are,
however, better than this beginning. To understand the ancients (he
says justly enough) it was necessary to make a careful study of their
language. Further, the opposition of thought to the Schoolmen
naturally brought about a recoil from the barbarisms of Scholastic
style, and the anxiety to win over the general imprinted care and
elegance and vigour on preaching and writing. All this, he adds as
Its justly, turned to excess. Men began to “hunt more
denunciation after words than matter; more after the choiceness
of mere of the phrase and the round and clean composition
word-study.
of the sentence, and the sweet falling of the
clauses, and the varying and illustration of their words with tropes
and figures, than after the weight of matter, worth of subject,
soundness of argument, life of invention, or depth of judgment.” The
Ciceronianism of Osorius, Sturm, “Car of Cambridge,” and even
Ascham, receives more or less condemnation; and Erasmus is, of
course, cited for gibes at it. On this text Bacon proceeds to enlarge
in his own stately rhetoric, coolly admitting that it “is a thing not
hastily to be condemned, to clothe and adorn the obscurity even of
philosophy itself with sensible and plausible elocution.” But he very
quickly glides off into his usual denunciations of the schoolmen. Nor
have I found anything else in this First Book really germane to our
purpose; for one cannot cite as such the desultory observations on
patronage of literature (among other branches of learning) which fill
a good part of it.
The Second Book is somewhat more fruitful in quantity, if not very
much; but the quality remains not very different. The opening
“Address to the King” contains, in an interesting first draft (as we
may call it), the everlasting grumble of the scientific man, that
science is not sufficiently endowed, the further grumble at mere
book-learning, the cry for the promotion—by putting money in its
purse—of research. The Second and Third Chapters contain some
plans of books drawn up in Bacon’s warm imaginative way, especially
a great series of Histories, with the History of England for their
centre. And then we come, in the Fourth Chapter, to Poesy.
But except for Bacon’s majestic style (which, however, by accident
or intention, is rather below itself here) there is absolutely nothing
Its view of novel. The view (which, as we have seen, all the
Poetry. Elizabethan critics adopted, probably from the
Italians)—the view is that poetry is just a part of learning licensed in
imagination; a fanciful history intended to give satisfaction to the
mind of man in things where history is not; something particularly
prevalent and useful in barbarous ages; divisible into narrative,
representative and allusive; useful now and then, but (as Aristotle
would say) not a thing to take very seriously. Yet poetry, a vinum
dæmonum at the worst, a mere illusion anyhow, is still, even as
such, a refuge from, and remedy for, sorrow and toil. Of its form, as
distinguished from its matter, he says,[264] “Poetry is but a character
of style, and belongeth to arts of speech, and is not pertinent for the
present.” He attempts no defence of it as of other parts of learning,
because “being as a plant that cometh of the lust of the earth
without a formal seed, it hath sprung up and spread abroad more
than any other kind.” And he turns from it to philosophy, with the
more than half-disdainful adieu, “It is not good to stay too long in
the theatre.”
We might almost quit him here with a somewhat similar leave-
taking; but for his great reputation some other places shall be
Some obiter handled. At XIV. 11 there are some remarks on the
dicta. delusive powers of words; at XVI. 4, 5 some on
grammar and rhetoric, including a rather interesting observation, not
sufficiently expanded or worked out, that “in modern languages it
seemeth to me as free to make new measures of verses as of
dances”; in XVIII. a handling of strictly oratorical rhetoric, with a
digression to these “Colours of Good and Evil” which interested
Bacon so much; in XX. another descant on the same art; in XXI. a
puff of the Basilikon Doron; in XXXII. observations on the moral
influence of books; in XXXV. some general observations on literature;
and, just before the close, a well-known and often-quoted eulogy,
certainly not undeserved, of the eloquence of the English pulpit for
forty years past.
If it were not for the singular want of a clear conception of literary
Criticism, which has prevailed so long and so widely, it would hardly
be necessary to take, with any seriousness at all, a man who has no
The whole of more than this to say on the subject.[265] It is most
very slight assuredly no slight to Bacon to deny him a place in
importance. a regiment where he never had the least ambition
to serve. That he was himself a great practitioner of literature, and
so, necessarily if indirectly, a critic of it in his own case, is perfectly
true; the remarks which have been quoted above on the Ciceronians
show that, when he took the trouble, and found the opportunity, he
could make them justly and soundly. But his purpose, his interests,
his province, his vein, lay far elsewhere. To him, it is pretty clear,
literary expression was, in relation to his favourite studies and
dreams, but a higher kind of pen-and-ink or printing-press. He
distrusted the stability of modern languages, and feared that studies
couched in them might some day or other come to be unintelligible
and lost to the world. This famous fear explains the nature and the
limits of his interest in literature. It was a vehicle or a treasury, a
distributing agent or a guard. Its functions and qualities accorded: it
was to be clear, not disagreeable, solidly constructed, intelligible to
as large a number of readers as possible. The psychological
character and morphological definition of poetry interested him
philosophically. But in the art and the beauty of poetry and literature
generally, for their own sakes, he seems to have taken no more
interest than he did in the coloured pattern plots in gardens, which
he compared to “tarts.” To a man so minded, as to those more
ancient ones of similar mind whom we have discussed in the first
volume, Criticism proper could, at the best, be a pastime to be half
ashamed of—a “theatre” in which to while away the hours; it could
not possibly be a matter of serious as well as enthusiastic study.
Between Bacon and Ben may be best noticed the short Anacrisis
or Censure of Poets, Ancient and Modern,[266] by Sir William
Stirling’s Alexander, Earl of Stirling. It has received high
Anacrisis. praise;[267] but even those who think by no means ill
of Aurora, may find some difficulty in indorsing this. It is simply a
sort of “Note,” written, as the author says, to record his impressions
during a reading of the poets, which he had undertaken as
refreshment after great travail both of body and mind. He thinks
Language “but the apparel of poesy,” thereby going even further
than those who would assign that position to verse, and suggesting
a system of “Inarticulate Poetics,” which he would have been rather
put to it to body forth. He only means, however, that he judges in
the orthodox Aristotelian way, by “the fable and contexture.” A
subsequent comparison of a poem to a garden suggests Vauquelin
de la Fresnaye (v. supra, p. 129), whom he may have read.
Alexander is a sort of general lover in poetry; he likes this in Virgil,
that in Ovid, that other in Horace; defends Lucan against Scaliger,
even to the point of blaming the conclusion of the Æneid; finds “no
man that doth satisfy him more than that notable Italian, Torquato
Tasso”; admits the historical as well as the fictitious poetic subject;
but thinks that “the treasures of poetry cannot be better bestowed
than upon the apparelling of Truth; and Truth cannot be better
apparelled to please young lovers than with the excellences of
poetry.” Disrespectful language neither need nor should be used of
so slight a thing, which is, and pretends to be, nothing more than a
sort of table-book entry by a gentleman of learning as well as
quality. But, if it has any “importance” at all, it is surely that of being
yet another proof of the rapid diffusion of critical taste and practice,
not of stating “theory and methods considerably in advance of the
age.” If we could take extensively his protest against those who
“would bound the boundless liberty of the poet,” such language
might indeed be justified; but the context strictly limits it to the very
minor, though then, and for long before and after, commonly
debated, question of Fiction v. History in subject.
Save perhaps in one single respect (where the defect was not
wholly his fault), Ben Jonson might be described as a critic armed at
Ben Jonson: all points. His knowledge of literature was extremely
his wide, being at the same time solid and thorough.
equipment. While he had an understanding above all things
strong and masculine, he was particularly addicted, though in no
dilettante fashion, to points of form. His whole energies, and they
were little short of Titanic, were given to literature. And, lastly, if he
had not the supremest poetic genius, he had such a talent that only
the neighbourhood of supremacy dwarfs it. Where he came short
was not in a certain hardness of temper and scholasticism of
attitude: for these, if kept within bounds, and tempered by that
enthusiasm for letters which he possessed, are not bad equipments
for the critic. It was rather in the fact that he still came too early for
it to be possible for him, except by the help of a miracle, to
understand the achievements and value of the vernaculars. By his
latest days, indeed,[268] the positive performance of these was
already very great. Spain has hardly added anything since, and Italy
not very much, to her share of European literature; France was
already in the first flush of her “classical” period, after a long and
glorious earlier history: and what Ben’s own contemporaries in
England had done, all men know. But mediæval literature was shut
from him, as from all, till far later; he does not seem to have been
much drawn to Continental letters, and, perhaps in their case, as
certainly in English, he was too near—too much a part of the
movement—to get it into firm perspective.
In a sense the critical temper in Jonson is all-pervading. It breaks
out side by side with, and sometimes even within, his sweetest
lyrics; it interposes what may be called parabases in the most
His Prefaces, unexpected passages[269] of his plays. The Poetaster
&c. is almost as much criticism dramatised as The
Frogs. But there are three “places,” or groups of places, which it
inspires, not in mere suggestion, but with propriety—the occasional
Prefaces, or observations, to and on the plays themselves, the
Conversations with Drummond, and, above all, the at last fairly
(though not yet sufficiently) known Discoveries or Timber.
To piece together, with any elaboration, the more scattered critical
passages would be fitter for a monograph on Jonson than for a
History of Criticism. The “Address to the Readers” of Sejanus, which
contains a reference to the author’s lost Observations on Horace, his
Art of Poetry (not the least of such losses) is a fair specimen of
them: the dedication of Volpone to “the most noble and most equal
sisters, Oxford and Cambridge,” a better. In both, and in numerous
other passages of prose and verse, we find the real and solid,
though somewhat partial, knowledge, the strong sense, the
methodic scholarship of Ben, side by side with his stately, not
Euphuistic, but rather too close-packed style, his not ill-founded, but
slightly excessive, self-confidence, and that rough knock-down
manner of assertion and characterisation which reappeared in its
most unguarded form in the Conversations with Drummond.
The critical utterances of these Conversations are far too
interesting to be passed over here, though we cannot discuss them
The in full. They tell us that Ben thought all (other)
Drummond rhymes inferior to couplets, and had written a
Conversation treatise (which, again, would we had!) both against
s.
Campion and Daniel (see ante). His objection to
“stanzas and cross rhymes” was that “as the purpose might lead
beyond them, they were all forced.” Sidney “made every one speak
as well as himself,” and so did not keep “decorum” (cf. Puttenham
above). Spenser’s stanzas and matter did not please him. Daniel was
no poet. He did not like Drayton’s “long verses,” nor Sylvester’s and
Fairfax’s translations. He thought the translations of Homer
(Chapman’s) and Virgil (Phaer’s) into “long Alexandrines” (i.e.,
fourteeners) were but prose: yet elsewhere we hear that he “had
some of Chapman by heart.” Harington’s Ariosto was the worst of all
translations. Donne was sometimes “profane,” and “for not keeping
of accent deserved hanging”; but elsewhere he was “the first poet of
the world in some things,” though, “through not being understood,
he would perish.”[270] Shakespeare “wanted art”: and “Abram Francis
(Abraham Fraunce) in his English Hexameters was a fool.” “Bartas
was not a poet, but a verser, because he wrote not fiction.” He
cursed Petrarch for redacting verses to sonnets, “which were like
Procrustes’ bed.” Guarini incurred the same blame as Sir Philip: and
Lucan was good in parts only. “The best pieces of Ronsard were his
Odes.” Drummond’s own verses “were all good, but smelled too
much of the schools.” The “silver” Latins, as we should expect,
pleased him best. “To have written Southwell’s ‘Burning Babe,’ he
would have been content to destroy many of his.”
These are the chief really critical items, though there are others
(putting personal gossip aside) of interest; but it may be added, as a
curiosity, that he told Drummond that he himself “writ all first in
prose” at Camden’s suggestion, and held that “verses stood all by
sense, without colours or accent” (poetic diction or metre), “which
yet at other times he denied,” says the reporter, a sentence ever to
be remembered in connection with these jottings. Remembering it,
there is nothing shocking in any of these observations, nor anything
really inconsistent. A true critic never holds the neat, positive,
“reduced-to-its-lowest-terms” estimate of authors, in which a
criticaster delights. His view is always facetted, conditioned. But he
may, in a friendly chat, or a conversation for victory, exaggerate this
facet or condition, while altogether suppressing others; and this
clearly is what Ben did.
For gloss on the Conversations, for reduction to something like
system of the critical remarks scattered through the works, and for
the nearest approach we can have to a formal presentment of Ben’s
critical views, we must go to the Discoveries.[271]
The fact that we find no less than four titles for the book—Timber,
Explorata, Discoveries, and Sylva—with others of its peculiarities, is
explained by the second fact that Jonson never published it. It never
The appeared in print till the folio of 1641, years after its
Discoveries. author’s death. The Discoveries are described as
being made “upon men and matter as they have flowed out of his
daily reading, or had their reflux to his peculiar notions of the times.”
They are, in fact, notes unnumbered and unclassified (though
batches of more or fewer sometimes run on the same subject), each
with its Latin heading, and varying in length from a few lines to that
of his friend (and partly master) Bacon’s shorter Essays. The
influence of those “silver” Latins whom he loved so much is
prominent: large passages are simply translated from Quintilian, and
for some time[272] the tenor is ethical rather than literary. A note on
Perspicuitas—elegantia (p. 7) breaks these, but has nothing
noteworthy about it, and Bellum scribentium (p. 10) is only a satiric
exclamation on the folly of “writers committed together by the ears
for ceremonies, syllables, points,” &c. The longer Nil gratius protervo
libro (pp. 11, 12) seems a retort for some personal injury, combined
with the old complaint of the decadence and degradation of poetry.
[273]
There is just but rather general stricture in Eloquentia (p. 16) on
the difference between the arguments of the study and of the world.
“I would no more choose a rhetorician for reigning in a school,” says
Ben, “than I would choose a pilot for rowing in a pond.”[274] Memoria
(p. 18) includes a gird at Euphuism. At last we come to business.
Censura de poetis (p. 21), introduced by a fresh fling at Euphuism,
in De vere argutis, opens with a tolerably confident note, “Nothing in
our age is more preposterous than the running judgments upon
poetry and poets,” with much more to the same effect, the whole
being pointed by the fling, “If it were a question of the water-
rhymer’s[275] works against Spenser’s, I doubt not but they would
find more suffrages.” The famous passage on Shakespeare follows:
and the development of Ben’s view, “would he had blotted a
thousand,” leads to a more general disquisition on the differences of
wits, which includes the sentence already referred to. “Such [i.e.,
haphazard and inconsistent] are all the Essayists, even their master
Montaigne.” The notes now keep close to literature throughout in
substance, though their titles (e.g., Ignorantia animæ), and so forth,
may seem wider. A heading, De Claris Oratoribus (p. 26), leads to
yet another of the purple passages of the book—that on Bacon, in
which is intercalated a curious Scriptorum catalogus, limited, for the
most part, though Surrey and Wyatt are mentioned, to prose writers.
And then for some time ethics, politics, and other subjects, again
have Ben’s chief attention.[276]
We return to literature, after some interval (but with a parenthetic
glance at the poesis et pictura notion at p. 49), on p. 52, in a curious
unheaded letter to an unnamed Lordship on Education, much of
which is translated directly from Ben Jonson’s favourite Quintilian;
and then directly accost it again with a tractatule De stilo et optimo
scribendi genere, p. 54, hardly parting company thereafter. Ben’s
prescription is threefold: read the best authors, observe the best
speakers, and exercise your own style much. But he is well aware
that no “precepts will profit a fool,” and he adapts old advice to
English ingeniously, in bidding men read, not only Livy before
Sallust, but also Sidney before Donne, and to beware of Chaucer or
Gower at first. Here occurs the well-known dictum, that Spenser “in
affecting the ancients writ no language; yet I would have him read
for his matter.” A fine general head opens with the excellent version
of Quintilian, “We should not protect our sloth with the patronage of
Difficulty,” and this is followed by some shrewd remarks on diction—
the shrewdest being that, after all, the best custom makes, and ever
will continue to make, the best speech—with a sharp stroke at
Lucretius for “scabrousness,” and at Chaucerisms. Brevity of style,
Tacitean and other, is cautiously commended. In the phrase (Oratio
imago animi), p. 64, “language most shows a man,” Ben seems to
anticipate Buffon, as he later does Wordsworth and Coleridge, by
insisting that style is not merely the dress, but the body of thought.
[277]
All this discussion, which enters into considerable detail, is of the
first importance, and it occupies nearly a quarter of the whole book.
It is continued, the continuation reaching till the end, by a separate
discussion of poetry.
It is interesting, but less so than what comes before. A somewhat
acid, though personally guarded, description of the present state of
the Art introduces the stock definition of “making,” and its corollary
that a poet is not one who writes in measure, but one who feigns—
all as we have found it before, but (as we should expect of Ben) in
succincter and more scholarly form. Yet the first requisite of the poet
is ingenium—goodness of natural wit; the next exercise of his parts
—“bringing all to the forge and file” (sculpte, lime, cisèle!); the third
Imitation—to which Ben gives a turn (not exactly new, for we have
met it from Vida downwards), which is not an improvement, by
keeping its modern meaning, and understanding by it the following
of the classics. “But that which we especially require in him is an
exactness of study and multiplicity of reading.” Yet his liberty is not
to be so narrowly circumscribed as some would have it. This leads to
some interesting remarks on the ancient critics, which the author
had evidently meant to extend: as it is, they break off short.[278] We
turn to the Parts of comedy and tragedy, where Ben is strictly
regular—the fable is the imitation of one entire and perfect action,
&c. But this also breaks off, after a discussion of fable itself and
episode, with an evidently quite disconnected fling at “hobbling
poems which run like a brewer’s cart on the stones.”
These Discoveries have to be considered with a little general care
Form of the before we examine them more particularly. They
book. were, it has been said, never issued by the author
himself, and we do not know whether he ever would have issued
them in their present form. On the one hand, they are very carefully
written, and not mere jottings. In form (though more modern in
style) they resemble the earlier essays of that Bacon whom they so
magnificently celebrate, in their deliberate conciseness and
pregnancy. On the other hand, it is almost impossible to doubt that
some at least were intended for expansion; it is difficult not to think
that there was plenty more stuff of the same kind in the solidly
constructed and well-stored treasuries of Ben’s intelligence and
erudition. It is most difficult of all not to see that, in some cases, the
thoughts are co-ordinated into regular tractates, in others left loose,
as if for future treatment of the same kind.
Secondly, we should like to know rather more than we do of the
Its date. time of their composition. Some of them—such as
the retrospect of Bacon, and to a less degree that of
Shakespeare—must be late; there is a strong probability that all date
from the period between the fire in Ben’s study, which destroyed so
much, and his death—say between 1620 and 1637. But at the same
time there is nothing to prevent his having remembered and
recopied observations of earlier date.
Thirdly, it is most important that we rightly understand the
Mosaic of old composition of the book. It has sometimes been
and new. discovered[279] in these Discoveries, with pride, or
surprise, or even scorn, that Ben borrowed in them very largely from
the ancients. Of course he did, as well as something, though less,
from the Italian critics of the age immediately before his own.[280]
But in neither case could he have hoped for a moment—and in
neither is there the slightest reason to suppose that he would have
wished if he could have hoped—to disguise his borrowings from a
learned age. When a man—such as, for instance, Sterne—wishes to
steal and escape, he goes to what nobody reads, not to what
everybody is reading. And the Latins of the Silver Age, the two
Senecas, Petronius, Quintilian, Pliny, were specially favourites with
the Jacobean time. In what is going to be said no difference will be
made between Ben’s borrowings and his original remarks: nor will
the fact of the borrowing be referred to unless there is some special
critical reason. Even the literal translations, which are not
uncommon, are made his own by the nervous idiosyncrasy of the
phrase, and its thorough adjustment to the context and to his own
vigorous and massive temperament.
Of real “book-criticism” there are four chief passages, the brief
flings at Montaigne and at “Tamerlanes and Tamerchams,” and the
longer notices of Shakespeare and Bacon.
The flirt at “all the Essayists, even their master Montaigne,” is
especially interesting, because of the high opinion which Jonson
elsewhere expresses of Bacon, the chief, if not the first, English
Essayist of his time, and because of the fact that not a few of these
very Discoveries are “Essays,” if any things ever were. Nor would it
The fling at be very easy to make out a clear distinction, in
Montaigne; anything but name, between some of Ben’s most
favourite ancient writers and these despised persons. It is, however,
somewhat easier to understand the reason of the condemnation.
Jonson’s classically ordered mind probably disliked the ostentatious
desultoriness and incompleteness of the Essay, the refusal, as it
were out of mere insolence, to undertake an orderly treatise. Nor is
it quite impossible that he failed fully to understand Montaigne, and
was to some extent the dupe of that great writer’s fanfaronade of
promiscuousness.
The “Tamerlane and Tamercham”[281] fling is not even at first sight
at Tamerlane, surprising. It was quite certain that Ben would
seriously despise what Shakespeare only laughed at
—the confusion, the bombast, the want of order and scheme in the
“University Wits”—and it is not probable that he was well enough
acquainted with the even now obscure development of the earliest
Elizabethan drama to appreciate the enormous improvement which
they wrought. Nay, the nearer approach even of such a dull thing as
Gorboduc to “the height of Seneca his style,” might have a little
bribed him as it bribed Sidney. He is true to his side—to his division
of the critical creed—in this also.
The train of thought—censure of the vulgar preference—runs clear
from this to the best known passage of the whole, the section De
the Shakespeare Nostrat. It cannot be necessary to
Shakespeare quote it, or to point out that Ben’s eulogy, splendid
Passage, as it is, acquires tenfold force from the fact that it is
avowedly given by a man whose general literary theory is different
from that of the subject, while the censure accompanying it loses
force in exactly the same proportion. What Ben here blames, any
ancient critic (perhaps even Longinus) would have blamed too: what
Ben praises, it is not certain that any ancient critic, except Longinus,
would have seen. Nor is the captious censure of “Cæsar did never
wrong but with just cause” the least interesting part of the whole.
The paradox is not in our present texts: and there have, of course,
not been wanting commentators to accuse Jonson of garbling or of
forgetfulness. This is quite commentatorially gratuitous and puerile.
It is very like Shakespeare to have written what Ben says: very like
Ben to object to the paradox (which, pace tanti viri, is not
“ridiculous” at all, but a deliberate and effective hyperbole); very like
the players to have changed the text; and most of all like the
commentators to make a fuss about the matter.
What may seem the more unstinted eulogy of Bacon is not less
and that on interesting. For here it is obvious that Ben is
Bacon. speaking with fullest sympathy, and with all but a
full acknowledgment of having met an ideal. Except the slight stroke,
“when he could spare or pass by a jest,” and the gentle insinuation
that Strength, the gift of God, was what Bacon’s friends had to
implore for him, there is no admixture whatever in the eulogy of
“him who hath filled up all numbers,[282] and performed that in our
tongue which may be compared or preferred to insolent Greece or
haughty Rome.” Indeed it could not have been—even if Ben Jonson
had not been a friend, and, in a way, follower of Bacon—but that he
should regard the Chancellor as his chief of literary men. Bacon,
unluckily for himself, lacked the “unwedgeable and gnarled” strength
of the dramatist, and also was without his poetic fire, just as Ben
could never have soared to the vast, if vague, conceptions of
Bacon’s materialist-Idealism. But they were both soaked in
“literature,” as then understood; they were the two greatest masters
of the closely packed style that says twenty things in ten words: and
yet both could, on occasion, be almost as rhetorically imaginative as
Donne or Greville. It is doubtful whether Bacon’s own scientific scorn
for words without matter surpassed Jonson’s more literary contempt
of the same phenomenon. Everywhere, or almost everywhere, there
was between them the idem velle et idem nolle.
A limited précis, however, and a few remarks on special points,
cannot do the Discoveries justice. The fragmentary character of the
notes that compose it, the pregnant and deliberately “astringed”
style in which these notes are written, so that they are themselves
the bones, as it were, of a much larger treatise, defy such
treatment. Yet it is full of value, as it gives us more than glimpses
“Of what a critic was in Jonson lost,”
or but piecemeal shown. We shall return, in the next chapter, to his

relative position; but something should be said here of his intrinsic
character.
He does not, as must have been clearly seen, escape the
General “classical” limitation. With some ignorance,
character of doubtless, and doubtless also some contempt, of
the book. the actual achievements of prose romance, and with
that stubborn distrust of the modern tongues for miscellaneous
prose purposes, which lasted till far into the seventeenth century, if
it did not actually survive into the eighteenth, he still clings to the
old mistakes about the identity of poetry and “fiction,” about the
supremacy of oratory in prose. We hear nothing about the “new
versifying,” though no doubt this would have been fully treated in his
handling of Campion and Daniel: but had he had any approval for it,
that approval must have been glanced at. His preference for the
(stopped) couplet[283] foreshadowed that which, with beneficial

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Protein Nucleic Acid Interactions Structural Biology RSC

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Principles of Nucleic Acid Structure Stephen Neidle

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Chapter 1 Overview of Protein Conjugation.................................................................................. 1

Chapter 2 Review of Protein and Nucleic Acid Chemistry........................................................... 9

Chapter 3 Reagents Targeted to Specific Functional Groups...................................................... 35

3.4 Reagents Directed toward Carboxyl Groups.................................................... 43

Chapter 4 How to Design and Choose Cross-Linking Reagents................................................. 53

Chapter 5 Homobifunctional Cross-Linking Reagents................................................................ 81

Chapter 6 Heterobifunctional Cross-Linkers............................................................................. 191

6.2.1 Amino- and Sulfhydryl-Group–Directed Cross-Linkers................. 191

Chapter 7 Multifunctional Cross-Linking Reagents.................................................................. 239

Chapter 8 Monofunctional and Zero -Length Cross -Linking Reagents.................................... 265

8.3.5.1 Transglutaminase............................................................... 282

Chapter 9 General Approaches for Chemical Cross-Linking.................................................... 297

9.6 Conditions for Cleavage of Cross-Linked Complexes................................... 313

Chapter 10 Analysis of Cross-Linked Products........................................................................... 321

Chapter 11 Applications of Chemical Cross-Linking to the Study of Biological

11.6 Determination of Nucleic Acid Interaction and Nucleic

Chapter 12 Applications of Chemical Conjugation in the Preparation

12.3 Preparation of Immunogens........................................................................... 366

Chapter 13 Application of Chemical Conjugation for the Preparation of Immunotoxins

Chapter 14 Application of Chemical Conjugation to Solid-State Chemistry..............................409

14.3.4 Activation of Amines........................................................................ 414

Appendix A: Amino Group–Directed Homobifunctional Cross-Linkers..................................... 447

(A) (B) (C)

FIGURE 1.2 Hedgehog signaling pathway in Drosophila.

Inactive PKA Regulatory Free active

methods, however, chemical cross-linking provides detailed characterization of protein–protein

“The multitudinous seas incarnadine.”

Then he passes to feet, of which he practically allows only the iamb;

“Ye dainty nymphs that in this blessed brook.”

It is enough to say that he succeeds in stripping all three of every

“Of what a critic was in Jonson lost,”

or but piecemeal shown. We shall return, in the next chapter, to his

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9.6 Conditions for Cleavage of Cross-Linked Complexes................................... 313

12.3 Preparation of Immunogens........................................................................... 366

14.3.4 Activation of Amines........................................................................ 414