Buffers

• General rule:
buffering region is
± 1 pKa of buffer

• Example Tris:
Polyprotic Acids

O
HO P OH
OH
 Amino Acid Structure
• Amino acids are the building blocks of peptides
and proteins, the majority of which are
composed of 20 commonly occurring ones.
• The properties of amino acids that make them
suitable for the varied roles of proteins is that:
1. They have the capacity to polymerize forming
the peptide bond.
2. They have novel-acid base properties.
3. They have varied structural and chemical
functionality.
4. They are chiral.
• Amino acids are composed of a tetrahedral
arrangement of 4 substituents around a central
carbon atom, called the alpha carbon.
• All (except proline) have a common carboxyl
group (the acid), and an alpha hydrogen. They
differ only in their R substituent.
• Additional carbon in R groups are designated
, , , and .
 Amino Acid Classification
• Knowledge of the chemical properties of the common amino acids is
central to an understanding of biochemistry. Remember structure, pKa
and letter codes.
• They can be divided in five main classes based on the properties of the R
groups.

1. Non-polar, aliphatic R groups

2. Aromatic R groups
3. Polar, uncharged R groups
4. Positively charged R groups
5. Negatively Charged R groups

• Specifically on polarity, tendency to interact with water at biological pH

(~7.0). They vary widely from non-polar and hydrophobic (water insoluble)
to highly polar and hydrophilic (water soluble).
 Non-polar, Aliphatic R Groups

• Gly has the simplest structure, Although it is formally non-polar, its very small side chain makes no real
contribution to hydrophobic interactions.
• Ala, Val, Leu, and Ile tend to cluster together within proteins stabilizing protein structure by means of
hydrophobic interactions.
• Met is one of two amino acids containing sulfur. R group is a thiol ether.
• The secondary amino group of Pro residues is held in a rigid conformation that reduces the structural
flexibility of polypeptide regions containing Pro.
 Amino Acid Classification
• Knowledge of the chemical properties of the common amino acids is
central to an understanding of biochemistry. Remember structure, pKa
and letter codes.
• They can be divided in five main classes based on the properties of the R
groups.

1. Non-polar, aliphatic R groups

2. Aromatic R groups
3. Polar, uncharged R groups
4. Positively charged R groups
5. Negatively Charged R groups

• Specifically on polarity, tendency to interact with water at biological pH

(~7.0). They vary widely from non-polar and hydrophobic (water insoluble)
to highly polar and hydrophilic (water soluble).
 Aromatic R Groups
• Phe, Tyr and Trp are
relatively hydrophobic.
All can participate in
hydrophobic
interactions.
• Tyr and Trp are
significantly more polar
than Phe because of
Tyr hydroxyl group and
the nitrogen of the Trp
indole ring.
• The hydroxyl group of
Tyr can form hydrogen
bonds and it is
important functional
group in some
enzymes.
 Absorption of UV Light of Aromatic AA
• Trp and Tyr, and to a much lesser extent Phe, absorb UV light. This
account for the characteristic strong absorbance of light by most proteins
at a wavelength of 280 nm.

• Trp molar  is 5 times that of Tyr and 50 times that of Phe.

 Amino Acid Classification
• Knowledge of the chemical properties of the common amino acids is
central to an understanding of biochemistry. Remember structure, pKa
and letter codes.
• They can be divided in five main classes based on the properties of the R
groups.

1. Non-polar, aliphatic R groups

2. Aromatic R groups
3. Polar, uncharged R groups
4. Positively charged R groups
5. Negatively Charged R groups

• Specifically on polarity, tendency to interact with water at biological pH

(~7.0). They vary widely from non-polar and hydrophobic (water insoluble)
to highly polar and hydrophilic (water soluble).
 Polar, Uncharged R Groups

• The R groups of these AA

is more hydrophilic
because they contain
functional groups that form
hydrogen bonds.

• Polarity of Ser and Thr is

due to their hydroxyl group.
• Polarity of Asn and Gln due
to the amide group.

• Polarity of Cys due to the

sulfhydryl group.
Charged R
groups
 Cysteine Oxidation

Don’t need to know the DTT, TCEP and

-mercaptoethanol structures but you have
to know what they are for. You have
to know cysteine and cystine.

• Cys is readily oxidized to form a covalently linked dimeric amino acid called cystine.
• The disulfide-linked residues are strongly hydrophobic.
• Disulfide bonds play a special role in the structures of some proteins.
• Some proteins (including the majority of enzymes) should be kept in a reducing
environment to keep the Cys reduced. Common reagents used are -
mercaptoethanol, dithiothreitol (DTT or Cleland’s reagent), and TCEP
 Working with Cysteine
Covalent modification:

O H2 O
-
O OOC C C S
NH3+
+
H3N CH C O- N CH2CH3 N CH2CH3
CH2
O O
SH
N-ethylmaleimide

O-
O O C
O H2 H2 O
+ - H2
H3N CH C O CH C S C C HI
I C C
O- O-
CH2 NH3+
O-
SH Iodoacetamide
O C
H2
CH C S NO2
Covalent modificationand cysteine quantification:
O NH3+ COO-
+
O2N S S NO2 H2N CH C O-
CH2
-
OOC COO- -
S NO2
SH Absorbs at 412 nm
-
COO
• Cysteine concentration in peptides or proteins can be determined from the absorbance and
Beer’s Law (ε412=11,400 M-1 cm-1). This reagent is called Ellman’s reagent.
• You don’t have to know these structures and mechanisms but you have to know what these
reagents are for.
 Try to do this problem
• An unknown peptide has molar extinction coefficient at
280 nm of 10,000 M-1 cm-1.
• A 1 mL solution of this peptide in a 1 cm cuvette has
an absorbance at 280 nm of 0.5.
• Upon addition of Ellman’s reagent to the 1 mL of
peptide, the solution turns dark yellow and the
absorbance at 412 nm (1 cm cuvette) is 1.14 (ε412=
11,400 M-1 cm-1) .
• Calculate the number of Cys present assuming that
the volume has not change after addition of Ellman’s
reagent.

• Answer will be given in the next lecture and will be on

the lecture notes.
 Less Common Amino Acids

• You are not supposed to know their structures but you should
be able to recognize that they are not among the 20 most
common amino acids.
 Zwitterionic Amino Acid
• An amino acid in water exists
predominantly as the dipolar ion
or zwitterion.

• A zwitterion can act as either an

acid or a base

• Compounds with this dual nature

are amphoteric and are often
called ampholytes
pH=1 pH=7 pH=13
O O O

+ + - -
H3N CH C OH H3N CH C O H2N CH C O

CH3 CH3 CH3

Net charge: +1 0 -1
Cation form Zwitterion (neutral) Anionic form
 AA Titration

Titration curves provides

experimental determination
of:

1. Measurements of pKa of
ionizing groups.
2. It shows the region of
buffering power. Glycine
has two one 2.34 ± 1 pH
and the other 9.60 ± 1 pH.
pI=(pK1 + pK2) x 1/2
AA Titration-II
 A Peptide

• Amino acids in a peptide are called

residues.

• The amino acid at the end with free

-NH2 is the amino-terminal (N-
terminal) residue and the one with
free -COOH is the carboxyl-
terminal (C-terminal) residue.

• Peptides (as well as proteins) have

their own characteristic titration
curve and pI.
 Chemical stability of peptides-I

• At neural pH, Keq for hydrolysis of peptide is favored BUT t1/2 is in years so that even
if the reaction is thermodynamically favored is kinetically extremely slow at pH 7.0. At
this pH peptide bond is stable on a physiological time scale.
• At acid pH (6N HCl, 24h 110 °C) and basic pH (4h, in NaOH 110 °C) peptide bonds
is unstable and these reactivity are taken advantage in sequencing of peptides.

You don’t need to know these reactions’ mechanisms but you should know that a
peptide bond is labile to acid and base.
Chemical stability of peptides-II

• Ser, Thr, Cys and Arg

degrade during basic
conditions.

• Acid hydrolysis destroys

several amino acids:
-Trp and Cys are totally
destroyed
-Asn and Gln are hydrolyzed to
the corresponding carboxylic
acids.
 Problem: pI of Peptides

Glu-His-Trp-Gly-Leu-Arg-Pro-Gly-Lys Net

pH=1 +1 / +1 / / / +1 / / +1 / +4
pH=7 +1 -1 / / / / +1 / / +1 -1 +1
pH=14 / -1 / / / / / / / / -1 -2

• pI is between pH 7 and pH 14. You can approximate the pi by looking at groups that change ionization state from 7 to 14:
amino-terminus, Lys side chain and Arg side chain:
pI= (9.7 + 10.5 + 12.5)/3 =10.9

• Or you could see that after pH 7 you need to deprotonate one group to get a net charge of zero. The groups that has pKa
closer to pH 7 that can be deprotonated is the amino-terminus (pKa 9.7).

pH=10 / -1 / / / / +1 / / +1 -1 0

Now find the two groups that ionize near pH 10: amino-terminal (pKa 9.7) and Lys (pKa 10.5):

pI= (9.7 + 10.5)/2 =10.1