Productivity On the Go

Asus Exclusive Store IT Mall (The…

Chandigarh 10:30AM–8:30PM

The word “enzyme” was first introduced by Kuhne in 1878. It is

derived from the original Greek word enzyme (Gr. en-in, zyme-
leaven), which means “in yeast”.

In 1896, Buchner succeeded in extracting from the yeast cells a

substance that was active in fermentation. This substance was
later called zymase and represents a part of the enzyme system
involved in fermentation. In 1926, Professor J.B. Sumner iso­-
lated from jack beans, by means of acetone, the enzyme urease
in crystalline form.

Definition:
An enzyme may be defined as a complex biological catalyst that
is produced by a living organism in its cells to regulate the
various physiological processes of the body. Enzymes
functional outside the living cells are called exoenzymes, e.g.,
enzymes present in diges­tive juices, lysozyme of tears.
Enzymes functional inside living cells are known as
endozymes, e.g., enzymes of Krebs cycle, enzymes of glycolysis,
etc.
The substance on which an enzyme acts is called the
“substrate” and generally speaking, the enzyme itself is named
after the substrate by adding the suffix, ‘ase’ to that of
substrate. Thus, for example, proteases are a group of enzymes
acting upon proteins, lipases are a group of enzymes acting
upon lipid substances and maltase is the name of enzyme
acting upon maltose.

ADVERTISEMENTS:

Laptop for Every Need

Asus Exclusive Store…

Chandigarh 10:30AM–…

Sometimes the name of an enzyme indicates the nature of the

reaction it brings about. For example, invertase which breaks
sucrose into glucose and fructose, brings about inversion (this
is a process in which the raw material showing one type of
optical rotation gives out end products that shows the opposite
type of optical rotation).

Nomenclature:
A scrutiny of the enzyme nomenclature reveals that in many
cases, it is both inconsistent as well as misleading. Also
instances are not lacking where different biochemists gave
different names for the same enzyme. This anomaly has been
removed by the International Commission on Enzymes in its
report in 1961.
The Commission recognised that each enzyme should consist
of: (1) name of the substrate and (2) a word ending in ‘ase’
specifying one kind of catalytic reaction as in succinic dehy­-
drogenase, pyruvate transaminase. This nomenclature is
precise and systematic, though in some cases, it is long and
tongue-twisting. It is for this reason the trivial names are
retained with official sanction but only with reference to their
systematic names.

The modern system of enzyme classification was introduced by

International Union of Biochem­istry (IUB) in 1961. It groups
enzymes into the following six categories.

1. Oxidoreductases:
ADVERTISEMENTS:

They take part in oxidation and reduction reactions or transfer

of electrons. Oxidoreductases are of three types—oxidases,
dehydrogenases and reductases, e.g., cytochrome oxi­dase
(oxidises cytochrome), succinate dehydrogenase, nitrate
reductase.

2. Transferases:
They transfer a group from one molecule to another e.g.,
glutamate-pyruvate transaminase (transfers amino group from
glutamate to pyruvate during synthesis of alanine). The
chemical group transfer does not occur in the Free State.

3. Hydrolases:

They break up large molecules into smaller ones with the help
of hydrogen and hydroxyl groups of water molecules. The
phenomenon is called hydrolysis. Digestive enzymes belong to
this group, e.g., amylase (hydrolysis of starch), sucrase, and
lactase.

4. Lyases:

The enzymes cause cleavage, removal of groups without

hydrolysis, addition of groups to double bonds or reverse, e.g.,
histidine decarboxylase (breaks histidine to histamine and
CO2), aldolase (fructose-1, 6-diphosphate to dihydroxy acetone
phosphate and glyceraldehyde phosphate).

5. Isomerases:

The enzymes cause rearrangement of molecule structure to

effect isomeric changes. They are of three types, isomerases
(aldose to ketose group of vice-versa like glucose 6-phosphate to
fructose 6-phosphate), epimerases (change in position of one
constituent or carbon group like xylu­lose phosphate to ribulose
phosphate) and mutases (shifting the position of side group like
glucoses- phosphate to glucose-l-phosphate).

6. Ligases:

(Synthetases). The enzymes catalyse bonding of two chemicals

with the help of energy obtained from ATP, e.g, phosphenol
pyruvate PEP carboxylase (combines phosphenol pyru­vate
with carbon dioxide forming oxaloacetate accompanied by
hydrolysis of ATP.)

The modern system of enzyme nomenclature introduced by

International Union of Biochemis­try (IUB) envisages a method
of giving four numbers to any given enzyme, the first number
indicating the main class into which the enzyme falls, the
second and third indicates the subclass and sub­classes
respectively and the fourth is the serial number of the enzyme
in its particular sub-class; the four numbers are separated by
points.

Thus malic dehydrogenase is given the enzyme commission

number (Ec. No. 1) 1.1.1.37. The first 1 indicates that the
enzyme is an Oxidoreductase, the second 1 indicates that the
enzyme acts on CH-OH group of donors and third 1 indicates
that in the reaction which the enzyme promotes, NAD or NADP
functions as an acceptor molecule, 37 which is the last number
in the serial number given to this particular enzyme is the
group characterised by properties that 1.1.1 indicate.
Chemical Nature of Enzymes:
All enzymes are proteinaceous in nature (Sumner, 1926) with
the exception of recently discov­ered RNA enzymes. Some
enzymes may additionally contain a non-protein group.

ADVERTISEMENTS:

On the basis of differences in chemical nature, the enzymes

may be described as follows:

(i) Simple Enzymes:

Some enzymes are simple proteins, i.e., on hydrolysis, they

yield amino acids only. Digestive enzymes such as pepsin,
trypsin and chymotrypsin are of this nature.

(ii) Conjugate Enzymes:

It is an enzyme which is formed of two parts – a protein part

called apoenzyme (e.g., flavoprotein) and a non-protein part
named cofactor. The complete conjugate enzyme, consisting of
an apoenzyme and a cofactor, is called holoenzyme.

ADVERTISEMENTS:

There can be an enzymatic activity only when both

components (apoenzyme and cofactor) are present together.
The cofactor is sometimes a simple divalent metallic ion (e.
£.,Ca, Mg, Zn, Co, etc), and sometimes a nonprotein organic
com­pound. However, some enzymes require both kinds of
cofactors. If the cofactor is firmly bound to the apoenzyme, it is
called prosthetic group.

For example, cytochromes are the enzymes that possess

porphyrins as their prosthetic groups. If, instead of being more
or less permanently bound to the apoenzyme the cofactor
attaches itself to the apoenzyme only at the time of reaction, it
is called a coenzyme.

(iii) Metallo-enzymes:

The metal cofactors involved in enzymic reactions are both

monovalent (K+) and divalent cations (Mg++, Mn++, Cu++). These
may be loosely held by the enzyme, or as in some cases, go into
the composition of the molecule itself. If the metal forms part
of the molecule, as iron of haemoglobin or cytochrome is, the
enzymes are called metallo-enzymes.

(iv) Isoenzymes (Isozymes):

At one time it was believed that an organism has only a single

enzyme for a given step of a metabolic reaction. It was later
discovered that a substrate may be acted upon by a number of
variants of an enzyme producing the same product.

The multiple molecular forms of an enzyme occurring in the

same organism and having a similar substrate activity are
called isoenzymes or isozymes. Over 100 enzymes are known
to have isoenzymes. Thus a-amylase of wheat endosperm has
16 isozymes, lactic dehydrogenase has 5 isoenzymes in man,
while alcohol dehydrogenase has 4 isozymes in maize.
Isoenzymes differ in activity optima and inhibition.
The most thoroughly studied isozyme is lactic dehydrogenase
(LDH) which occurs in five possible forms in organs of most
vertebrates as observed by starch gel electrophoretic
separation. Two basically different types of LDH occur. One
type, which is inhibited strongly by relatively low
concentrations of pyruvate, predominates in the heart and is
called heart LDH.

ADVERTISEMENTS:

The other type, less easily inhibited by pyruvate, occurs in

many skeletal muscles and is thus called muscle LDH. The heart
LDH consists of 4 identical subunits, which are called H
subunits. The muscle LDH consists of 4 identical M subunits.
The two types of subunits, H and M, have different amino acid
compositions, enzyme kinetics and immunological properties.
These subunits in different combinations produce 5
isoenzymes.

They are thus useful to organism in adapting to varied

environmental conditions.

Mechanism of Enzyme Action:

The enzyme promotes a given reaction, but itself remains

unchanged at the end of the reaction. In 1913, Michaelis and
Menten proposed that an intermediate enzyme-substrate
complex is formed during the enzymic activity. The following
scheme may be written to illustrate concept:
Enzymes are biological catalysts which accelerate the rate of
reaction by altering the kinetic properties. Thus, the enzyme (E)
exercises its catalytic role on the substrate (S) by forming an
enzyme- substrate complex (ES) by a reversible reaction where
K1 is the rate constant for the formation of ES, and K2 is the rate
constant for the dissociation of ES to E and S.

After the formation of ES, substrate (S) is converted into the

products, thus making enzyme (E) available for further
combination with more substrate. The rate of conversion of ES
to the products of the reaction may be indicated by the
constant K3.

ADVERTISEMENTS:

Each enzyme-catalyzed reaction has a characteristic Km value,

which is the Michalies-Menten constant, which is a measure of
the tendency of the enzyme and the substrate to combine with
each other.

In this way the Km value is an index of the affinity of the

enzyme for its particular substrate. Greater the affinity of an
enzyme for its substrate, the lower the Km value.

Enzymes Reduces the Energy of Activation:

Energy of activation is that minimal amount of energy which is

required of a molecule to take part in a reaction. The effect of
enzymes is to lower the activation energy requirements,
thereby promot­ing appreciable reaction rates at lower
temperatures than would be possible otherwise.
The Catalytic Sites:

Enzymes are much larger compared to the substrate molecules.

In an enzyme-substrate therefore, the substrate is in contact
with only a very small area of the enzymic surface. This part of
the enzyme comprising amino acid resi­dues and peptide bonds
that are in physical contact with the substrate but essential for
cata­lytic activity put together constitute an active site,
presently referred as the catalytic site.

Ex­cluding the catalytic site, the rest of the enzyme molecule

may be necessary for maintaining the correct three-
dimensional conformation of the catalytic site or it may just be
there without any functional role.

ADVERTISEMENTS:

The structure of a catalytic site has been studied in some

enzymes. It is either a crevice on the enzyme as in papain and
ribonuclease or a deep pit as in carbonic anhydrase. What­ever
the shape of the catalytic site may be, it is believed that the
correct substrate binds with the catalytic site producing a
substrate-catalytic site complex.

The term productive binding is often ap­plied to this complex.

In productive binding, both the enzymes and substrates show
conformational changes with a reduction in activation energy
so that the substrate is converted into a product.
Theories of Enzyme Action:
1. Lock and Key Hypothesis:

The enzyme-substrate complex first hypothesized by Emil

Fischer in about 1884, assumed a rigid lock-and-key union
between the two. The portion of the enzyme to which the
substrate (or substrates) combines as it undergoes conversion
to a product is called the active site.

If the active site were rigid and specific for a given substrate,
reversibility of the reaction would not occur, because the
structure of the product is different from that of the substrate
and would not fit well.

2. Induced-Fit Theory:

As contrasted to a rigidly arranged active site of Fischer, Daniel

E. Koshland (1973) found evi­dence that the active site of
enzymes can be induced by close approach of the substrate (or
product) to undergo a change in conformation that allows a
better combination between the two.

ADVERTISEMENTS:
This idea is now widely known as the induced-fit theory and is
illustrated below. Apparently, the structure of the sub­strate is
also changed during many cases of induced fit, thus allowing a
more functional enzyme- substrate complex.

Properties of Enzymes:

1. The catalytic nature of the enzyme has been already

discussed in details earlier.

2. Reversibility:

Theoretically, all enzyme controlled reactions are reversible.

Reversibility is, however, dependent upon energy
requirements, availability of reactant, concentration of end
prod­ucts and pH. If the chemical potential of reactants is very
high compared to that of the products, the reaction might
proceed only towards products formation, because of the
chemical law of mass action. Most decarboxylation and
hydrolytic reactions are irreversible.

ADVERTISEMENTS:
The same enzyme facilitates forward and backward movement
of a reaction if only it is possible thermodynamically. A
convincing example is seen in the pathways of respiration and
photosynthesis. The enzymes of glycolysis and pentose
phosphate pathway dissimilate glucose. Some of these enzymes
work in the reverse direction in photosynthesis and build
glucose from carbon dioxide and water.

3. Heat sensitivity:

All enzymes are heat sensitive or thermolabile. Most enzymes

operate optimally between 25°-35°C. They become inactive at
freezing temperatures and denatured at 50°-55° C. However,
thermal algae and bacteria are an exception. Their enzymes
remain functional even at 80°C. Enzymes of seeds and spores
are also not denatured at 60°-70°C.

4. pH-sensitive:

Each enzyme functions at a particular pH, e.g., pepsin (2 pH),

sucrase (4-5 pH), trypsin (8.5 pH). A change of pH makes the
enzymes ineffective.

5. Specificity of actions:

Enzymes show specificity towards the substrates on which they

exercise their catalytic role. This unique property of the
enzymes is decided by: (1) the structural configuration of the
substrate molecule, (2) the conformation of the enzyme and (3)
the active or catalytic sites on the enzyme. The substrate
specificity of enzymes is of two kinds: group specificity and
stereo-specificity.

Enzymes usually show group specificity i.e., they attack only a

group of chemically related com­pounds. The group specificity
may be a relative group specificity, in which case the enzyme
functions on a number of homologous substrates.

Thus, hexokinase transfers phosphate group from ATP to at

least 23 hexoses or their derivatives like glucose, mannose,
fructose, and glucosamine. Some of the group specific enzymes
exhibit an absolute group specificity, which means the enzyme
acts only on a single compound and not its homologues.
Mannose, glucokinase and fructokinase are involved in
phosphorylations of the hexoses, mannose, glucose and
fructose respectively.

Enzymes also show stereo-specificity towards the substrate and

it is exhibited with both optical and geometric isomers.

(i) If the enzyme shows optical specificity, it acts on either

dextro (D) or laevo (L) isomer of the compounds. Thus, D.
aminoacid oxidase oxidises only D. amino-acids and L.
aminoacid oxidases react only with L. aminoacids.
(ii) The geometrical specificity is exhibited towards the cis and
trans isomers. Fumaric and malic acids are two geometrical
isomers. Fumaric hydratase acts on only the trans-isomer
fumaric acid but not on the cis-isomer malic acid.

6. Enzyme inhibition:

Substances or compounds that decrease the rate of an enzyme-

catalyzed reaction are known as inhibitors and the
phenomenon is described as enzyme-inhibition. There are
three types of inhibitions.

(i) Competitive inhibition:

When a compound competes with a substrate for the active site

on the enzyme protein and thereby reduces the catalytic
activity of that enzyme, the compound is consid­ered to be a
competitive inhibitor. Inhibition by such structural analogues
(called antimetabolites), which is reversed by simply adding
more substrate to the reaction mixture, is known as
competitive inhibition.

For example, succinate dehydrogenase readily oxidises succinic

acid to fumaric acid. If increas­ing concentrations of malonic
acid, which closely resembles succinic acid in structure, are
added, succinic dehydrogenase activity falls greatly.

The inhibition can now be reversed by increasing in turn the

concentration of the substrate succinic acid. The amount of
inhibition in this type of inhibition is related to (i) inhibitor
concentra­tion, (ii) substrate concentration, and relative
affinities of inhibitor and substrate. The inhibitory effect is
reversible.

Whether an inhibitor is competitive or not can be found out by

constructing the Lineiveaver- Burk Plot. Competitive inhibitors
alter Km of the enzyme because they occupy the active sites.
They do not, however, alter the Vmax or maximum velocity of
the reaction.

(ii) Non-competitive inhibition:

The type of inhibition that cannot be reversed by increasing the

substrate concentration is called non-competitive inhibition.
The inhibitor combines rather strongly with a site on the
enzyme other than the active site and this effect is not
overcome by simply raising the substrate concentration.

The amount of inhibition in this type of inhibition is related to

(a) inhibitor concentration, and (b) inhibitors affinity for the
enzyme. The substrate concentration has no effect on this
system, and non-competitive inhibitors alter the Vmax and not
the Km of the enzyme.

Cyanide, azide and heavy metal like silver, mercury, lead, etc
are some examples of non-competi­tive inhibitors that combine
with or destroy essential sulfhydryl groups or the metal
component of the enzymes.

(iii) Feedback (end-product) inhibition:

When end-product of a reaction serves to prevent the

formation of one of its own precursors by inhibiting the action
of the enzyme catalyzing the very- reaction, the inhibition is
called feedback inhibition.

The inhibition of conversion of A to B by X would be such an

inhibition. Here X, the ultimate product of the reaction, serves
to prevent the formation of one of its own precursors (B) by
inhibiting the action of enzyme a’ which catalyzes the change
from A to B.

In this instance, enzyme ‘a’ can be called the pacemaker since

the entire sequence is effectively regulated by it. An actual
example is the formation of cytidine triphosphate (CTP) from
aspartic acid and carbamyl phosphate in E. coli.

As a critical concentration of CTP is built up, the triphosphate

slows down its own formation by inhibiting the enzyme,
aspartate transcarbamylase (ATCase), which catalyzes the
pacemaker step of its own syn­thesis. When the concentration
of triphosphate is sufficiently lowered by metabolic utilization,
inhibi­tion is released, and its synthesis is renewed.

Factors Affecting Enzyme Action and Enzyme Kinetics:

1. Enzyme Concentration:

The rate of a biochemical reaction rises with the increase in

enzyme concentration upto a point called limiting or saturation
point. Beyond this, increase in enzyme concentration has little
effect.

2. Substrate concentration:

The first satisfactory mathematical analysis of the effect of

substrate concen­tration on the reaction velocity of enzyme-
catalysed re­action was made by Michaelis and Menten (1913).
With fixed enzyme concentration, an increase of substrate will
result at first in a very rapid rise in the velocity or reac­tion
rate.

As the substrate concentration continues to increase, however,

the increase in the rate of reaction begins to slow down until,
with a large substrate con­centration, no further change in the
velocity is observed. The velocity of the reaction obtained at
this high substrate concentration is defined as the maximum
velocity (Vm) of the enzyme-catalyzed reaction under the
specified conditions and the initial re­action velocity obtained
with substrate concen­trations below the saturation level is
called V.

The substrate concentration required to yield half the

maximum velocity (Vm/2) can be readily determined from
above figure and is an important constant in the enzyme
kinetics. It de­fines the Michaelis constant or Km. In other
words, K is defined as the substrate concentration when V= ½
Vm-Under carefully defined conditions of temperature, pH, and
ionic strength of the buffer, this constant Km approximates the
dissociation constant of an enzyme-substrate complex. The
recip­rocal of Km or 1/Km, approximates the affinity of an
enzyme for its substrate.

Kinetics of Enzyme Action:

The Michaelis constant K is of considerable importance since it

provides the mode of action of an enzyme catalyzing a
reaction. It should be noted that at low substrate concentration
the relation of velocity to substrate is almost linear and obeys
the first-order kinetics, i.e., the rate of the reaction A–>B is
directly propor­tional to the substrate concentration [A].

V = K’ [A] low [substrate]

Where V is the observed velocity of the reaction at

concentration [A] and K’ is the specific rate constant. At high
substrate concentration, however, the velocity of the reaction is
maximum and is independent of the substrate [A]; hence it
obeys the zero-order kinetics.
Vm = K’ Saturating [Substrate]

The Michaelis-Menten equation which describes this

relationship and also satisfactorily ex­plains curve, is as follows:

V = Vm[S]/Km +[S]

Where V = initial reaction velocity at given substrate

concentration [S]

Km = Michaelis constant, moles/litre.

Vm = Maximum velocity at saturated substrate concentrations

[S] = Substrate concentration in moles / litre

Determination of Km of an enzyme reaction by Michaelis-

Menten equation is in practice diffi­cult. An outcome of this
equation called Line-weaver-Burk plot is often used for such
determination.

1. Temperature:

An enzyme is active within a nar­row range of temperature. The

temperature at which an enzyme shows its highest activity is
called optimum tem­perature. Enzyme activity decreases above
and below this temperature. As catalyst they show an increased
reactiv­ity with temperature but their proteinaceous nature
makes them susceptible to thermal denaturation above the
opti­mum temperature.

2. pH:

The pH at which the maximum enzyme ac­tivity occurs varies

considerably from one enzyme to another. This is known as the
pH optimum. Any minor shift to either direction tends to lower
the enzyme activ­ity considerably. Since enzymes are proteins,
pH changes normally affect the ionic character of the amino
and carboxylic acid groups on the protein surface and
therefore markedly affect the catalytic nature of an enzyme.

3. Hydration:

Enzyme functions maximally under the enhanced kinetic

activity of the substrate as the continuous phase is higher. That
is why the seeds which have low water content register a
minimal enzymic activity though the substrates abound in
them. On germination, however, the enzy­mic activity rises
sharply and this is due to the absorption of water and
consequent promotion of kinetic activity of substrate
molecules.

Coenzymes:

In cellular physiology many enzymatic reactions are completed

in the presence of coenzymes. These are compounds which
function like the enzymes i.e., they speed up the biological
reactions, but they are not proteins like the true enzymes.

Definition:

A coenzyme may be defined as a particular kind of cofactor,

i.e., a non protein organic com­pound, or a carrier molecule
functioning in conjunction with a particular enzyme.

If the cofactor is firmly bound to the apoenzyme, it is called a

prosthetic group; and if, instead of being more or less
permanently bound to the apoenzyme, the organic cofactor
attaches itself to the enzyme protein only at the time of
reaction, it is called a coenzyme.

In cellular processes sometimes hydrogen atoms or electrons

are removed from one compound and transferred to another.
In all such cases a specific enzyme catalyzes the removal, but a
specific coenzyme must also be present to carry out the
transfer. The coenzyme temporarily joins to, or accepts the
removed group of atoms and may subsequently hand over
them to another acceptor compound.

Chemical Nature of Coenzymes:

The majority of coenzymes are chemical derivatives of the

nucleotides. More specifically, in most coenzymes the nitrogen
base portion of the nucleotides is substituted by another
chemical unit. This unit itself is usually a derivative of a
particular vitamin. The following coenzymes are important in
cellular physiology.

(i) Flavin derivatives or Flavin nucleotides (FMN and FAD)

(ii) Pyridine derivatives or Pyridine nucleotides (NAD and

NADP).

(iii) Coenzyme A

(iv) Coenzyme Q

(iv) Cytochromes
(vi) Thiamine pyrophosphate

Here only two coenzymes are described.

1. Flavin Nucleotides or Flavoproteins:

A large group of respiratory enzymes use as their cofactor one

of the two derivatives of riboflavin (vitamin B2). They are flavin
mononucleotide (FMN) and flavin adenine nucleotide (FAD).

Structure:

Riboflavin is a compound consisting of a ribose protein, and a

flavin portion, the latter being a complex triple ring structure.
In cells, a phosphate group is linked to riboflavin resulting in a
nucleotide like complex known as flavin mononucleotide
(FMN) or riboflavin monophosphate. If FMN joins to AMP, a
dinucleotide known as flavin adenine dinucleotide (FAD) is
formed.
Functions:

The combination of either FMN or FAD with an apoenzyme is

called flavoprotein (FP). Flavoproteins catalyzed the removal of
hydride ion (H–) and hydrozen ion (H+) from a metabolite. In
these coenzymes, it is the flavin portion of the molecule that
provides the specific place for temporary attachment of
hydrogen.

FMN + MH2 ——–> FADH2 + M

FMN + MH2——–> FMNH2 + M

In this reaction MH, represents a substrate, FADH, is the

reduced form of FAD, and FMNH2 is the reduced form of FMN.
An important source of hydrogen for this reaction is the
reduced pyridine nucleotide.

H+ + NADH + FAD ——–> NAD+ + FADH2

In all the cases reduced flavoproteins pass on their electrons to

the cytochromes.

2. Coenzyme Q:
This enzyme is a quinone, known as ubiquinone, and is mainly
found in the mitochondria but also in microsome and cell
nuclei, etc.

Structure:

The coenzyme Q or ubiquinone consists of a quinone with a

side chain whose length varies with the source of the
mitochondria. In most animal tissues the quinone possesses 10
isoprenosid units in its side chain and is called coenzyme Q10.

Function:

The coenzyme Q is a necessary component of the electron

transport chain in the mitochondria. It serves as an additional
hydrogen carrier between the flavin coenzymes (FAD and
FMN) and the cytochromes.

Q + FADH2——-> QH2 + FAD

Reduced (QH2) transfers its electrons to cytochrome b in the

mitochondria.

Related Articles:
1. Holoenzyme Components: (1) The Core Enzyme And (2) The
Sigma Factor
2. Enzyme-Linked Immnoabsorbent Assay (ELISA) Methods for
Detecting Antigen | Immunology