Chemical and Enzymatic Methods Revealed Basic Antibody Structure

Our knowledge of basic antibody structure was derived from a variety of experimental observations. When the 7-globulin fraction of serum is separated into high- and low-molecular-weight fractions, antibodies of around 150,000-MW, designated as immunoglobulin G (IgG) are found in the low-molecular-weight fraction. In a key experiment, brief digestion of IgG with the enzyme papain produced three fragments, two of which were identical fragments and a third that was quite different (Figure 4-3). The two identical fragments

Heavy chain Light chain (i,Y,a,S, or e KorX

Heavy chain Light chain (i,Y,a,S, or e KorX

Igg Molecular Weight Light Chain

Antigen binding

Biological activity

Antigen binding

-s

s-

0

a

to

u

-s

s-

-s

s-

0

rC)

a

u

-s

s-

COO"

Biological activity

COO-

COO"

FIGURE 4-2

Schematic diagram of structure of immunoglobulins derived from amino acid sequencing studies. Each heavy and light chain in an immunoglobulin molecule contains an amino-terminal variable (V) region (aqua and tan, respectively) that consists of 100-110 amino acids and differs from one antibody to the next. The remainder of each chain in the molecule—the constant (C) regions (purple and red)—exhibits limited variation that defines the two light-chain subtypes and the five heavy-chain subclasses. Some heavy chains (7, 8, and a) also contain a praline-rich hinge region (black). The amino-terminal portions, corresponding to the V regions, bind to antigen; effector functions are mediated by the other domains. The (x and € heavy chains, which lack a hinge region, contain an additional domain in the middle ofthe molecule.

Go to www.whfreeman.com/immunology Immunoglobulins

Animation

L chain

L chain

Basic Immunoglobulin Structure

H chain

Fc fragments

Mercaptoethanol reduction

H chain

Papain digestion

Fc fragments

Papain digestion

Mercaptoethanol reduction

H chains

H chains

FIGURE 4-3

Prototype structure of IgG, showing chain structure and interchain disulfide bonds. The fragments produced by various treatments are also indicated. Light (L) chains are in gray and heavy (H) chains in blue.

(each with a MW of 45,000), had antigen-binding activity and were called Fab fragments ("fragment, antigen binding"). The other fragment (MW of 50,000) had no antigen-binding activity at all. Because it was found to crystallize during cold storage, it was called the Fc fragment ("fragment, crystallizable"). Digestion with pepsin, a different pro-teolytic enzyme, also demonstrated that the antigen-binding properties of an antibody can be separated from the rest of the molecule. Pepsin digestion generated a single 100,000-MW fragment composed of two Fab-like fragments designated the F(ab')2 fragment, which binds antigen. The Fc fragment was not recovered from pepsin digestion because it had been digested into multiple fragments.

A key observation in deducing the multichain structure of IgG was made when the molecule was subjected to mercap-toethanol reduction and alkylation, a chemical treatment that irreversibly cleaves disulfide bonds. If the sample is chro-matographed on a column that separates molecules by size following cleavage of disulfide bonds, it is clear that the intact 150,000-MW IgG molecule is, in fact, composed of subunits. Each IgG molecule contains two 50,000-MW polypeptide chains, designated as heavy (H) chains, and two 25,000-MW chains, designated as light (L) chains (see Figure 4-3).

Antibodies themselves were used to determine how the enzyme digestion products—Fab, F(ab')2, and Fc—were related to the heavy-chain and light-chain reduction products.

This question was answered by using antisera from goats that had been immunized with either the Fab fragments or the Fc fragments of rabbit IgG. The antibody to the Fab fragment could react with both the H and the L chains, whereas antibody to the Fc fragment reacted only with the H chain. These observations led to the conclusion that the Fab fragment consists of portions of a heavy and a light chain and that Fc contains only heavy-chain components. From these results, and those mentioned above, the structure of IgG shown in Figure 4-3 was deduced. According to this model, the IgG molecule consists of two identical H chains and two identical L chains, which are linked by disulfide bridges. The enzyme papain cleaves just above the interchain disulfide bonds linking the heavy chains, whereas the enzyme pepsin cleaves just below these bonds, so that the two proteolytic enzymes generate different digestion products. Mercaptoethanol reduction and alkylation allow separation of the individual heavy and light chains.

Was this article helpful?

+1 0
Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

Get My Free Ebook


Responses

  • sergio
    What enzymes cleaves disulfide bonds?
    8 years ago
  • RITVA
    What is the effect of mercaptoethanol reduction on products of enzymatic digestion of antibodies?
    3 years ago
  • duncan
    What are the production of enzymatic digestion of antibody with mercaptoethanol reduction?
    3 years ago
  • deborah
    How immumoglobulin structure is revealed?
    3 years ago
  • nazario
    Which enzyme is used to study of the structure Immunoglobulin?
    1 year ago

Post a comment