Immunoglobulin Structure Analysis Protocol

Papain and Pepsin

Proteolytic enzymes (proteases) cleave polypeptide sequences. These enzymes have been intensely used to elucidate through dissection, the structure of antibody molecules. In addition, after protein subunit dissection, validation of which parts of the molecule are responsible for its various functions can be determined. These proteases cleave at specific amino acid sites. The enzymes papain and pepsin are two widely used proteases that have been used to study the structure of antibody molecules. Limited digestion with papin cleaves the antibody molecule into three fragments. Two fragments are identical and contain the binding activity. These fragments are termed the Fab fragments, Fragment antigen binding, because they are the arms of the antibody molecule and contain the light chains paired with the VH and CH1 domains of the heavy chains. The other fragment contains no antigen-binding activity but is able to crystallize readily, and is therefore called the Fc fragment. The Fc fragment is the paired CH2 and CH3 domains of the heavy chains and is the part of the antibody molecule that interacts with the effector molecules and cells. (1,4)

Immunoglobulin Fragments

The pattern of fragments depends on where the protease cleaves the antibody molecule in relation to the disulfide bonds that link the two heavy chains, which lie in the hinge region. Papain cleaves the antibody on the amino-terminal side of the disulfide bonds. In contrast, pepsin cleaves in the same general area of the antibody as papin, however on the carboxyl-terminal side of the disulfide bonds producing a fragment, the F(ab')2 fragment, which contains the two arms of antibody remain. The remaining heavy chain part is cut into several small fragments. MOreover, the F(ab)2 fragment has exactly the same antigen-binding properties of as the original intact antibody, but is unable to interact with any effector molecules or cells and there is a potential therapeutic application. (1,2,3)

 

Experimental Analysis of IgG Protein Structure

Human myeloma IgG1 protein can be structural analyzed through proteolysis with papain and pepsin, followed by running these and markers and controls on an SDS-PAGE. The samples were run in duplicate, with one set being run under reducing conditions and the other set, under non-reducing conditions. Beta-mercaptoethanol was utilized within the one half of samples.

The choice of acrylamide concentration utilized is vital to good separation. For proteins between 10 and 200 kDa molecular weights, an acrylamide gel concentration of 8-12% is suitable.

References:

1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing. 4th ed., New York, USA

2. Delves, P. and Roitt, I. 1999. Encyclopedia of Immunology: Academic Press Inc., 2nd ed., San Diego, USA

3. 1994. Current Protocols in Molecular Biology. Volume 2. John Wiley & Sons Inc., USA

4. Cruse, J. and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA