Question

1. Papain and pepsin are not the only enzymes that can digest antibodies. Find one example of another enzyme that can digest antibodies and answer the following. (~150-200 words total)

1. Describe any similarities or differences in the theoretical antibody fragments generated using this enzyme as compared to pepsin and papain. Use a figure to support your answer.
2. What results would you expect to see in a haemagglutination assay and on non-reducing and reducing SDS-PAGE gels after digesting rabbit anti-SRBC IgG using this enzyme? Include simple diagrams to illustrate your answer.

Answer 1

Antibodies are powerful tools for protein and molecular detection and purification.Papain and pepsin are not the only enzymes that can digest antibodies.One example of another enzyme that can digest antibodies is Ficin.

a) Ficin digestion: Mouse IgG1 fragments

Ficin is a thiol protease that can digest mouse monoclonal IgG1 into either F(ab')2 or Fab fragments, depending on the concentration of cysteine included. Ficin will generate F(ab')2 in the presence of 4mM cysteine. Fab fragments result with ficin in the presence of 25mM cysteine.

Mouse IgG1 Fab and F(ab')2 preparation by ficin digestion and fragmentation.

Whereas digestion of antibodies from Pepsin and Papain are different from Ficin in many ways:-

Papain digestion: Fab from IgG

Papain is a nonspecific, thiol-endopeptidase that has a sulfhydryl group in the active site, which must be in the reduced form for activity. When IgG molecules are incubated with papain in the presence of a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragment and one Fc fragment

. When Fc fragments are of interest, papain is the enzyme of choice because it yields an intact 50,000-dalton Fc fragment.

Antibody Fab preparation by papain digestion and fragmentation.

Pepsin digestion: F(ab')2 from IgG

Pepsin is a nonspecific endopeptidase that is active only at acid pH. It is irreversibly denatured at neutral or alkaline pH. Digestion by the enzyme pepsin normally produces one F(ab')2 fragment and numerous small peptides of the Fc portion. The resulting F(ab')2 fragment is composed of two disulfide-connected Fab units. The Fc fragment is extensively degraded, and its small fragments can be separated from F(ab')2 by dialysis, gel filtration or ion exchange chromatography.

Antibody F(ab')2 preparation by pepsin digestion and fragmentation.

Some other differences and similarities between the 3 enzymes are as followed:-

Properties	Ficin	Papain	Pepsin
Type protease	Thiol-type protease	Thiol-type protease	Acid-type protease
MW	26,000	23,000	35,000
Isoelectric point pI	1.5	1.5	11
pH optimum	6.5 (4 to 9.5)	6.5 (4 to 9.5)	1 (1 to 5)
A280 at 1%	21	25	14.7

b) Results expected to see in a haemagglutination assay and on non-reducing and reducing SDS-PAGE gels after digesting rabbit anti-SRBC IgG using the enzyme Ficin are:-

Ficin cleavage produces F(ab')2 fragments of nearly identical size to those obtained from IgG by pepsin but with immunoreactivities and affinities comparable to those of intact IgG1 antibody. By increasing the concentration of cysteine activator, Fab antigen-binding fragments can be generated. The integrity of the resultant antigen-binding fragments is aided by the neutral pH conditions of the ficin digestion. Ficin is the preferred protease for production of fragments of murine monoclonal IgG1. Although F(ab')2 can be generated from mouse IgG1 using pre-activated papain, efficiency and reproducibility with papain are difficult to obtain.

Immobilized ficin (Ficin Agarose Resin) enables better control of the digestion reaction than free ficin, as it results in antibody fragments that are free of autodigestion products. In addition, use of ficin resin allows complete and rapid removal of ficin from the antibody fragment products.