Antibody fragmentation by pepsin

Background

Pepsin, along with trypsin and chymotrypsin, is one of the major proteases of the digestive system. The active enzyme results from autocatalysis of its precursor pepsinogen in the presence of hydrochloric acid (HCl). Pepsin exhibits maximal activity at low pH (around 2.0) and is inactive at neutral pH (between 6.5 and 8.0). Alkaline pH (above 8.0) leads to irreversible denaturation. The preferential cleavage pattern involves aromatic (phenylalanine, tryptophan and tyrosine) as well as some hydrophobic residues in specific conditions.

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Digestion of immunoglobulins

Digestion of immunoglobulins by pepsin occurs below the hinge region of the heavy chains, resulting in a F(ab’)2 fragment which is composed of two Fab fragments still linked together by the remaining portion of their respective heavy chains at the hinge region.

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.

Use of F(ab’)2 fragments

F(ab’)2 fragments share many advantages with Fab fragments, but unlike the latter, F(ab’)2 fragments remain divalent and can be used to bind and precipitate antigens.

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Pepsin digestion of immunoglobulins

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