A two-stage cleavage of rabbit gamma-globulin by a water-insoluble papain preparation followed by cysteine.

The cleavage into large fragments of both normal and immune rabbit y-globulin by cysteine-activated papain was reported by Porter (1, 2). In the case of immune y-globulin the antibody activity was found associated with two of the three principal fragments isolated. Nisonoff et al. (3) have carried out the cleavage of rabbit immune y-globulin into similar fragments with a sedimentation constant of about 3.5 S by peptic digestion followed by reduction with cysteine. In this paper we describe an analogous two-stage cleavage of normal and immune rabbit -y-globulin by the consecutive action of a water-insoluble papain derivative and a reducing agent. The water-insoluble papain was prepared by a procedure similar to that employed by Bar-Eli and Katchalski (4) for the preparation of a water-insoluble trypsin. The use of water-insoluble papain permitted the termination of proteolysis at any given instant by filtration or centrifugation. The insoluble papain derivative, which could easily be freed of reducing agents, was found to act on proteins in the presence of negligible amounts of cysteine. It was thus possible to carry out the digestion of y-globulin without reduction of substrate or products. Immune y-globulin, hydrolyzed briefly under such conditions, although rendered susceptible to fragmentation by reduction, showed no alteration in its rate of sedimentation or in its ability to precipitate antigen. Antigen-antibody precipitates containing the papain-treated antibody dissolved on reduction to yield soluble complexes between antigen and fragments of antibody.