The procedure for the immunization of rats with renin was reinvestigated and optimal conditions for the formation of large amounts of antirenin within 3 weeks were established. This made possible the investigation of the effect on antigenicity of hog renin of treatment by enzymatic, chemical, immunochemical and physical procedures, known to exert profound effects on the structure of proteins, on the activity of other enzymes and on the immunologic specificity of antigens. These procedures included the digestion of renin by trypsin, papain, or pepsin, treatment with acid or alkali, exposure to carboxymethylation at various pH levels, reduction by various thiol compounds, depolymerization by urea, the detergent action of sodium dodecylsulfate, immunologic inactivation by antirenin, the blockade of functional groups by gold chloride and p-chloromercuribenzoate, nitroguanidination, and irradiation by x-rays or ultrasound.

Some of these treatments completely inactivated hog renin with respect to its enzymatic activity but spared to a variable extent the antigenic activity. Antirenin to unmodified hog renin is capable of inactivating the enzymatic activity of the renin of many animals but not of man. In no case was the antigenicity of modified hog renin so altered as to induce the formation of antirenin also capable of inactivating human renin.

Certain new properties of renin were established in the course of this study. It was shown that renin possesses an unusually high resistance to digestion by proteolytic enzymes. It can be exposed to reduction by cysteine without loss of activity, and subsequent exposure to atmospheric oxygen does not lead to its reoxidation. Exposure of renin to carboxymethylation in neutral solution results in complete inactivation of the pressor producing activity, but this activity can be protected by treatment with various thiol compounds. A reactive thiol group probably is not involved directly in the catalytic activity of renin. Renin which has been completely inactivated as a pressor agent by an excess of antirenin is still effective in the production of antirenin.

Various observations have been summarized in the proceedings of a recent symposium (6) which suggest that the suppression of antibody formation against antigen complexed with excess antibody is a general phenomenon. This does not apply to the renin-antirenin complex produced in vitro, for the product of this reaction remains antigenic, and retains the property of inducing the formation of antirenin. The choice of the species for immunization was found to be a determinant in the efficiency of the resulting antirenin to cross-react with renin of different species.

A distinction between enzymatically and antigenically active sites has become possible since complex formation, which abolishes the antigenicity of renin, retains its catalytic (pressor) activity (2), whereas various procedures which abolish the enzymatic activity of renin fail to destroy its ability to induce the formation of antirenin (Table V).

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