Isolation, crystallization, and properties of calotropins DI and DII from Calotropis gigantea

Abstract

The latex of madar plants, Calotropis gigantea, contained at least five proteases, two of which designated as calotropins DI and DII were isolated and crystallized. The crystalline preparations were homogeneous by ion exchange chromatography, polyacrylamide gel and sodium dodecyl sulfate-gel electrophoresis, and sedimentation in the ultracentrifuge. They contained a single sulfhydryl group and the enzymic activity depended on it. The physicochemical properties of both calotropins were similar to those of other plant cysteine proteases. As in the case of papain, their sedimentation coefficients were independent of protein concentration. The average molecular weights calculated from sedimentation and diffusion coefficient values, Archibald experiment, amino acid composition, and sodium dodecyl sulfate-gel electrophoresis were 23,800 for calotropin DI and 24,200 for calotropin DII. Their amino acid compositions were very similar with minor differences in individual amino acids. Like papain they were devoid of carbohydrate moieties. In the enzymic properties both calotropins were found to be indistinguishable. Hydrolysis of azoalbumin by each enzyme was optimal in the pH range 7.5–8.0 at 55 °C. Their caseinolytic activities were about one-third as active as papain or ficin. They, unlike papain and ficin, showed no measurable activity toward synthetic substrates such as benzoyl- l-arginine ethyl ester, p-tosyl- l-arginine methyl ester, carbobenzoxy glycine p-nitrophenyl ester, benzoyl- dl-arginine p-nitroanilide, and benzoyl- l-arginine amide.