PURIFICATION OF CHYMOTRYPSIN INHIBITORS FROM LARVAL HEMOLYMPH OF THE SILKWORM, BOMBYX-MORI

Chymotrypsin inhibitors were purified from larval hemolymph of Bombyx mori. The purification steps involved ammonium sulfate fractionation (30-70%) and liquid chromatography on Butyl-Toyopearl 650M, Sephadex G-75, concanavalin A-Sepharose, Mono-Q, and Mono-S columns. Fourteen isoforms of chymotrypsin inhibitor were resolved in hybrid crosses: ten isoforms from c60 x n511, eight from k03 x FJ1, and nine from i10 x p50. Chromatographic and electrophoretic studies on the inhibitors suggested that they were expressed co-dominantly and could be classified into three molecular-size groups: 8-13 kDa, 40 kDa, and 42 kDa. CI-b4, CI-13a, CI-13b, and CI-13c in the first group, CI-3 in the second group, and CI-8 in the third group were present in all of the hybrids. Inhibitors in the third size group had an affinity for concanavalin A-Sepharose. It was found that CI-13 was composed of three different inhibitors.