Serine proteinase from Cucurbita ficifolia seeds

A new serine proteinase was isolated from Cucurbita ficifolia seeds by the purification procedure, which includes: extraction, salting out with ammonium sulphate, chromatography on CM-cellulose. Sephacryl S-300 gel filtration and h.p.l.c. on DEAE-2SW TSK column. The enzyme was homogeneous both in native and SDS PAGE. Three independent methods showed its molecular mass to be approximately 77 kDa. The enzyme was inhibited by specific serine proteinase organic inhibitors, and was active in the presence of inhibitors specific for other proteinase classes. Surprisingly, squash proteinase exhibited a very high and broad pH optimum with a maximum at 10.7. It hydrolysed many different peptide bonds in B-chain of insulin and was able to cleave four bonds in endogenous serine proteinase inhibitor (CMTI).