PURIFICATION OF ENZYMATICALLY ACTIVE KALLIKREIN HK2 FROM HUMAN SEMINAL PLASMA

Kallikrein hK2 is a member of the human glandular kallikrein family wh ich includes prostate-specific antigen (PSA) and pancreatic-renal kall ikrein. The purpose of this work was to isolate and characterize for t he first time the enzymatically active form of the hK2 protein startin g from the PCI-hK2 complex isolated from human seminal plasma (Deperth es, D., Chapdelaine, P., Tremblay, R.R., Brunet, C., Berton, J., Heber t, J., Lazure, C. and Dube, J.Y. (1995) Biochim. Biophys. Acta 1245, 3 11-316). That complex was dissociated by an incubation at alkaline pH and final purification was achieved by C-18 reverse phase HPLC. The pu rified material contained a 27 kDa band by SDS gel electrophoresis and had the expected NH2-terminal amino acid sequence of hK2. It hydrolyz ed synthetic chromogenic substrates containing eaters of lysine and ar ginine but not of phenylalanine. Furthermore, hK2. formed molecular co mplexes with alpha(2)-antiplasmin, alpha(1)-antichymotrypsin, antithro mbin III and alpha(2)-macroglobulin but not with alpha(1)-antitrypsin. In conclusion, the new findings of the present paper are that the PCI -hK2 complex can be dissociated by mild procedures, that the free hK2 protein can be purified thereafter by standard HPLC procedures, that t he recovered free hK2 is a trypsin-like enzyme and that it can form mo lecular complexes with many of the major serum proteinase inhibitors.