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Purification and Characterization of Human Kallikrein 11, a Candidate Prostate and Ovarian Cancer Biomarker, from Seminal Plasma

Authors' Affiliations: ¹ Department of Pathology and Laboratory Medicine, Mount Sinai Hospital and ² Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Requests for reprints: Eleftherios P. Diamandis, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada. Phone: 416-586-8443; Fax: 416-586-8628; E-mail: ediamandis{at}mtsinai.on.ca.

Purpose: Preliminary data suggest that hK11 is a novel serum biomarker for prostate and ovarian cancer. To examine the enzymatic characteristics of hK11, we purified and functionally characterized native hK11 from seminal plasma.

Experimental Design: hK11 was purified from seminal plasma by immunoaffinity chromatography and characterized by kinetic analysis, electrophoresis, Western blots, and mass spectrometry.

Results: hK11 is present in seminal plasma at concentrations ranging from 2 to 37 µg/mL. Using immunoaffinity chromatography and reverse-phase high-performance liquid chromatography, we purified hK11 to homogeneity. In seminal plasma, hK11 is present as a free enzyme of 40 kDa. About 40% of hK11 is enzymatically active, whereas the rest is inactivated by internal cleavage after Arg¹⁵⁶ (Genbank accession no. AF164623), which generates two peptides of 20 kDa, connected by internal disulfide bonds. Purified hK11 possesses trypsin-like activity and cleaves synthetic peptides after arginine but not lysine residues. It does not cleave chymotrypsin substrates. Antithrombin, ₁-antichymotrypsin, ₂-antiplasmin, and ₁-antitrypsin have no effect on hK11 activity and do not form complexes with hK11 in vitro. The strongest inhibitor, APMSF, completely inhibited hK11 activity at a concentration of 2.5 mmol/L. Aprotinin and an hK11-specific monoclonal antibody inhibited hK11 activity up to 40%. Plasmin is a strong candidate for cleaving hK11 at Arg¹⁵⁶.

Conclusion: This is the first report on purification and characterization of native hK11. We speculate that hK11, along with other kallikreins, proteases, and inhibitors, participates in a cascade enzymatic pathway responsible for semen liquefaction after ejaculation.

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