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Purification of Human Kallikrein 6 from Biological Fluids and Identification of its Complex with ₁-Antichymotrypsin

¹ Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada.

² Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5G IL5 Canada.

^aAddress correspondence to this author at: Mount Sinai Hospital, Department of Pathology and Laboratory Medicine, 600 University Ave., Toronto, Ontario, M5G 1X5 Canada. Fax 416-586-8628; e-mail ediamandis{at}mtsinai.on.ca.

Background: Human kallikrein 6 (hK6) is significantly increased in serum in many patients with ovarian cancer and may have a role in amyloid precursor processing and Alzheimer disease. The forms of hK6 in biological fluids are poorly characterized.

Methods: hK6 protein was immunoaffinity-purified and positively identified by Western blotting, N-terminal sequencing, and mass spectrometry. hK6 in cerebrospinal fluid (CSF), milk, ascites, and serum was size-fractionated by chromatography and then measured by a highly sensitive and specific immunoassay. Hybrid assays were performed to detect the possible interactions between hK6 and proteinase inhibitors in CSF, milk, ascites fluid, and serum.

Results: N-Terminal sequencing identified hK6 in the proform in both CSF and milk. hK6 exists in two forms in milk and ascites fluid: a free form with a molecular mass of 25 kDa and a higher molecular mass form. Hybrid sandwich assays (capture antibody for hK6 and detection antibody for inhibitors), utilizing a panel of known serine protease inhibitors, indicated that ₁-antichymotrypsin forms a complex with hK6 in milk and ascites fluid. Only the free form of hK6 was detected in CSF and serum.

Conclusions: hK6 exists mainly as a proenzyme in milk and CSF. A fraction of this enzyme is partially complexed with ₁-antichymotrypsin in milk and ascites fluid of ovarian cancer patients.

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