فعاليت آنزيمي و ويژگي سوبسترايي آنزيم كاليكرين انساني 13

Background: hK 13 is one of the enzymes that encode by hK13 gene from the kallikrein family gene. It has been reported that this enzyme is found in the seminal plasma, ascites fluid of the patients with ovarian cancer and the breast milk. In this study, we aimed to detect the enzymatic activity and the substrate specificity of the hK13, which may be used in the future for the diagnosis and management of the cancer patients. Materials and Methods: The pichia pastoris culture supernatant, with incorporated hK13 recombinant DNA,was collected by FPLC for the purification of the hK13. The fluorescence was measured by Wallace Victor fluorometer and the Km was calculated by non-linear regression analysis using the Enzyme Kinetics Module 1.1 (Sigma plot, SSPS Inc). Results: The KcatlKm for Val-Pro-Arg-AMC and Phe-Ser-Arg-AMC was higher than that of the other substrates. hK13 was unable to cleave the peptide bonds from carboxyl side of the lysine effectively. The KcatlKm value for Val-Pro-Arg-AMC hydrolysis by trypsin was 1.5 times more than that of the hydrolysis of the same substrate by hK13. Conclusion: Among the 6 fluorogenic substrates, the tripeptide Val-Pro-Arg-AMC was the best substrate with the highest value of KcatlKm for hK13. So hK13 may be used as a tumor marker for identification of patients with cancer. The practical application of this suggestion needs to use advanced clinical laboratory techniques, especially substrates for hK13 and to eliminate the interference of the enzyme activity in the circulation.