Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide Substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized oil a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based oil the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring Of plasmin reaction oil the electrode Could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k(cat)/K-m value was 0.063 mu M-1 s(-1). Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system. (C) 2008 Elsevier Inc. All rights reserved.