New Insights on Plasmin Long Term Stability and the Mechanism of Its Activity Inhibition Analyzed by Quartz Crystal Microbalance

Abstract

We used the research quartz crystal microbalance (RQCM) to monitor regulatory effects of plasmin and trypsin in the presence of their inhibitor α2-antiplasmin. The gold surface of quartz crystals was modified with a β-casein layer that served as a substrate for protease digestion. The addition of plasmin or trypsin as well as their mixtures with α2-antiplasmin resulted in an increase of resonant frequency, f, and in a decrease of motional resistance, Rm, depending on the molar ratio of protease: antiplasmin. At equimolar concentrations of protease and α2-antiplasmin (5 nM:5 nM) full inhibition of protease activity took place. Monitoring of plasmin activity on an hourly and daily basis revealed a prominent effect of autolysis and decrease of plasmin activity in freshly activated samples. The degree of inhibition as well as plasmin half-life (t1/2 = 2.48 ± 0.28 days) connected with its degradation was determined.

Application of Multiharmonic QCM-D for Detection of Plasmin at Hydrophobic Surfaces Modified by β-Casein

“…Trypsin cleaves proteins from the C side of a lysine or arginine, while plasmin preferentially cleaves the lysine bond. As a result, trypsin appears to cleavage β-casein more than plasmin, with 15 potential cleavage sites versus 11 in the case of plasmin [49]. However, the most interesting finding is that the trypsin exclusive cleavage sites are preferentially found in the highly hydrophobic carboxyl terminal region of casein.…”

A General, Label-Free and Homogeneous Electrochemical Strategy for Probing of Protease Activity and Screening of Inhibitor

“…Traditional methods, such as enzyme-linked immunosorbent assay (ELISA), gel electrophoresis and high-performance liquid chromatography (HPLC), always involve tedious separation/washing steps, complicated equipment and/or low sensitivity for protease detection [ 2 , 3 ]. To overcome these shortcomings, various novel methods have been developed recently, including mass spectrometry, fluorescence, colorimetric assays, quartz crystal microbalance, surface plasma resonance (SPR), surface-enhanced Raman scattering (SERS), and electrochemical, photoelectrochemical or electrochemiluminescent assays [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. As a typical homogeneous assay, fluorescence methods have been broadly applied because of their distinguish merits of high sensitivity and fast response.…”