A Study of the Structure of Trypsin-Like Serine Proteinases: 1. Study of Mini-Plasminogen Activation Using Tryptophan Fluorescence

Abstract

Abstract—It has been shown that the curve of the time dependence of tryptophan fluorescence during plasminogen activation by urokinase is well correlated with kinetic curves of activation, which were plotted according to both direct measurements of amidolytic activity of the forming plasmin and the analysis of the plasminogen cleavage products. Three curves represent the intercorrelation within a wide range of the pH and temperature change. We hypothesized that the fluorescence shift is caused by the changes in the Trp215 (Chymotrypsin numbering) side chain environment of activated plasmin. Plasmin activation via proteolytic cleavage of plasminogen induces rotation of the Trp215 side chain with subsequent translocation of the benzene ring of Trp215 from negatively charged (Asp194 and Glu143) to positively charged (Arg175) neighborhood. Our findings show that this rotation is not caused by the indol ring displacement from the substrate recognition pocket, which is provoked by the inhibitor or substrate binding. It has also been demonstrated that the conformation of plasminogen (at least relevant to Trp215 in the substrate recognition pocket) is not sensitive to the pH or temperature changes, while changes in fluorescence spectrum of plasmin correlate with its amidolytic activity.