Examination of protease effects on fluorescent peptid and protein substrates modified with pyrenyl derivatives by fluorometric methods

Spectrofluorometric methods are widely used for examination of activities of many various enzymes. Monitoring of enzyme activity in fluorometric examinations are performed by evaluation of changes of the emission signals obtained directly from the substrates of enzymes or from the fluorescent derivatives of their substrates. Thus it can sensitively be succeeded that the formation of Enzyme – Substrate complexes, conformational changes and enzymatic reaction kinetics by evaluation of fluorometric data. Many fluorescent organic substances, such as pyrene, fluorescein, rhodamin, anthracene, etc. can be covalently bound to the functional groups such as –COOH, -NH2, -OH, -SH existing in alkyl side chains of L-α-amino acids by the way of proper chemical changes performed in their structures. In this study, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat Bovine Serum Albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analysed by using the Exponential Series Method (ESM) to obtain the changes of fluorescence lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the proteases, the fluorescence lifetime distributions exhibited specific structures, which are attributed to the different activities of the proteases. This phenomenon opens the door to new assay methods which would be developed to determine and to distinguish the activities of proteases which have key roles in many diseas like cancer, AIDS, SARS, etc.