Assessment of Proteolysis by Pyrylium and Other Fluorogenic Reagents

Abstract

Aims:: We aim to evaluate the potential application of amine reactive fluorogenic reagents for estimating enzymatic proteolysis. Background:: Proteolytic enzymes play important roles in regulating many physiological processes in living organisms. Objectives:: Assessment of protein degradation by using reagents for protein assay techniques. Methods:: We have assayed samples at the start and after 30-60 minutes incubation with trypsin by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents and NanoOrange as non-amine reactive dye. Results:: All BSA prepared samples with trypsin have shown significantly higher fluorescence intensity (FI) versus controls (which reflects proteolysis) when assayed by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents. However, same samples assayed with NanoOrange as non-amine reactive reagent did not show any significant variation between samples containing trypsin and controls. Conclusion:: These results are confirming reliability of highly sensitive protein assays utilizing amine reactive fluorogenic reagents for general estimation of proteolysis.