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.

Keywords: CBQCA; Proteolysis; chromeo P503; nanoOrange.; pyrylium; spectrofluorometry.