A Novel Fluorometric Method for the Selective Determination of Pro-Gly and Pro-Gly-Pro

Fluorescence (FL) derivatization reactions have often been used for the selective determination of bioactive peptides. Herein, a sensitive and selective fluorometric method has been developed for Pro-Gly and Pro-Gly-Pro using a derivatizing reagent 3,4-dihydroxybenzoic acid (3,4-DHBA). In the presence of borate buffer (pH 8.0) and sodium periodate, peptides were reacted with 3,4-DHBA at 37 °C for 30 min. The resulting FL intensity was measured by spectrofluorometer with the excitation wavelength of 450 nm and the emission wavelength of 535 nm. Different reaction conditions such as concentrations of the reagents, reaction time and pH were optimized to develop the method. Under the optimized conditions, a linear relationship was obtained between FL intensity and peptide concentration from 5–30 µM with a lower detection limit of 5 µM. We found that 3,4-DHBA showed strong preference for Pro-Gly and Pro-Gly-Pro amongst all the peptides tested and no other biogenic substances such as amino acids or proteins produced any FL. The reaction is selective, sensitive and simple which can be applied for the determination of peptides as biomarkers in biological samples or for the assay of various protease activities.