Sensitive and selective determination of peptides,PG and PGP,using a novel fluorogenic reagent 4-chlorobenzene-1,2-diol

A novel fluorometric method was developed for the sensitive and selective detection of Pro-Gly (PG) and Pro-Gly-Pro (PGP) using 4-chlorobenzene-1,2-diol (4-CBD) as a fluorogenic reagent. The reaction was performed at 37°C for 30 min in the presence of a borate buffer (pH 7.0) and sodium periodate. The resulting fluorescence intensity was measured using a spectrofluorometer with excitation and emission wavelengths of 450 nm and 535 nm. To obtain a stable fluorescent signal and maximise its intensity, different reaction conditions such as the concentrations of the reagents, the reaction time, and the pH were optimised. Under the optimised conditions, a linear relationship was obtained between fluorescence intensity and peptide concentrations from 1.0–40.0 µmol L^?1 with a limit of detection of 1.0 µmol L^?1 (S/N = 3). Both PG and PGP generated a strong signal out of all the peptides tested and no other biogenic substances such as amino acids or proteins produced any fluorescence. The reaction thus developed is simple, rapid, selective, and sensitive. It can be applied to the determination of peptides as biomarkers or substrates.