Improved fluorometric assay of histidine and peptides having NH2-terminal histidine using o-Phthalaldehyde

o-Phthalaldehyde (OPT) reacts with many biogenic compounds such as spermidine, histamine, histidine and peptides with NH₂-terminal histidine, yielding intensely fluorescent condensation products. This communication examines the reaction conditions for the OPT-induced fluorescence of histidine and peptides with NH₂-terminal histidine for the purpose of improving the sensitivity as well as the specificity of the assay of these compounds. Reaction with OPT at pH 11.2–11.5 and at 40°C for 10 min was found to be optimal for histidine. After cooling, the fluorescence was read at $\text{360}\text{440}\text{nm}$ (uncorrected instrument values). The method measures as little as 4–5 ng/ml. Peptides with NH₂-terminal histidine were found to interfere with the assay whereas histamine, histidinol and spermidine did not. The optimum reaction and assay conditions for the OPT-induced fluorescence of the histidyl-dipeptides varied markedly from one peptide to another. As a group peptides with NH₂-terminal histidine are best assayed by condensation with OPT at pH 11.8 at room temperature and with a reaction time of 30 min. Fluorescence should be read before as well as after acidification to pH 2.5. Details are given for the assay of individual histidyl-dipeptides.