Kinetics of appearance of hemorphins from bovine hemoglobin peptic hydrolysates by a direct coupling of reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry

Reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry was used to improve the preparation of three opioid peptides (Leu–Val–Val–hemorphin-7, Val–Val–hemorphin-7 and Val–Val–hemorphin-4) resulting from bovine hemoglobin peptic hydrolysates. Optimal conditions for the preparation of these peptides were determined thanks to their kinetic studies of appearance in the course of peptic hydrolyses as a function of degree of hydrolysis of hemoglobin. We propose a low degree of hydrolysis (3%) to prepare Leu–Val–Val–hemorphin-7, a mean degree of hydrolysis (11%) to prepare Val–Val–hemorphin-7 and a high degree of hydrolysis (21%) to prepare Val–Val–hemorphin-4.