Kinetics of appearance of four hemorphins from bovine hemoglobin peptic hydrolysates by HPLC coupled with photodiode array detection.

The kinetics of appearance of hemorphins during peptic hydrolysis of bovine hemoglobin was investigated by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array detector. The degree of hydrolysis (DH) of hemoglobin by pepsin was determined and different defined DH of hydrolysates were obtained. The analysis of these hydrolysates by HPLC coupled with a photodiode array detector allowed us to identify and quantify the hemorphins in every hydrolysate and to determine the quantitative evolution of hemorphins as a function of DH. It indicated that hemoglobin was a direct precursor of LVV-hemorphin-5 and LVV-hemorphin-7. These peptides were demonstrated to be secondary substrates for pepsin to generate VV-hemorphin-5 and VV-hemorphin-7. Moreover, LVV-hemorphin-7 was more stable towards pepsin than LVV-hemorphin-5. The affinity of pepsin towards some peptidic bonds was also demonstrated.