Solvent effect on kinetics of appearance of neokyotorphin, VV-haemorphin-4and a bradykinin-potentiating peptide in the course of peptic hydrolysis of bovine haemoglobin

The kinetics of appearance of bioactive peptides (neokyotorphin, VV-haemorp hin-4 and a bradykinin-potentiating peptide) were investigated in the cours e of the hydrolysis of haemoglobin by pepsin at 23 degrees C in 0.1 M sodiu m acetate buffer at pH 4.5, Isolation of these peptides was performed in on e step by C-4 reversed-phase HPLC. We have shown that neokyotorphin, and VV -haemorphin-4 are two final peptides and that the bradykinin-potentiating p eptide is an intermediate peptide. At pH 4.5, LVV-haemorphin-7, VV-haemorph in-7 and VV-haemorphin-4 appeared successively in the course of the peptic hydrolysis. At: pH 2, the optimum pH of the pepsin hydrolysis, VV-haemorphi n-4 was not detected. The effect of the composition of the solvent on the p eptic hydrolysis was studied to improve the preparation of these active pep tides, The kinetics of appearance of these peptides were compared in the pr esence of 20% (v/v) ethanol, a stabilizing solvent of haemoglobin and in th e presence of urea, a denaturant agent. The best conditions for preparing n eokyotorphin and VV-haemorphin-4 were to achieve the peptic reaction in the buffer alone or in the presence of urea at a high degree of hydrolysis. Fo r the preparation of the bradykinin-potentiating peptide, the hydrolysis of haemoglobin in the urea-containing buffer at a moderate degree of hydrolys is of 10% was suitable.