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

Abstract

The kinetics of appearance of bioactive peptides (neokyotorphin, VV-haemorphin-4 and a bradykinin-potentiating peptide) were investigated in the course of the hydrolysis of haemoglobin by pepsin at 23 degrees C in 0.1 M sodium acetate buffer at pH 4.5. Isolation of these peptides was performed in one 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 peptide is an intermediate peptide. At pH 4. 5, LVV-haemorphin-7, VV-haemorphin-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-haemorphin-4 was not detected. The effect of the composition of the solvent on the peptic hydrolysis was studied to improve the preparation of these active peptides. The kinetics of appearance of these peptides were compared in the presence of 20% (v/v) ethanol, a stabilizing solvent of haemoglobin and in the presence of urea, a denaturant agent. The best conditions for preparing neokyotorphin 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. For the preparation of the bradykinin-potentiating peptide, the hydrolysis of haemoglobin in the urea-containing buffer at a moderate degree of hydrolysis of 10% was suitable.