Kinetic study of the appearance of an anti-bacterial peptide in the course of bovine haemoglobin peptic hydrolysis.

The kinetics of the alpha (1-23) peptide, which is the first anti-bacterial peptide to be isolated from a haemoglobin hydrolysate, was studied in the course of peptic hydrolysis at pH 4.5 and 23 degrees C in an homogeneous-phase system. A one-step reversed-phase HPLC coupled with photodiode array detector method was applied to identify and isolate this anti-bacterial peptide. The kinetics of peptide appearance were investigated in acetate buffer alone and in urea as a haemoglobin-denaturing agent. Two different mechanisms, 'one-by-one' for native haemoglobin hydrolysis and 'zipper' for denatured haemoglobin hydrolysis, were observed. Whatever the haemoglobin state, native or denatured, and whatever the hydrolytic mechanism, one-by-one or zipper, the anti-bacterial alpha (1-23) peptide is a transient peptide. To prepare the alpha (1-23) peptide it is suitable to hydrolyse haemoglobin in the presence of urea at a corrected degree of hydrolysis (DH(c)) of 13.5%. The amount of peptide produced in the presence of urea was twice as high as for the hydrolysis of native haemoglobin. The yields of alpha (1-23) peptide with respect to haemoglobin at the optimal DH(c) values were 55 and 25% respectively.