Improvement of Staphylococcus aureus-V8-protease hydrolysis of bovine haemoglobin by its adsorption on to a solid phase in the presence of SDS: peptide mapping and obtention of two haemopoietic peptides

Hydrolysis of bovine haemoglobin by the V8 protease from Staphylococcus aur eus (EC 3.4.21.19) was studied in the presence of sos in a homogeneous-phas e and in a solid-phase system. In both cases, hydrolyses were performed at 37 degrees C, in 50 mM phosphate buffer, pH 6.0, containing 0.1% SDS. Solid -phase hydrolyses were carried out with haemoglobin adsorbed on a negativel y charged hydrophobic support, namely Amberlyst 15Wet (Rohm and Haas). The, peptides were isolated from the hydrolysates by reverse-phase HPLC and ana lysed for their amino acid composition on a Waters Pico-Tag column, confirm ed by second-order derivative spectrometry or by MS. A peptide map of the h ydrolysates was drawn up, and numerous new cleavages in haemoglobin chains were observed, especially after Asp. This study showed that SDS permitted a dramatic improvement in the hydrolysis of whole haemoglobin by vs protease in both homogeneous-phase and solid phase systems after adsorption of haem oglobin on to an anionic support. Moreover, in the heterogeneous phase, all the theoretical cleavage sites of V8 protease, Asp as well as Glu bonds, w ere hydrolyeed, except for four sites which were resistant owing to strong interactions with the support. These results led to us obtain two haemopoie tic peptides, namely peptide alpha (Leu(76)- Pro-Gly-Ala-Leu-Seu-Glu(82)) a nd peptide beta (Lys(94)-Leu-His-Val-Asp-Pro-Glu(100)). These active peptid es have never before been prepared from bovine haemoglobin, and they may ha ve great potentialities in biotechnology.