Antibacterial activity of a pepsin-derived bovine hemoglobin fragment.

Peptic digestion of bovine hemoglobin yields a fragment with antibacterial activity. This peptide was purified to homogeneity by a two-step procedure including anion exchange chromatography and preparative reversed-phase HPLC. Mass determination and fragmentation indicated that this peptide corresponded to the 1-23 fragment of the alpha chain of hemoglobin. The minimum inhibitory concentration and mode of action of this peptide towards Micrococcus luteus strain A270 were determined. Hemolytic assay, interaction with liposomes, and study of its structure in solution were also performed.

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 aureus (EC 3.4.21.19) was studied in the presence of SDS in a homogeneous-phase 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 negatively charged hydrophobic support, namely Amberlyst 15Wet (Rohm and Haas). The peptides were isolated from the hydrolysates by reverse-phase HPLC and analysed for their amino acid composition on a Waters Pico-Tag column, confirmed by second-order derivative spectrometry or by MS. A peptide map of the hydrolysates 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 V8 protease in both homogeneous-phase and solid phase systems after adsorption of haemoglobin on to an anionic support. Moreover, in the heterogeneous phase, all the theoretical cleavage sites of V8 protease, Asp as well as Glu bonds, were hydrolysed, except for four sites which were resistant owing to strong interactions with the support. These results led to us obtain two haemopoietic peptides, namely peptide alpha (Leu(76)-Pro-Gly-Ala-Leu-Ser-Glu(82)) and peptide beta (Lys(94)-Leu-His-Val-Asp-Pro-Glu(100)). These active peptides have never before been prepared from bovine haemoglobin, and they may have great potentialities in biotechnology.

Selectivity modification of chymotryptic hydrolysis of haemoglobin by its adsorption on a solid phase.

A change of selectivity of the chymotryptic hydrolysis of haemoglobin was evidenced when the protein was adsorbed on to a negatively charged hydrophobic support. The hydrolysis in heterogeneous phase improved the obtaining of positively charged and hydrophobic peptides as carriers of water-insoluble molecules. Haemoglobin adsorption on Amberlyst 15Wet was carried out in 0.1 M Tris/HCl buffer at pH 6.0. Chymotryptic hydrolysis was performed for 72 h at 37 degrees C in the same buffer. In solution, the presence of SDS was necessary to achieve the complete hydrolysis of haemoglobin chains, whereas it was not needed when haemoglobin was previously adsorbed on to the resin. The hydrolysis proceeded more slowly in heterogeneous phase than in homogeneous solution because of the diffusional restrictions but, at the end of the hydrolysis, the peptide populations were very different, as shown by reversed-phase HPLC. Moreover their functional properties were different too, since the haemoglobin hydrolysate obtained by heterogeneous catalysis had a better solubilizing ability towards the water-insoluble molecule, protoporphyrin IX, a photosensitizer for photodynamic therapy. A time-course study of the hydrolysis was performed to follow the evolution of a marker peptide (1-14alpha), which allowed us to explain the change in the selectivity of the chymotryptic reaction. This change could be due to a slowing down of the cut-off of some sites interacting with the support.