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.
Vercaigne-Marko
D
D
Laboratoire de Technologie des Substances Naturelles, Institut Universitaire de Technologie 'A' de Lille 1, BP 179, 59653 Villeneuve d'Ascq Cédex, France.
Kosciarz
E
E
Nedjar-Arroume
N
N
Guillochon
D
D
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology; D002417:Cattle; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D012697:Serine Endopeptidases / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013211:Staphylococcus aureus / Q000201:enzymology
D006454:Hemoglobins; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase