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.
Kosciarz
E
E
Laboratoire de Technologie des Substances Naturelles, IUT "A" de Lille 1, BP 179, 59 653 Villeneuve d'Ascq Cedex, France.
Vercaigne-Marko
D
D
Scellier
N
N
Nedjar-Arroume
N
N
Guillochon
D
D
D000327:Adsorption; D001671:Biochemistry / Q000379:methods*; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011524:Protoporphyrins / Q000737:chemistry / Q000378:metabolism
D006454:Hemoglobins; D010446:Peptide Fragments; D011524:Protoporphyrins; C028025:protoporphyrin IX; D002918:Chymotrypsin; C117608:alpha-chymotrypsin