FRACTIONATION AT PILOT-PLANT SCALE OF AN HEMOGLOBIN HYDROLYSATE BY STRONG ANIONIC-EXCHANGE CHROMATOGRAPHY - APPLICATION TO THE PREPARATION OF AN AMPHIPHILIC PEPTIDE

The development and the scale-up of high performance anion chromatogra phy to obtain 1 milligram to 1 gram yields of a peptide fraction from a complex peptic haemoglobin hydrolysate is described here. The chroma tographic conditions were developed using a 1 cm(3) Mono Q analytical column and progressively scaled-up to a 6 dm(3) Q Sepharose Fast Flow column. For easy recovery of peptide and easy adjustment of conditions for final purification, a volatile buffer, ethanolamine/HCl buffer 20 mmol dm(-3), pH 10.5, was employed; desalting was carried out by a pi lot-plant scale electrodialysis which permitted the elimination of 99 % NaCl without important loss of peptide (less than 15 %). A combinati on of these techniques with reverse phase HPLC proved a useful strateg y for fractionation of a complex peptide mixture and enabled pure pept ides to be obtained in sufficient quantities for further analyses and biological tests. The example of preparation and purification of an am phiphilic peptide is described. Its ability to solubilize an insoluble photosensitizer, protoporphyrin IX, was determined in order to study its utilization as a carrier for photochemotherapy. (C) 1998 SCI.