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Biotechnology and Applied Biochemistry (2001) 34, (173–181) (Printed in Great Britain)

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A simple method for the two-step preparation of two pure haemorphins from a total haemoglobin peptic hydrolysate by conventional low-pressure chromatographies

Luc Choisnard, David Durand, Dominique Vercaigne-Marko¹, Naima Nedjar-Arroume, Pascal Dhulster and Didier Guillochon

Laboratoire de Technologie des Substances Naturelles IUT ''A'' de Lille 1, BP 179, 59653 Villeneuve d'Ascq cedex, France

Key words: cation-exchange chromatography, hydrophobic-interaction chromatography, opioid peptide.

Abbreviations used: RP-HPLC, reversed-phase HPLC; LVVh7, LVV-haemorphin-7; VVh4, VV-haemorphin-4; VVh7, VV-haemorphin-7; MALDI, matrix-assisted laser-desorption ionization.

¹To whom correspondence should be addressed (e-mail Dominique.Vercaigne@univ-lille1.fr).

The development of a simple purification method for two haemorphins, VV-haemorphin-7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) and VV-haemorphin-4 (Val-Val-Tyr-Pro-Trp-Thr), from a total peptic haemoglobin hydrolysate is described. Cation-exchange chromatography on CM-Sephadex was used as a pre-fractionation step for the hydrolysate. VV-haemorphin-7 and VV-haemorphin-4 were eluted in two different fractions. The second and final purification step was hydrophobic-interaction chromatography on phenyl-Sepharose, which allowed the isolation of the two pure haemorphins. Haemoglobin and globin hydrolysates were compared as starting materials. For easy recovery of haemorphins and easy adjustment of conditions for final purification, a volatile buffer, ammonium acetate buffer, pH 6.5, was employed. This process, which allowed the preparation of pure haemorphins from a total protein hydrolysate, could be scaled up and used in the food industry.

Received 1 June 2001/13 September 2001; accepted 17 September 2001

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