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Involvement of histidine-32 in the biological activity of α-lactalbumin

par Prieels, Jean-Paul ;Bell, John Ellis;Schindler, Melvin;Castellino, Francis F.J.;Hill, Robert R.L.
Référence Biochemistry, 18, 9, page (1771-1776)
Publication Publié, 1979

Article révisé par les pairs

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Résumé :

Reaction of either native or carboxymethylated bovine and human α-lactalbumins with diethyl pyrocarbonate at pH 6.1 results in the ethoxyformylation of up to two histidine residues in the bovine and one histidine residue in the human protein. At low concentrations (1 mg/mL) of bovine α-lactalbumin, modification of up to one histidine residue per molecule did not affect biological activity; however, modification of a second residue resulted in complete loss of activity. At higher concentrations (46 mg/mL), modification of a single histidine residue resulted in complete loss of activity. Carboxymethylated bovine α-lactalbumin, containing primarily 1,3-dicarboxymethylhistidine-68 and 3-carboxymethylhistidine-32, was found to incorporate one ethoxyformyl group per molecule on reaction with diethyl pyrocarbonate, and the modified derivative was totally inactivated. Proton magnetic resonance spectroscopy indicated that only histidine-32 was ethoxyformylated under conditions in which the biological activity of either native or carboxymethylated bovine α-lactalbumin was lost. Ultraviolet and circular dichroic difference spectra of either native or carboxymethylated bovine α-lactalbumin indicated that ethoxyformylation produced no gross conformational changes. Specificity studies with model compounds including histidine and its N-methyl and Ncarboxymethyl derivatives indicated that the N(1) nitrogen of the imidazole group is specifically ethoxyformylated by diethyl pyrocarbonate. These studies, in conjunction with those on α-lactalbumin and its carboxymethyl derivatives, suggest that the N(l) nitrogen of the imidazole group of histidine-32 in bovine α-lactalbumin is intimately involved in the interaction of α-lactalbumin with UDP-galactose N-acetylglucosamine β1→4-galactosyltransferase in the formation of lactose synthase. © 1979 American Chemical Society.