Modification of bovine alpha-lactalbumin with N-bromosuccinimide and 2-hydroxy-5-nitrobenzylbromide.
Reaction of alpha-lactalbumin at pH 7 in aqueous solution with either 2-hydroxy-5-nitrobenzylbromide or N-bromosuccinimide yields derivatives in which only 2 of the 4 tryptophan residues are modified. All 4 residues of tryptophan are modified under the similar conditions in 8 M urea. Structural analysis of the modified derivatives revealed that tryptophans 26 and 118 are the sole reactive residues and that tryptophan 118 reacts more rapidly than tryptophan 26. The fluorescence of alpha-lactalbumin modified to varying extents with N-bromosuccinimide indicates that tryptophan 118 is exposed to solvent whereas tryptophan 26 is in a more hydrophobic environment. The chemical reactivities and fluorescence properties of tryptophans 26 and 118 are consistent with the proposed conformations of alpha-lactalbumin based on its similarity with egg white lysozyme. The kinetic properties of both derivatives of alpha-lactalbumin containing up to 2 modified residues indicate that each derivative has decreased affinity for the galactosyltransferase but that at saturating concentrations, Km and Vmax for lactose synthesis are unchanged from those of native alpha-lactalbumin.
D015077:2-Hydroxy-5-nitrobenzyl Bromide / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D002417:Cattle; D005260:Female; D007700:Kinetics; D007768:Lactalbumin / Q000032:analysis / Q000378:metabolism; D007788:Lactose Synthase / Q000378:metabolism; D008892:Milk / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013388:Succinimides; D014364:Tryptophan / Q000032:analysis; D014508:Urea
D000596:Amino Acids; D010446:Peptide Fragments; D013388:Succinimides; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D014364:Tryptophan; D014508:Urea; D007768:Lactalbumin; D007788:Lactose Synthase; D001974:Bromosuccinimide