The modification of the tryptophan residues of bovine α-lactalbumin with 2-hydroxy-5-nitrobenzyl bromide and with dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide . I. Characterisation of the modified protein

Abstract

1. 1.|The reaction of α-lactalbumin with 2-hydroxy-5-nitrobenzyl bromide and its water-soluble derivative, dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide have been examined. 2. 2.|At pH 2.7 α-lactalbumin was exceptionally reactive. At low molar ratios of reagent to protein, three of the four tryptophan residues reacted, namely Trp 26, Trp 104 and Trp 118. Under the conditions used, all of the incorporated 2-hydroxy-5-nitrobenzyl (HNB) group could be accounted for as the two possible monosubstituted products of tryptophan. The amount of disubstituted tryptophan was very low and there was no evidence for the modification of amino acids other than tryptophan or for inter- or intra-molecular cross linkages. At very high molar ratios of reagent to protein, all of the tryptophan residues were disubstituted. 3. 3.|At pH 6.0, both of the tryptophan reagents were less specific than at pH 2.7 and a histidine residue reacted (His 32) in addition to Trp 26, Trp 104 and Trp 118. Further, of the HNB group incorporated, about 30% was labile at pH 8.5. 4. 4.|A general method for the isolation in high yields of tryptophan-containing peptides labelled with the 2-hydroxy-5-nitrobenzyl group is also described.