The modification of the tryptophan residues of bovine α-lactabumin with 2-hydroxy-5-nitrobenzyl bromide and with dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide II. Effect on the specifier protein activity

Abstract

The effects on the function and conformation of α-lactalbumin after reaction with 2-hydroxy-5-nitrobenzyl bromide and its water soluble derivative dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide have been studied. Three of the four tryptophan residues of α-lactalbumin (Trp 26, Trp 104 and Trp 118) were accessible to the tryptophan reagents and the reaction mixtures were complex. The labelling process could not be made more selective by changing the experimental conditions and to determine the importance of each of the accessible tryptophan residues an attempt was made to separate α-lactalbumin labelled uniquely at Trp 26, Trp 104 or Trp 118. A partial separation was possible by ion-exchange chromatography and three preparations thus isolated were subjected to kinetic and conformational studies. The results obtained suggest that when Trp 26, Trp 104 or Trp 118 is alkylated with the 2-hydroxy-5-nitrobenzyl group, α-lactalbumin loses its specifier protein activity. The labelling of tryptophan did not cause a gross conformational change but a minor change may have occurred.