Inter- and intramolecular interactions of alpha-lactalbumin. X. Effect of acylation of tyrosyl and lysyl side chains on molecular conformations.

Of the 4 tyrosyl residues of bovine ar-lactalbumin, 2 were found to be quite reactive with N-acetylimidazole; the third group is less so, and the fourth is only weakly reactive. Acylation of the two most reactive groups results in a reversible increase in the quantum yield of a tryptophan residue or residues which emit maximally at 350 rnp. At relatively high concentrations of reagent, in addition to acylation of tyrosyl residues, six amino groups react giving rise to quenching of ’ the fluorescence of tryptophan emitting maximally below 320 mp and enhancement of fluorescence from groups which emit maximally at 350 ml.r. Thus, acylation of the two tyrosyl groups perturbs tryptophan residues which are freely in contact with the medium, whereas acplation of amino groups, by contrast, appears to involve tryptophan residues which are not freely in contact with the solvent. Changes in circular dichroism were observed on acylation of bovine cy-lactalbumin in the 250 to 300 rnp side chain band system, in the 208 rnp band, and in the 212 to 230 mp region. These circular dichroism changes, which depend in a complex way on the nature of the groups acylated, appear to be due primarily to alteration of the environment of the side chains in the protein. Changes in fluorescence and circular dichroism properties observed on acylation of amino groups were shown to be similar to those observed on acid denaturation. It is likely that those processes, as well as alkaline denaturation, bring about essentially the same local conformational change which gives rise to increased freedom of rotation of tyrosyl and tryptophyl side chains. The pH dependence of the conformational transitions, as well as the chemical reactivity of