Inter- and Intramolecular Interactions of α-Lactalbumin

Of the 4 tyrosyl residues of bovine α-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 mµ. 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 mµ and enhancement of fluorescence from groups which emit maximally at 350 mµ. Thus, acylation of the two tyrosyl groups perturbs tryptophan residues which are freely in contact with the medium, whereas acylation 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 α-lactalbumin in the 250 to 300 mµ side chain band system, in the 208 mµ band, and in the 212 to 230 mµ 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 side chains, suggests that breaking of charge pairs (amino and carboxylic acid groups) may initiate the structural change. Inspection of a recently proposed model for bovine α-lactalbumin indicates the proximity of a number of lysyl, glutamyl, and aspartyl side chains, in accord with the above hypothesis. The model further indicates that Tyr-36 and Trp-118, and Tyr-103 and Trp-104 are sufficiently close, respectively, to explain the changes in fluorescence on acylation of tyrosyl groups. The environments of Trp-60 and perhaps Trp-104, located in a crevice-like region of the molecule, are such that they may be the low wave length emitting groups normalized on acylation of amino groups.