Environment of tryptophan residues in alpha-lactalbumin [cattle, rat]

The environment of tryptophan residues in α-lactalbumin was determined by comparative study of bovine, human, and guinea-pig α-lactalbumins in terms of solvent perturbation and denaturation. The results were interpreted on the basis of the fact that Trp-26 and Trp-60 of bovine α-lactalbumin are replaced by other amino acids in human and guinea-pig α-lactalbumins, respectively, and on the assumption that the three α-lactalbumins have similar conformations. Solvent perturbation measurements using ethylene glycol, glycerol, and dimethylsulfoxide as perturbants showed almost the same degree of exposure of tryptophan residues among the three α-lactalbumins at pH 7, and 25°C and 2°C, suggesting that Trp-26 is buried in the interior of the molecule and is inaccessible to solvent, and that Trp-60 is almost buried. A cationic detergent, cetyldimethylbenzylammonium chloride (CDBA) caused an abnormal difference spectrum of α-lactalbumin, which has a major positive peak at about 302 nm characteristic of the native structure of α-lactalbumin. The similarity of the spectrum of α-lactalbumin to that of lysozyme was considered to suggest that α-lactalbumin has a cleft-like region homologous with the active site cleft of lysozyme. By comparison of the ratio of the At value of the main peak to that for N-acetyl-L-tryptophan ethyl ester among the three α-lactalbumins, it was found that two tryptophan residues, Trp-60 and either Trp-104 or Trp-118, are located in this region. The denaturations induced by guanidine hydrochloride, acid, and heat were followed by difference spectroscopy. The magnitude of the denaturation blue shift at 293 nm of human α-lactalbumin was found to be the smallest, which indicates that Trp-26 contributes to the blue shift and is thus in a nonpolar environment in the native state. The positive peak at about 303 nm observed for bovine and human α-lactalbumins is greatly reduced for guinea-pig α-lactalbumin, and it was considered that Trp-60 is close to charged groups. These results are consistent with the proposed conformations of α-lactalbumin based on its similarity to hen egg-white lysozyme, and support the validity of the proposed molecular models.