TEMPERATURE-DEPENDENCE OF THE PHOSPHORESCENCE QUANTUM YIELD OF VARIOUS ALPHA-LACTALBUMINS AND OF HEN EGG-WHITE LYSOZYME

The radiative quantum yield, PHI(p)o of the triplet state of human alp ha-lactalbumin (HLA) has been measured in the temperature range betwee n 6 K and the softening point of the aqueous glass (approximately 150 K). PHI(p)o has little temperature dependence below approximately 30 K , but above this it decreases sharply with increasing temperature. The unusual temperature dependence is fitted by a phenomenological two-st ate model in which the phosphorescence originates primarily from a don or, tryptophan (Trp) 104, and an acceptor, Trp 60, the populations of which are coupled by a thermally activated triplet-triplet energy tran sfer process. The model assumes that the acceptor (Trp 60) triplet sta te undergoes radiationless deactivation by a proximal disulfide residu e, while the donor (Trp 104) has no such extrinsic quencher. The decre ase of PHI(p)o with increasing temperature is accounted for by the the rmally activated triplet-triplet energy transfer process. The disulfid e quenching rate constant itself is assumed to be temperature independ ent, in accord with recent measurements of simple disulfide quenching in long chain snake venom neurotoxins (Schlyer, B. D., E. Lau, and A. H. Maki. 1992. Biochemistry. 31:4375-4383; Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). We find that the phosphoresc ence quenching in HLA occurs with an activation energy of 97 cm-1, whi ch we associate with a barrier to the energy transfer process. The dat a are fit well by the model if we assume a value for the temperature-i ndependent disulfide quenching constant of k(Q) > 3 s-1 that is consis tent with recent measurements on indole-disulfide model systems (Li, Z ., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371 ). Simil ar results are reported for bovine alpha-lactalbumin (BLA) and for hen egg-white lysozyme (HEWL) that contains the structural equivalents of Trp 104 and Trp 60 of HLA. HLA provides the best agreement with calcu lations since it is the simplest, lacking Trp 26, a residue not consid ered in the model, that probably contributes significantly to the phos phorescence of BLA, guinea pig alpha-lactalbumin (GPLA), and HEWL. GPL A, which contains Trp 104 but lacks Trp 60, shows qualitatively less t hermally induced phosphorescence quenching than HLA, BLA, and HEWL, th us supporting the postulated quenching model.