Photophysics, photochemistry and energetics of UV light induced disulphide bridge disruption in apo-α-lactalbumin.

ABSTRACT

Continuous 295 nm excitation of whey protein bovine apo-α-lactalbumin (apo-bLA) results in an increase of tryptophan fluorescence emission intensity, in a progressive red-shift of tryptophan fluorescence emission, and breakage of disulphide bridges (SS), yielding free thiol groups. The increase in fluorescence emission intensity upon continuous UV-excitation is correlated with the increase in concentration of free thiol groups in apo-bLA. UV-excitation and consequent SS breakage induce conformational changes on apo-bLA molecules, which after prolonged illumination display molten globule spectral features. The rate of tryptophan fluorescence emission intensity increase at 340 nm with excitation time increases with temperature in the interval 9.3-29.9°C. The temperature-dependent 340 nm emission kinetic traces were fitted by a 1st order reaction model. Native apo-bLA molecules with intact SS bonds and low tryptophan emission intensity are gradually converted upon excitation into apo-bLA molecules with disrupted SS, molten-globule-like conformation, high tryptophan emission intensity and red-shifted tryptophan emission. Experimental Ahrrenius activation energy was 21.8 ± 2.3 kJ x mol(-1). Data suggests that tryptophan photoionization from the S(1) state is the likely pathway leading to photolysis of SS in apo-bLA. Photoionization mechanism(s) of tryptophan in proteins and in solution and the activation energy of tryptophan photoionization from S(1) leading to SS disruption in proteins are discussed. The observations present in this paper raise concern regarding UV-light pasteurization of milk products. Though UV-light pasteurization is a faster and cheaper method than traditional thermal denaturation, it may also lead to loss of structure and functionality of milk proteins.