Tryptophan-mediated denaturation of β-lactoglobulin A by UV irradiation

beta-Lactoglobulin A, a genetic variant of one of the main whey proteins, was irradiated at 295 nm for 24 h. After irradiation, 18% of the protein was denatured (determined by reverse-phase chromatography). The fluorescence spectrum of the irradiated protein was red-shifted compared to that of the native protein, indicating a change in protein folding. Sulfhydryl groups, which are buried in native beta-lactoglobulin, were exposed following irradiation and became available for quantification using the Ellman assay. The quantity of exposed sulfhydryls increased, but the number of total sulfhydryl groups decreased. Gel permeation chromatography showed that some protein aggregation occurred during irradiation. Fourier transform infrared (FTIR) spectroscopy of irradiated beta-lactoglobulin revealed changes in the secondary structure, comparable to that of early events during heat-induced denaturation. There was evidence for some photo-oxidation of tryptophan.