Illumination of goat alpha-lactalbumin (GLA) with 280 or 295 nm light results in tryptophan-mediated photolysis of disulfide bonds within the protein. The photolysis is not dependent on the absence or presence of Ca(2+) and is observed as well on illumination of native and of partially unfolded GLA. However, photolysis of native GLA results in a partial unfolding of the protein. The latter phenomenon is most clearly observed on fluorescence measurements at low temperatures (near 3 degrees C). The photolysis induces some dimerization and oligomerization, but most GLA molecules remain monomeric. To obtain more information about the reaction products, the illuminated protein is treated with iodoacetamide to label the free thiol groups, it is fragmented with trypsin, and the fragments are analyzed by mass spectrometry. Via this approach, we observe that the cleavage of disulfide bonds is restricted to Cys6-Cys120 and Cys73-Cys91 bonds. The photolytic cleavage of either of these disulfide bonds results in the formation of a single free thiol, a phenomenon restricted to Cys120 and Cys91, respectively. We also found indications that a thioether linkage is formed between Cys73 and Trp60. The alkylsulfenylation of Trp60 presumably results from a combination of primary thiyl and tryptyl radicals.

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Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.

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