Photoexcitation of tryptophan groups induces reduction of two disulfide bonds in goat alpha-lactalbumin.

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.

Structural basis for difference in heat capacity increments for Ca(2+) binding to two alpha-lactalbumins.

Thermodynamic parameters for the unfolding of as well as for the binding of Ca(2+) to goat alpha-lactalbumin (GLA) and bovine alpha-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25 degrees C. Among the different parameters available, the heat capacity increments (Delta C(p)) offer the most direct information for the associated conformational changes of the protein variants. The Delta C(p) values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective Delta C(p) values for the binding of Ca(2+) are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca(2+)-dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca(2+)-binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in Delta C(p) values for the binding of Ca(2+) is presumably in part related to this difference in charge distribution.