At pH 8.6 and low ionic strength, unblocked (i.e. containing 1 free SH) bovine albumin (BA) shows 3 denaturation peaks by differential scanning calorimetry (DSC); these correspond to the unfolding of discrete regions within BA. In contrast, blocked BA, prepared by reaction of the free SH with L-cystine, exhibits only 2 peaks, which have denaturation temperatures (Td's) corresponding to those of the second and third transitions seen with unblocked BA. We have demonstrated that this observation is a manifestation of the linkage between protein denaturation and disulfide linked dimer formation, which involves S-S bond formation between free SH groups of fully and/or partially unfolded (but not native) monomers. In blocked BA, 2 regions have the same Td with the third region having a higher Td (i.e. Td(1) = Td(2) < Td(3)). In unblocked BA, Td(1) is shifted downward from Td(2) due to the stabilization of the partially and fully unfolded species through coupling between the unfolding and dimerization equilibria whereas the other Td's are essentially unperturbed. We have begun a study of human albumin (HA) at pH 7.0. At low ionic strength, both unblocked and blocked HA show 3 thermal transitions by DSC (with 2 transitions below 80 ~C); however, urea unfolding shows only 2 transitions by UV difference spectroscopy (UV-DS) and only 1 transition by changes in intrinsic tryptophan (trp) fluorescence, which corresponds to the transition observed by UV-DS at lower urea concentrations. Monitoring the heat induced unfolding of the unblocked and blocked proteins up to 80 ~C by means of circular dichroism, UV-DS, and intrinsic trp fluorescence also shows 2 transitions, which can be detected by taking the first derivative of the progress curve and which correlate with Td(1) and Td(2) determined by DSC. The result of the fluorescence experiments is unanticipated since HA contains a single trp in domain II. However, crystallographic data show contacts between it and residues in other domains thereby making the perturbation of this chromophore during the unfolding of different regions feasible. These data also suggest burial of the trp; this is affirmed by our inability to oxidize this group with N- bromosuccinimide. Recently, we commenced a study of the effects of the binding of Cl- on thermal denaturation. Albumin undergoes a number of pH induced transitions (i.e. N-F, F-E, N-B, and B-A), and a major goal of this work is to elucidate the regions of the molecule involved in these transitions and the energetics of the interactions among these regions.
