Human albumin (HA) is a single chain comprised of three major domains. Under physiological conditions, the thermal denaturation of HA occurs in a highly cooperative manner, i.e. a single transition is observed by DSC, whereas at lower ionic strength, the denaturation becomes highly non-cooperative, i.e. two well separated, major transitions occur. The binding of halide anion appears to promote the conversion of the protein from a non-cooperative form to a highly cooperative form. The effects of increasing concentrations of Cl-, I-, and Br- on the thermal denaturation of HA now reveal that order of efficiency in promoting the conversion is: I- > Br- > Cl-. We are attempting to determine the relative affinity of native HA for these anions to correlate these with the ability to promote the conversion. Furthermore, we are attempting to identify the regions of the molecule that give rise to the two discrete, thermally induced transitions at low ionic strength. Alpha-1 proteinase inhibitor (A1-PI) is the physiologically important inhibitor of neutrophil elastase and is a single chain, containing an important beta sheet (A sheet) and reactive site loop. During inhibition, the loop undergoes a specific cleavage, but A1-PI remains tightly bound to the protease. In the free (uncomplexed) cleaved form, the newly formed polypeptide tail is inserted between central strands of the A sheet with the cleaved ends distal thereby resulting in a major conformational change and loss of inhibitory function. Without cleavage, the reactive site loop is partially inserted into the A sheet in the intact form. The intact form can be induced to undergo a rearrangement with the full insertion of the loop into the A sheet to generate the locked form or with the full insertion of the loop from one molecule into the A sheet of another to form dimers or higher polymers. Heating and cooling or treatment with low concentrations of denaturant generates these additional forms, which also exhibit no inhibitory activity. The guanidine (Gu) induced unfolding of A1-PI is biphasic with intermediate(s) at 1.5 M Gu. We have been able to measure some rates of partial unfolding (0->1.5 M Gu/1 rate) and refolding (7->0 M Gu/2 rates) and (1.5->0 M Gu/2 rates). We have discovered a slow equilibration of intermediates that takes place in 1.5 M Gu and have detected 3 polymer intermediates and 2 monomer intermediates (intact and locked?). In O M Gu, no such equilibration of folded species occurs thereby affecting the reversibility of the first Gu induced transition. We will attempt to characterize these intermediates thermodynamically, kinetically, and structurally.
