Anion‐induced stabilization of human serum albumin prevents the formation of intermediate during urea denaturation

The unfolding of human serum albumin (HSA), a multidomain protein, by urea was followed by far‐UV circular dichroism (CD), intrinsic fluorescence, and ANS fluorescence measurements. The urea‐induced transition, which otherwise was a two‐step process with a stable intermediate at around 4.8 M urea concentration as monitored by far‐UV CD and intrinsic fluorescence, underwent a single‐step cooperative transition in the presence of 1.0 M KCl. The free energy of stabilization $\bf ( \Delta \Delta G_D^{H_2 O})$ in the presence of 1 M KCl was found to be 1,090 and 1,200 cal/mol as determined by CD and fluorescence, respectively.The salt stabilization occurred in the first transition (0–5.0 M urea), which corresponded to the formation of intermediate (I) state from the native (N) state, whereas the second transition, corresponding to the unfolding of I state to denatured (D) state, remained unaffected. Urea denaturation of HSA as monitored by tryptophan fluorescence of the lone tryptophan residue (Trp214) residing in domain II of the protein, followed a single‐step transition suggesting that domain(s) I and/or III is (are) involved in the intermediate formation. This was also confirmed by the acrylamide quenching of tryptophan fluorescence at 5 M urea, which exhibited little change in the value of Stern‐Volmer constant. ANS fluorescence data also showed single‐step transition reflecting the absence of accumulation of hydrophobic patches. The stabilizing potential of various salts studied by far‐UV CD and intrinsic fluorescence was found to follow the order: NaClO4 > NaSCN >Na2SO4 >KBr >KCl >KF. A comparison of the effects of various potassium salts revealed that anions were chiefly responsible in stabilizing HSA. The above series was found similar to the electroselectivity series of anions towards the anion‐exchange resins and reverse of the Hofmeister series, suggesting that preferential binding of anions to HSA rather than hydration, was primarily responsible for stabilization. Further, single‐step transition observed with GdnHCl can be ascribed to its ionic character as the free energy change associated with urea denaturation in the presence of 1.0 M KCl (5,980 cal/mol) was similar to that obtained with GdnHCl (5,870 cal/mol). Proteins 2000;40:29–38. © 2000 Wiley‐Liss, Inc.