Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 m guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions. Keywords: acid denaturation; human serum albumin (HSA); molten globule; pH.Folding of a protein from a structureless denatured state to an ordered biologically active native state is considered to be a highly complex process because of the lack of information about the folding intermediates formed in the folding pathway. This process is even more complex for multidomain proteins, in which each domain may be capable of refolding independently [1]. Keeping in view information on the formation of the native biologically functional structure in the primary sequence [2,3], previous studies have aimed to increase our understanding of the denatured state of proteins [4] and the role of segment± segment interactions and the interactions between the amino acid side chains with the surrounding medium [5,6] and also to characterize the refolding intermediates [7±9]. The process of protein folding from a denatured state to its native state depends on the type of denatured state, as each method of denaturation is considered to be a distinct process yielding different products [4,10]. It has been shown in several cases that denatured proteins contain some residual structure and therefore are not completely unfolded [11,12]. A comparison of different methods of denaturation showed that the most completely unfolded form can be obtained in either 9 m urea or 6 m guanidine hydrochloride (GdnHCl) [10]. On the other hand, acid denaturation of some proteins results in a denatured state that is often less unfolded than the completely unfolded form obtained in high concentrations of urea and GdnHCl, far from a random coil [4,13,14]. ...

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 (DeltaDelta G(H2O)D) 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 (Trp(214)) 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: NaClO(4) > NaSCN >Na(2)SO(4) >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).

Avian egg whites are a rich source of protein inhibitors of proteinases belonging to all four mechanistic classes. Ovomucoid and ovoinhibitor are multidomain Kazal-type inhibitors with each domain containing an actual or putative reactive site for a serine proteinase. Cystatin is a cysteine proteinase inhibitor, while ovostatin inhibits proteinases of all four mechanistic classes. In this review we have summarized the general features, isolation, inhibitory mechanism and evolutionary aspects of these inhibitors.

A peak in 3D-fluorescence spectra of proteins, often assigned to backbone emission, is shown to be due to aromatic residues.

Interaction of flavokawain B (FB), a multitherapeutic flavonoid from Alpinia mutica with the major transport protein, human serum albumin (HSA), was investigated using different spectroscopic probes, i.e., intrinsic, synchronous, and three-dimensional (3-D) fluorescence, circular dichroism (CD), and molecular modeling studies. Values of binding parameters for FB-HSA interaction in terms of binding constant and stoichiometry of binding were determined from the fluorescence quench titration and were found to be 6.88 × 10(4) M(-1) and 1.0 mol of FB bound per mole of protein, respectively, at 25 °C. Thermodynamic analysis of the binding data obtained at different temperatures showed that the binding process was primarily mediated by hydrophobic interactions and hydrogen bonding, as the values of the enthalpy change (ΔH) and the entropy change (ΔS) were found to be -6.87 kJ mol(-1) and 69.50 J mol(-1) K(-1), respectively. FB binding to HSA led to both secondary and tertiary structural alterations in the protein as revealed by intrinsic, synchronous, and 3-D fluorescence results. Increased thermal stability of HSA in the presence of FB was also evident from the far-UV CD spectral results. The distance between the bound ligand and Trp-214 of HSA was determined as 3.03 nm based on the Förster resonance energy transfer mechanism. Displacement experiments using bilirubin and warfarin coupled with molecular modeling studies assigned the binding site of FB on HSA at domain IIA, i.e., Sudlow's site I.