Molten globule‐like state of human serum albumin at low pH

Abstract

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]. ...

Reversible two-step unfolding of heme–human serum albumin: a 1H-NMR relaxometric and circular dichroism study

“…Although the occurrence of a folding intermediate in the unfolding of HSA upon extensive glycation or lowering of pH was previously reported [52,53], this is the first evidence for a folding intermediate in heme-HSA stabilized by myristate binding. Moreover, it appears that heme binding is not crucial for the formation of the folding intermediate.…”

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

“…In order to assimilate and release molecules of quite distinct size/shape in the circulatory system, HSA has to possess a flexible structure. This was positively inferred from changes in the solvation dynamics of HSA, using the single tryptophan fluorescence as reporter, which correlated with the conformational transitions that occur upon pH changes [30].…”

Biochemical characterization of the interactions between doxorubicin and lipidic GM1 micelles with or without paclitaxel loading

“…15 Further studies revealed that the amount of albumin that ends up being associated with micelles reached saturation at 1/1 HSA-GM1 w/w ratio and also that at 55°C and at pH 3, where HSA is reversibly denatured and exposes part of its hydrophobic residues, the interaction is favored. [29][30][31] Considering these results together and the fact that Dox might end up located in more external domains of GM1 micelles, we wondered whether the incorporation of Dox into GM1 or Ptx-GM1 micelles could have any effect on their ability to interact with HSA.…”