Abstract
The misfolding and aggregation of proteins is involved in some of the most prevalent neurodegenerative disorders. The importance of human serum albumin (HSA) stems from the fact that it is involved in bio-regulatory and transport phenomena. Here the effect of acetonitrile (ACN) on the conformational stability of HSA and by comparison, ovalbumin (OVA) has been evaluated in the presence and absence of NaCl. The results show the presence of significant amount of secondary structure in HSA at 70% ACN and in OVA at 50% ACN, as evident from far-UV Circular Dichroism (CD) and Attenuated Total Reflection Fourier transformed infra red spectroscopy (ATR-FTIR). Tryptophan and 8-Anilino-1-Naphthalene-Sulphonic acid (ANS) fluorescence indicate altered tryptophan environment and high ANS binding suggesting a compact “molten globule”-like conformation with enhanced exposure of hydrophobic surface area. However, in presence of NaCl no intermediate state was observed. Detection of aggregates in HSA and OVA was possible at 90% ACN. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and ATR-FTIR. These aggregates exhibit increase Thioflavin T (Th T) fluorescence with a red shift of Congo red (CR) absorption spectrum. X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM) analysis confirmed the presence of fibrillar aggregates. Single cell gel electrophoresis (SCGE) assay of these fibrillar aggregates showed the DNA damage resulting in cell necrosis confirming their genotoxic nature. Some proteins not related to any human disease form fibrils in vitro. In the present study ACN gives access to a model system to study the process of aggregation.
Highlights
The process by which a linear sequence of amino acids folds into a discrete and functional three-dimensional protein is the most fundamental and universal example of biological self assembly
In the absence of NaCl, a bipartite response was observed with fluorescence intensity approaching a maximum in the presence of 50% v/v ACN (Unless otherwise indicated all ACN concentrations are present in percent v/v) and decreasing thereafter by a factor of eight at 90%
We suggest that the observed drop in fluorescence intensity after 70% ACN addition, could be due to either (i) exposure of tryptophan to the solvent and/or (ii) quenching of tryptophan intensity due to an increase in the number of phenylalanine, histidine, and disulfide residues in the proximity of tryptophan that quenches the emission upon aggregation mediated by both electrostatic and hydrophobic interactions
Summary
The process by which a linear sequence of amino acids folds into a discrete and functional three-dimensional protein is the most fundamental and universal example of biological self assembly. The ability of the side chains to form the tight and specific interactions like that of a native protein is an important final step in the protein folding pathway [3]. Nonspecific interactions among the side chains or inappropriately exposed hydrophobic surfaces of incompletely folded polypeptides result in coagulation, most widely viewed as protein aggregation. Production of misfolded or denatured proteins is potentially deleterious to cells because of their ability to co-aggregate with and thereby trap unrelated cellular proteins that may transiently display complementary surfaces [4,5]. Abnormal interactions has been proposed to underlie the toxicity associated with protein aggregates in many neurodegenerative disorders like Alzheimers, Parkinsons, Creutzfeldt-Jakob Disease, etc [6]
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