Intrinsic Fluorescence studies.

<p>(a) Relative intrinsic fluorescence intensity of OVA in absence (▪) and presence (♦) of NaCl as a function of increasing concentration of ACN, (b) Tryptophan fluorescence emission spectra of native OVA in 20 mM sodium phosphate buffer, pH 7.2 (curve 1); curves 2 and 3 represent OVA at 50% and 90% ACN respectively. OVA concentration was 4.44 µM and the path length was 1 cm. (c) Relative intrinsic fluorescence intensity of HSA in absence (▪) and presence (♦) of NaCl as a function of increasing concentration of ACN, (d) Tryptophan fluorescence emission spectra of native HSA in 20 mM sodium phosphate buffer, pH 7.2 (curve 1); curves 2 and 3 represent HSA at 70% and 90% ACN. HSA concentration was 3.03 µM and the path length was 1 cm. The fluorescence intensity measurement was carried out at an excitation wavelength of 280 nm.</p

Congo red assay of albumins.

<p>Congo red dye binding of OVA and HSA in the presence of 90% ACN. Concentration of OVA and HSA was 4.44 and 3.03 µM respectively and the path length was 1 cm. Absorbance was recorded in the range of 400 to 700 nm.</p

Deciphering Structural Intermediates and Genotoxic Fibrillar Aggregates of Albumins: A Molecular Mechanism Underlying for Degenerative Diseases

<div><p>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.</p> </div

CD studies.

<p>(a) Far-UV CD spectra of OVA in the presence of ACN in 20 mM sodium phosphate buffer, pH 7.2. Curve 1 shows native OVA, curve 2 and 3 corresponds to OVA at 50% and 90% ACN respectively; (b) Far-UV CD spectra of HSA in the presence of ACN in 20 mM sodium phosphate buffer, pH 7.2. Curve 1 shows native HSA, curve 2 represents HSA in presence of 40% ACN; curve 3 and 4 represents HSA at 70% and 90% ACN respectively. Concentration of OVA in the samples was 4.44 and HSA was 3.03 µM respectively and the path length was 0.1 cm.</p

SCGE assay.

<p>Images of plasmid DNA damage in native HSA treated lymphocytes (a) and lymphocytes treated with HSA aggregates (b). The concentration of aggregates was 50 µg/ml.</p

Rayleigh scattering measurements.

<p>OVA and HSA in the presence of 90% ACN. The excitation wavelength was 350 nm and emission was recorded in the wavelength range of 300–400 nm. Path length used was 1 cm and the protein concentration in the samples was 4.44 µM for OVA and 3.03 µM for HSA.</p

Stern-Volmer plots for acrylamide quenching of tryptophan fluorescence of albumins.

<p>Native OVA (•); HSA (▴) and NATA alone (♦) at pH 7 in presence of varying concentration of quencher. 90% ACN incubated OVA (○); HSA (<b>▵</b>) and NATA (<b>◊</b>). Values shown are the ratios of fluorescence in the absence of acrylamide (F₀) to the fluorescence at the given concentration of quencher (F). Protein concentration for OVA was 4.44 and for HSA was 3.03 µM. Path length for the study was 1 cm and the excitation wavelength was 295 nm.</p

Thioflavin T assay.

<p>Thioflavin T spectra of HSA in the presence of ACN. Curve 1 shows native protein; curves 2 and 3 represent HSA at 70% and 90% ACN respectively. Thioflavin T fluorescence was monitored at an excitation wavelength of 440 nm. Concentration of OVA and HSA for the analysis was 4.44 and 3.03 µM and the path length was 1 cm.</p