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Copper is an essential trace element for human biology where its metal dyshomeostasis accounts for an increased level of serum copper, which accelerates protein aggregation. Protein aggregation is a notable feature for many neurodegenerative disorders. Herein, we report an experimental study using two model proteins, bovine serum albumin (BSA) and human serum albumin (HSA), to elucidate the mechanistic pathway by which serum albumins get converted from a fully folded globular protein to a fibril and an amorphous aggregate upon interaction with copper. Steady-state fluorescence, time-resolved fluorescence studies, and Raman spectroscopy were used to monitor the unfolding of serum albumin with increasing copper concentrations. Steady-state fluorescence studies have revealed that the fluorescence quenching of BSA/HSA by Cu(II) has occurred through a static quenching mechanism, and we have evaluated both the quenching constants individually. The binding constants of BSA–Cu(II) and HSA–Cu(II) were found to be 2.42 × 10 4 and 0.05 × 10 4 M –1 , respectively. Further nanoscale morphological changes of BSA mediated by oligomers to fibril and HSA to amorphous aggregate formation were studied using atomic force microscopy. This aggregation process correlates with the Stern–Volmer plots in the absence of discernible lag phase. Raman spectroscopy results obtained are in good agreement with the increase in antiparallel β-sheet structures formed during the aggregation of BSA in the presence of Cu(II) ions. However, an increase in α-helical fractions is observed for the amorphous aggregate formed from HSA.

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The fluorescence emission properties of lysozyme immobilized at selected pH 5, 8.8 and 10.6 on chitosan/polystyrene sulfonate (CHI/PSS) multilayer membranes were investigated. The tryptophanyl fluorescence was selectively excited at 290 nm. The emission maximum and fluorescence intensity were found to be dependent on the pH of lysozyme solution. The fluorescence intensity was highest at pH 8.8. A slight red shift was observed as the pH changes from 5 to 10.6. Quenching of lysozyme fluorescence by iodide was used to study the effect of pH on the adsorption pattern of lysozyme to multilayers. For lysozyme (pH 10.6) adsorbed membrane, the fluorescence intensity was found to decrease progressively with quencher concentration. Almost complete quenching of fluorescence was observed when the iodide concentration was 1 M suggesting full accessibility of quencher to tryptophanyl residues. A linear Perrin plot was obtained suggesting a static mechanism for quenching under the experimental conditions. Fluorescence quenching studies were also carried out with lysozyme (pH 5) adsorbed membranes prepared by normal adsorption methods and also under ultrafiltration conditions. The quenching efficiency was different for the two sets of membranes. The fluorescence quenching studies reveals that adsorption pattern of proteins on multilayers strongly depend on pH and immobilization method.