Probing the Role of Cu(II) Ions on Protein Aggregation Using Two Model Proteins.

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.