Spectro-kinetic investigations on the release mechanism of lysozyme from layer-by-layer reservoirs.

The investigations of protein adsorption and release on interfaces aid in the elucidation of the protein-surface interaction mechanism, which has several applications in the biomedical area. The spectro-kinetic and morphological analysis of the release of lysozyme (Lyz) from chitosan/polystyrene sulphonate (CHI/PSS) multilayer immobilized at pHs 10.6, 8.8 and 5.0 shows that the extent of release strongly depends on the pH of Lyz loading and the ionic strength of the desorbing solution. When compared to pH 8.8, the release for pH 10.6 achieves equilibrium more rapidly. At loading pH 10.6, the release is surface-mediated, at pH 8.8, it is both surface- and bulk-mediated, while at pH 5.0 it is bulk mediated with minimal release. Lyz released for loading pH 10.6 retains its native secondary structure. Kinetic fitting suggests that high loading pH 8.8-10.6 and high release ionic strength (0.5-1.0 M NaCl) lead to burst release of Lyz from CHI/PSS multilayer. Surface morphology changes of multilayer interface upon Lyz loading and release are highlighted by SEM topography and AFM height distribution analysis. The present work indicates that CHI/PSS multilayer system can function as a reservoir for burst as well as controlled release of lysozyme by selecting the loading pH and ionic strength.

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 × 104 and 0.05 × 104 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 ß-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.

Fabrication of switchable protein resistant and adhesive multilayer membranes.

Fabrication of protein adhesive and resistant surfaces based on chitosan/polystyrene sulfonate (CHI/PSS) multilayer membranes is presented. Adsorption behavior of bovine serum albumin (BSA) and lysozyme to CHI/PSS multilayer was studied by simple adsorption method and under pressure driven (ultrafiltration) conditions. The protein incorporated membranes were characterized by FT-IR, UV-vis, SEM and AFM. The loading of proteins to the multilayer was found to be dependent on the nature of protein, pH, number of bilayers, methods of adsorption and time of adsorption. Simple adsorption resulted in BSA adhesive layers with some conformational changes at higher number of bilayers. Ultrafiltration leads to protein repellence at higher number of bilayers which is attributed to the presence of irremovable water. Lysozyme adsorption/sorption varied with pH. Surface coverage dominates at pH close to pI and at pH 5 under ultraflitration condition where as simple adsorption resulted in protein repellence at pI. The secondary structure of adsorbed lysozyme is preserved for a wide pH range (5-11). Desorption study of lysozyme adsorbed membranes at pH 8.8 was carried out to understand the adsorption/sorption of protein. Diffusion of the sorbed lysozyme from the inner layers to the surface is found to take place at lower concentrations of NaCl.