Abstract
Bovine serum albumin (BSA) is an important transport protein of the blood and its aggregation/fibrillation would adversely affect its transport ability leading to metabolic disorder. Therefore, understanding the mechanism of fibrillation/aggregation of BSA and design of suitable inhibitor molecules for stabilizing its native conformation, are of utmost importance. The qualitative and quantitative aspects of the effect of osmolytes (proline, hydroxyproline, glycine betaine, sarcosine and sorbitol) on heat induced aggregation/fibrillation of BSA at physiological pH (pH 7.4) have been studied employing a combination of fluorescence spectroscopy, Rayleigh scattering, isothermal titration calorimetry (ITC), dynamic light scattering (DLS) and transmission electron microscopy (TEM). Formation of fibrils by BSA under the given conditions was confirmed from increase in fluorescence emission intensities of Thioflavin T over a time period of 600 minutes and TEM images. Absence of change in fluorescence emission intensities of 8-Anilinonaphthalene-1-sulfonic acid (ANS) in presence of native and aggregated BSA signify the absence of any amorphous aggregates. ITC results have provided important insights on the energetics of interaction of these osmolytes with different stages of the fibrillar aggregates of BSA, thereby suggesting the possible modes/mechanism of inhibition of BSA fibrillation by these osmolytes. The heats of interaction of the osmolytes with different stages of fibrillation of BSA do not follow a trend, suggesting that the interactions of stages of BSA aggregates are osmolyte specific. Among the osmolytes used here, we found glycine betaine to be supporting and promoting the aggregation process while hydroxyproline to be maximally efficient in suppressing the fibrillation process of BSA, followed by sorbitol, sarcosine and proline in the following order of their decreasing potency: Hydroxyproline> Sorbitol> Sarcosine> Proline> Glycine betaine.
Highlights
The aggregation of proteins can take place under various environmental conditions, giving rise to higher order supramolecular structures including fibrillar forms [1,2]
Temperature dependent thioflavin T (ThT) binding kinetics experiments were carried out to monitor the fibrillation of Bovine serum albumin (BSA) at temperatures 298.15 K, 323.15 K, 328.15 K and 333.15 K
Our study suggests that hydrophobic effect and polar interactions play important role in the fibrillation process of BSA at 333.15 K under physiological pH
Summary
The aggregation of proteins can take place under various environmental conditions, giving rise to higher order supramolecular structures including fibrillar forms [1,2]. In recent time, active research is going on, in order to implement these fibrillar structures, generated in vitro, in the field of biomedicine and bionanotechnology [5]. These fibrils find their application from development of peptide nanotubes useful in bio-electrochemical sensor applications to creation of scaffolds applicable in tissue engineering and drug delivery [4]. The fibrils could be used for bacterial biofilm development [2]
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