Exploring the effects of L-arginine on protein aggregation and fibrillation
Date Issued
2011
Date
2011
Author(s)
Wang, Hsiang-Yun
Abstract
L -arginine is a type of semi essential amino acid, and does not cause any harm on human being due to its biologically compatible property. While several lines of evidence has indicated that L arginine can effectively reduce protein aggregation, the precise interacting mechanism regarding the aggregation inhibition induced by L- arginine still remains elusive.
The first part of this thesis is aimed at exploring the effects of L- arginine on the formation of amorphous aggregates using papain as the model protein system. Our data revealed that low concentration of guanidine hydrochloride (GdnHCl) could induce papain aggregation at acid condition (pH 2.0) with the highest level of aggregation observed at 0.7 M GdnHCl, which was taken as the aggregation condition used in our study. We found that, when a small amount of L- arginine (final concentration
0.02- 0.2M) were present in papain solution, the amount of papain aggregation was augmented. However L- arginine was observed to dose dependently attenuate the extent of papain aggregation upon futher increase of L- arginine (final concentration 0.3- 1.5M). Therefore, we postulate that, similar to GdnHCl, L- arginine not only perturbs the
structure of papain but also has hydrophobic interactions with papain.
In addition, in order to explore the ability of L- arginine to retain papain activity when its anti aggregation potency was observed, neutral pH and 65°C were used as the aggregation inductive condition. Our results demonstrated that, whereas 1M L-arginine was sufficient to decrease papain aggregation, only 10% of papain activity was retained. We hypothesize that papain active sites may exhibit higher conformational flexibility than the enzyme molecule as a whole, resulting in the finding that the loss of papain activity precedes noticeable conformational change of papain.
As for the study of protein ordered aggregation, we used bovine serum albumin (BSA) as model protein. When incubating BSA at 65°C for hours, there was a considerable reduction in the α helical content accompanied by increases in β- sheet content in the BSA solution containing immature fibrils. We found that L- arginine dose dependently reduced the thioflavin T (ThT) fluorescence of BSA. However, as
revealed by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and dynamic light scattering (DLS) results, L-arginin did not prevent amyloid- like fibril formation by BSA. We concluded that L -arginine competed against ThT for binding sites on BSA amyloid -like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for
potential inhibitors against amyloid fibrillation can give misleading results.
Subjects
L-arginine
aggregation
papain
guanidine hydrochloride (GdnHCl)
bovine serum albumin (BSA)
thioflavin T (ThT) fluorescence assay
Type
thesis
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