Refolding of chemically denatured alpha-amylase in dilution additive mode

Biochimica Et Biophysica Acta
Reza Khodarahmi, Razieh Yazdanparast

Abstract

Cyclodextrins (CDs) possess hydrophobic surfaces, which probably shield the hydrophobic surfaces of denatured proteins and prevent the direct interactions between the surfaces which are believed to be responsible for protein aggregation during refolding process. This probability was evaluated by studying the refolding process of denatured alpha-amylase in the presence and absence of alpha-CD, as a dilution additive agent. Our data indicate that in the presence of 100 mM alpha-CD in the refolding buffer, the extent of aggregation reduces by almost 90%. Spectrofluorometric analysis of the refolding intermediate(s) also indicates that the tertiary structure of the refolded alpha-amylase, in the presence of alpha-CD, is very similar to the tertiary structure of the native protein. However, this similarity was distorted upon addition of exogenous hydrophobic (aliphatic or aromatic) amino acids to the refolding buffer, meaning that the hydrophobic interactions between alpha-CD and the denatured protein play significant role in preventing aggregate formation. In addition, by weakening the extent of these hydrophobic interactions by adding polarity-reducing agent (e.g. ethylene glycol) to the refolding buffer, more aggregates were form...Continue Reading

Citations

May 3, 2008·Protein Science : a Publication of the Protein Society·Bryan F ShawGeorge M Whitesides
Nov 4, 2017·Journal of Agricultural and Food Chemistry·Anna C KrämerMichael J Davies

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