Refolding of chemically denatured a-amylase in dilution additive mode Reza Khodarahmi, Razieh Yazdanparast * Institute of Biochemistry and Biophysics, The University of Tehran, P.O. Box 13145-1384, Enghelab Street, Tehran 13145-1384, Iran Received 3 February 2004; received in revised form 1 June 2004; accepted 16 June 2004 Available online 4 July 2004 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 a-amylase in the presence and absence of a-CD, as a dilution additive agent. Our data indicate that in the presence of 100 mM a-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 a-amylase, in the presence of a-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 a-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 formed. In contrast, strengthening these interactions by enhancing the ionic strength of the refolding buffer made these hydrophobic interactions very strong. Therefore, a-CD could not depart from the protein/a-CD complex, as it usually does during the process of refolding. As a result, more aggregates were formed in the presence of a-CD compared to the corresponding control samples. D 2004 Elsevier B.V. All rights reserved. Keywords: a-CD; a-Amylase; Additive mode; Refolding 1. Introduction It is generally accepted that the refolding driving force of a protein resides in its amino acid sequence [1]. However, in vitro refolding of a protein from its denatured state to the native form hardly occurs by 100%. In other words, the refolding process is always accompanied by some degree of misfolding and also aggregation of the refolding intermedi- ates under the experimental conditions. From these obser- vations, it is certainly implied that external factors, besides intramolecular forces, play significant role(s) in the refold- ing processes [2,3]. The occurrence of misfolding and/or aggregation of proteins has been attributed to two main types of hydrophobic interactions [4]: the intramolecular interactions, which usually coordinate the spontaneous fold- ing of a polypeptide chain, and the intermolecular interac- tions between the partially refolded species, which are believed to be responsible for aggregation and inactivation of proteins in vitro situations [2–6]. These forces are believed to be absent in in vivo processes of refolding [2,3]. The proper refolding of proteins in vivo takes place under the influence of chaperones, a class of proteins which bind to the nonnative forms of proteins to prevent aggrega- tion and to promote the accurate refolding of the denatured proteins into their corresponding proper conformations [2,3,7]. Therefore, an accurate and full understanding of the folding/refolding principles and the chaperone assisted systems will aid in developing better experimental condi- tions to prevent or to reduce the extent of protein aggrega- tion and misfolding. This knowledge is also very necessary and helpful in biotechnology. Overexpression of proteins by genetically engineered organisms is often faced with the formation of inclusion bodies, aggregates of incompletely folded chains [8]. The use of these inclusion bodies in basic research or for the industrial aims necessitates the proper folding of these proteins into active forms. In this respect, the inclusion bodies are usually brought into solution by 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.06.010 Abbreviations: BSA, bovine serum albumin; a, h, g-CD, alpha, beta, gamma cyclodextrin; DNS, 3,5 dinitrosalicylic acid; GuHCl, guanidinium chloride; EG, ethylene glycol * Corresponding author. Tel.: +98-21-695-6976; fax: +98-21-640- 4680. E-mail address: yazdan@ibb.ut.ac.ir (R. Yazdanparast). www.bba-direct.com Biochimica et Biophysica Acta 1674 (2004) 175 – 181