Alcohol-induced versus anion-induced states of K-chymotrypsinogen A at low pH Faaizah Khan, Rizwan Hasan Khan *, Salman Muzammil 1 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India Received 16 March 2000; received in revised form 6 June 2000; accepted 8 June 2000 Abstract Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K 3 FeCN 6 , Na 2 SO 4 , KClO 4 and KCl) on the acid-induced state of K-chymotrypsinogen A, a predominantly L-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of K-helical structure. The order of effectiveness of alcohols was shown to be TFE s butanol s propanol s ethanol s methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K 3 FeCN 6 s Na 2 SO 4 s KClO 4 s KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Molten globule ; Alcohol-induced state ; Anion-induced state ; Circular dichroism 1. Introduction After proteins are synthesized on ribosomes they are e¡ectively and e¤ciently folded into their biolog- ically active form. The mechanism by which such an intricate process takes place still eludes protein chem- ists and molecular biologists. One of the major strat- egies followed in the study of protein folding is the characterization of conformational transition and folding intermediates [1,2]. The detailed description of partially folded states of proteins is often di¤cult due to the high cooperativity of the folding process; however, using mild denaturing conditions partially folded states of a number of proteins that are stable at equilibrium have been found [3,4]. Several studies have shown that the compactness and the amount of secondary structure of the intermediate state formed 0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII:S0167-4838(00)00129-1 * Corresponding author. Fax: +91-571-701081; E-mail : rizwanhkhan@hotmail.com 1 Also corresponding author. Biochimica et Biophysica Acta 1481 (2000) 229^236 www.elsevier.com/locate/bba