Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A atlow pH

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various a lcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) a nd salts (K3FeCN6, Na2SO4, KClO4 and KCl) on the acid-induced state of alph a-chymotrypsinogen A, a predominantly beta-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 increa se in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE > bu tanol I propanol > ethanol > methanol. ANS fluorescence data showed a decre ase in fluorescence intensity on alcohol addition, suggesting burial of hyd rophobic patches. The near-UV CD spectra showed disruption of tertiary stru cture on alcohol addition. No change in ellipticity was observed on additio n 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 ell ipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-li ke state, i.e. both increase in ellipticity at 222 nm and increase in ANS f luorescence, followed the order K3FeCN6 > Na2SO4 > KClO4 > KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disor dering of tertiary structure results suggested that the decrease in ANS flu orescence intensity may be attributed to the unfolding of the ANS binding s ites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfold ed state. Characterization of such partially folded states has important im plications for protein folding. (C) 2000 Elsevier Science B.V. All rights r eserved.