Comparative study of effects of polyols, salts, and alcohols on trichloroacetic acid-induced state of cytochrome c

A systematic investigation of the effect of polyethylene glycols, salts, and alcohols on the trichloroacetic acid (TCA)-induced state of ferricytochrome c was made using various spectroscopic techniques. Native cytochrome c (Cyt c) has a fluorescence maximum at 335 nm, whereas the TCA-induced state of Cyt c has a red shift of 7 nm with enhanced fluorescence intensity. The near- and far-UV CD spectra showed a significant loss of tertiary and secondary structure, although the protein is relatively less unfolded as compared with a conformation at pH 2.0. Addition of 70% (v/v) polyols to TCA (3.3 mM)-induced state of Cyt c resulted in increased 1-anilino-8-naphthalene sulfonate binding and increased mean residue ellipticity at 222 nm, indicating increase in compactness with enhanced exposure of hydrophobic surface area. Also, the stabilizing effect of salts and alcohols on the TCA-induced state was studied and compared with their effect on trifluoroacetic acid-unfolded state of Cyt c. Among all the polyols, salts, and alcohols studied, PEG-400, K3[Fe(CN)6], and butanol were the most efficient in inducing secondary structure in TCA-induced state as examined by the above-mentioned spectroscopic techniques. For salts, the efficiency in inducing the secondary structure followed the order K3[Fe(CN)6] > KClO4 > K2SO4 > KCl. For alcohols, this order was found to be as follows: butanol > propanol > ethanol > methanol.