Trichloroacetic acid-induced unfolding of bovine pancreatic ribonuclease. Existence of molten globule-like state.
The exposure of ribonuclease A to trichloroacetic acid was earlier shown to alter the conformation of the protein resulting in reduced enzymatic activity (Sagar, A. J., Subbiah, V., and Pandit, M. W. (1989) Biochim. Biophys. Acta 995, 144-150). We have studied the structure and enzymatic activity of ribonuclease A treated with trichloroacetic acid over a wide range of acid concentrations (0-40%). The far ultraviolet circular dichroism spectra of ribonuclease A, on exposure to acid concentrations less than 10%, indicated an exceptionally high degree of chiral structure. Exposure of ribonuclease A to acid concentrations between 10 and 30% resulted in the formation of a molecule with significant chiral structure (conventionally assigned to residual secondary structure) but reduced tertiary structure (characteristics very similar to those of molten globule). Increased binding of the hydrophobic probe 1-anilinonaphthalene-8-sulfonate to the enzyme treated with 15-30% acid, as compared with the untreated or completely unfolded protein, supported the existence of a state having characteristics of molten globule. Reversed phase high performance liquid chromatography corroborated the data obtained by circular dichroism as well as 1-anilino-naphthalene-8-sulfonate-binding studies. Beyond acid concentrations of 30%, the ribonuclease is completely denatured. The trichloroacetic acid-induced unfolding is shown to be completely reversible.
Kumar
T K
TK
Centre for Cellular and Molecular Biology, Hyderabad, India.
Subbiah
V
V
Ramakrishna
T
T
Pandit
M W
MW
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D014238:Trichloroacetic Acid / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D014238:Trichloroacetic Acid; C027132:1-anilino-8-naphthalenesulfonate; D012259:Ribonuclease, Pancreatic