TRICHLOROACETIC ACID-INDUCED UNFOLDING OF BOVINE PANCREATIC RIBONUCLEASE - EXISTENCE OF MOLTEN GLOBULE-LIKE STATE

The exposure of ribonuclease A to trichloroacetic acid was earlier sho wn to alter the conformation of the protein resulting in reduced enzym atic activity (Sagar, A.J., Subbiah, V., and Pandit, M.W. (1989) Bioch im. Biophys. Acta 995, 144-150). We have studied the structure and enz ymatic activity of ribonuclease A treated with trichloroacetic acid ov er a wide range of acid concentrations (0-40%). The far ultraviolet ci rcular dichroism spectra of ribonuclease A, on exposure to acid concen trations less than 10%, indicated an exceptionally high degree of chir al structure. Exposure of ribonuclease A to acid concentrations betwee n 10 and 30% resulted in the formation of a molecule with significant chiral structure (conventionally assigned to residual secondary struct ure) but reduced tertiary structure (characteristics very similar to t hose 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, suppor ted the existence of a state having characteristics of molten globule. Reversed phase high performance liquid chromatography corroborated th e data obtained by circular dichroism as well as 1-anilino-naphthalene -8-sulfonate-binding studies. Beyond acid concentrations of 30%, the r ibonuclease is completely denatured. The trichloroacetic acid-induced unfolding is shown to be completely reversible.