EFFECT OF PH AND DENATURANTS ON THE FOLDING AND STABILITY OF MURINE INTERLEUKIN-6

The conformation and stability of a recombinant mouse interleukin-6 (m IL-6) has been investigated by analytical ultracentrifugation, fluores cence spectroscopy, urea-gradient gel electrophoresis, and near- and f ar-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4. 0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence qua ntum yield was not due to unfolding of the molecule because the confor mation of mIL-6, as judged by both urea-gradient gel electrophoresis a nd CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentati on equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicoche mical studies. Quenching of tryptophan fluorescence (20%) also occurre d in the presence of 6 M guanidine hydrochloride upon going from pH 7. 4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Tr p-160, may be partially involved in the quenching of endogenous fluore scence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal reg ion of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide HI was 30% in the ra ndom coil conformation and 60% in the presence of alpha-helix-promotin g solvents. Replacement of His-3 3 with Ala-33 in peptide H1 alleviate d a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with t he indole ring of Trp-36 is a major determinant responsible for the qu enching of the endogenous protein fluorescence of mIL-6.