J. Biochem, 1983, Vol. 94, No. 1 51-61
© 1983 Japanese Biochemical Society
research-article |
1H Nuclear Magnetic Resonance Titration Curves and Microenvironments of Aromatic Residues in Bovine Pancreatic Ribonuclease A
Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113
The aromatic region of the NMR spectrum of bovine pancreatic nbonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37°C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results.
Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(
) and C(
) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C() and C() protons (two tyrosine residues), (3) not titratable and different chemical shifts for C() and C() protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C() and C() protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues.
The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 m NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.
1Present address: Department of Physiology, Medical College of Oita, P.O. Box 37, Oita 870-91.