Abstract
The ionization constants of 3 of the histidine residues of ribonuclease A have beenobtained at 5 temperatures from the nuclear magnetic resonance titration curves of the imidazole C2 proton resonances. Thermodynamic parameters derived from the ionization constants indicate that histidine residues 105 and 119 are fairly well exposed to solvent, while histidine residue 12 is in a somewhat more restricted environment. Measurements of the low pH inflection present in the titration curve of histidine-12 yield a large negative entropy value, indicating that the group givine rise to this inflection is also buried.
Highlights
The ionization constants of 3 of the histidine residues of ribonuclease A have been obtained at 5 temperatures from the nuclear magnetic resonance titration curves of the imidazole C2 proton resonances
Thermodynamic parameters derived from the ionization constants indicate that histidine residues 105 and 119 are fairly well exposed to solvent, while histidine residue 12 is in a somewhat more restricted environment
Thermodynamic parameters for the ionization of an individual histidine residue in a protein can be determined by measuring the temperature dependence of the NMR titration curve of the histidine C2 proton resonance (1, 2)
Summary
Ribonuclease A (Worthington Biochemical Corp., lot RAF lFC), obtained as a phosphate-free lyophilized powder, was used without further purification. Proton NMR spectra were recorded on a Varian Associates XL-100 spectrometer operating at 100 MHz and equipped with a pulse-Fourier transform accessory including a 620i computer. All chemical shifts are quoted downfield from external dimethylsulfoxide. Curve fitting was carried out as described previously (4, 8), using an expression for a simple proton association-dissociation equilibrium,?. Is the observed chemical shift, 6, is the chemical shift for the unprotonated form, and A is the chemical shift change upon protonation. Where an added inflection was present in the curve of H-3 the expression used was Equation 2, where A1 and A2 are the chemical shift changes for the low pH inflection (pK,) and the imidazole group (pK,), respectively
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