|Abstract or Summary
- The spectrophotometric titration of the tyrosine residues,
their reaction with N-acetylimidazole, and the effect of pH on the
thermally induced conformational transition of ribonuclease T₁ have
been investigated. The results of spectrophotometric titration in
0.20 M KCl/0.001 M sodium phosphate suggest that titration of the
tyrosine residues occurs in two steps, the first with an apparent
pK of 10.7, and the second with an apparent pK greater than 12.
About 8 of the 9 tyrosine residues titrate in the first step, which is
characterized by a sharp dependence of the degree of ionization on
pH. This behavior is suggestive of a cooperative titration. In 8 M
urea-0.20 M KCl/0.001 M sodium phosphate the tyrosine residues
titrate in one step with an apparent pK of 10.9. Acetylation of
ribonuclease T₁ in 0.02 M Veronal, pH 7.5, indicates that about
2 tyrosine residues are readily acetylated with a 60-180 fold molar excess of acetylimidazole, while other tyrosine residues react only
at much higher concentrations of acetylating reagent. In 8 M
urea-0.02 M Veronal, pH 7.5, about 8 of the 9 tyrosine residues
are readily acetylated. Studies on the thermal transition of ribonuclease
T₁ showed that, above about pH 4, the transition temperature
decreases with increasing pH. Thermal transition occurred
with the exposure of tryptophan and tyrosine residues to solvent.
The above properties of ribonuclease T₁ have been contrasted with
those of bovine pancreatic ribonuclease A.
The homogeneity of ribonuclease T₁ was investigated by high-speed
sedimentation equilibrium measurements, and the protein was
found to be homogeneous under the conditions used in the experiments.
Molecular weights of ribonuclease T₁ calculated from the
sedimentation equilibrium experiments agreed, within experimental
error, with the molecular weight based on the known amino acid
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