On the Structure of 41-Dinitrophenyl Ribonuclease A. Solvent Perturbation, Thermal Transition, Optical Rotatory Dispersion, and Binding Studies

TO further describe the inactivation of ribonuclease A by dinitrophenylation at lysine-41, solvent perturbation, thermal transition, optical rotatory dispersion, and binding properties were studied. Utilizing the hydantoin of e-dinitrophenyllysine as a model for a completely accessible dinitrophenyl moiety, solvent perturbation with 20% ethylene glycol revealed that the dinitrophenyl group in 41-dinitrophenyl ribonuclease A is at least paritally folded into the protein matrix. Thermal transition measurements provided substantiation, since a dinitrophenyl difference spectrum appeared on heat denaturation of the protein. Optical rotatory dispersion and thermal transition measurements also revealed possible small conformational differences between ribonuclease A and 41-dinitrophenyl ribonuclease A. 41-Dinitrophenyl ribonuclease A binds cytidine 3′-monophosphate but the binding affinity was found to be significantly less than with ribonuclease A at pH 5.5. An interpretation of the results is offered in which the lack of activity of 41-dinitrophenyl ribonuclease A is ascribed to diminished binding affinity and impaired catalytic efficiency that result from the lowered basicity of the anion-binding site and from small conformation changes.