Temperature-induced denaturation of ribonuclease S: a thermodynamic study.
In this paper the thermal denaturation of ribonuclease S, the product of mild digestion of ribonuclease A by subtilisin, is deeply investigated by means of DSC and CD measurements. It results that at whatever pH in the range 4-7.5 the process if fully reversible but not well represented by the simple two-state N<-->D transition. Actually, a two-state model that considers both unfolding and dissociation, NL<-->D + L*, well accounts for the main features of the process: the tail present in the low-temperature side of DSC peaks and the marked dependence of denaturation temperature on protein concentration. This mechanism is strictly linked to the exact stoichiometry of RNase S. An excess of the protein component of RNase S, the so-called S-protein, shifts the system toward a more complex behavior, that deserves a separate treatment in the accompanying paper [Graziano, G., Catanzano, F., Giancola, C., & Barone, G. (1996) Biochemistry 35, 13386-13392]. The thermodynamic analysis leads to the conclusion that the difference in thermal stability between RNase S and RNase A is due to entropic effects, i.e., a greater conformational flexibility of both backbone and side chains in RNase S. The process becomes irreversible at pH 8.0-8.5, probably due to side-reactions occurring at high temperature. Finally, the influence of phosphate ion on the stability of RNase A and RNase S at pH 7.0 is studied and explained in terms of its binding on the active site of ribonuclease. The analysis enables us to obtain an estimate of the apparent association constant and binding enthalpy also.
Catanzano
F
F
Department of Chemistry, University of Naples, Federico II, Italy.
Giancola
C
C
Graziano
G
G
Barone
G
G
D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008956:Models, Chemical; D010710:Phosphates / Q000378:metabolism; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D010710:Phosphates; D012260:Ribonucleases; C022542:ribonuclease S