TEMPERATURE-INDUCED DENATURATION OF RIBONUCLEASE-S - A THERMODYNAMIC STUDY

Citation
F. Catanzano et al., TEMPERATURE-INDUCED DENATURATION OF RIBONUCLEASE-S - A THERMODYNAMIC STUDY, Biochemistry, 35(41), 1996, pp. 13378-13385
Citations number
49
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
Biochemistry → ACNP
ISSN journal
0006-2960
Volume
35
Issue
41
Year of publication
1996
Pages
13378 - 13385
Database
ISI
SICI code
0006-2960(1996)35:41<13378:TDOR-A>2.0.ZU;2-0
Abstract
In this paper the thermal denaturation of ribonuclease S, the product of mild digestion of ribonuclease A by subtilisin, is deeply investiga ted by means of DSC and CD measurements. It results that at whatever p H in the range 4-7.5 the process is fully reversible but not well repr esented by the simple two-state N <-> D transition. Actually, a two-st ate model that considers both unfolding and dissociation, NL <-> D + L , well accounts for the main features of the process: the tail presen t in the low-temperature side of DSC peaks and the marked dependence o f denaturation temperature on protein concentration. This mechanism is strictly linked to the exact stoichiometry of RNase S. An excess of t he protein component of RNase S, the so-called S-protein, shifts the s ystem toward a more complex behavior, that deserves a separate treatme nt in the accompanying paper [Graziano, G., Catanzano, F., Giancola, C ., & Barone, G. (1996) Biochemistry 35, 13386-13392]. The thermodynami c analysis leads to the conclusion that the difference in thermal stab ility 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 influen ce 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 ribonucleases, The analysis enables us to obtain an estimate of the a pparent association constant and binding enthalpy also.