Abstract
The thermal denaturation of microbial Ribonuclease T1 (RNAase T1) as a function ofpH, was studied by means of DSC microcalorimetry. The midpoint denaturation temperatures, enthalpy changes and heat capacity changes of Ribonuclease T1 were compared with those obtained for pancreatic Ribonuclease A (RNAase A). It was found that the microbial T1 protein undergoes a more complex conformational transition than the simple two-state transition shown by Ribonuclease A. The hypothesis of the presence of a ‘molten globule’ form is discussed. The conformational stability of RNAase T1 is lower than that of RNAase A at highpH values. Indeed, the maximum stability of RNAase T1 occurs atpH ≈ 5, whereas that of RNAase A occurs atpH ≈ 8. AtpH=3.7 an irreversible aggregation phenomenon was indicated by the existence of a reproducible exothermic peak. The conformational transition of RNAase T1 is reversible in the range ofpH 4.5–7.0, whereas it becomes irreversible atpH≥8.0 as for RNAase A.
Zusammenfassung
Mittels DSC Mikrokalorimetrie wurde die thermische Denaturierung von mikrobieller Ribonuklease T1 (RNAase T1) als eine Funktion despH-Wertes untersucht. Die mittleren Denaturierungstemperaturen, Enthalpieänderungen und Wärmekapazitätsänderungen von Ribonuklease T1 wurden mit denen von pankreatischer Ribonuklease A (RNAase A) verglichen. Man fand, daß das mikrobielle Protein T1 einen komplizierteren Konformationsübergang zeigt, als die bei Ribonuklease A auftretende einfache Zweizustandsänderung. Es wird weiterhin die Hypothese des Auftretens einer “geschmolzenen Kügelchen” Form diskutiert. Die Konformationsbeständigkeit von RNAase T1 ist bei hohenpH-Werten niedriger als die von RNAase A. Die größte Stabilität von RNAase T1 liegt bei einempH-Wert von etwa 5, während die von RNAase A bei etwapH=8. Bei einempH-Wert von 3.7 wird durch einen reproduzierbaren exothermen Peak das Auftreten einer irreversiblen Aggregation angezeigt. Die Konformationsänderungen von RNAase T1 sind impH-Wertbereich 4.5 bis 7.0 reversibel, die von RNAase A sind irreversibel bei einempH-Wert von mindestens 8.0.
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This work was financed by the National Research Council (C.N.R.-Rome) and by Ministry of University and Scientific and Technological Research.
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Barone, G., Del Vecchio, P., Fessas, D. et al. Thermal denaturation of ribonuclease T1 a DSC study. Journal of Thermal Analysis 38, 2791–2802 (1992). https://doi.org/10.1007/BF01979753
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DOI: https://doi.org/10.1007/BF01979753