Über die thermische Denaturierung von trockener Ribonuclease /A Study of the Dry Thermal Denaturation of Ribonuclease

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Abstract
The irreversible thermal denaturation of dry ribonuclease in the temperature range between 105 and 140°C is a complex process, leading to a very large number of derivatives, different in conformation, enzymatic activity, and amino acid composition. By gelfiltration three groups of molecules can be separated. Two of them having low enzymatic activity are composed of larger molecules than the native one. The third group retaining most of the enzymatic activity is composed of molecules nearly unchanged in molecular volume. These molecules apparently unaltered in gelfiltration experiment are definitely different from the native molecules, as can be shown by the different “melting curves” measured optically by UV-absorption at 287 nm in the temperature range between 20 and 70 °C. The heterogenity and the change in conformation was further demonstrated by ion exchange chromatography yielding several enzymatically active derivatives of ribonuclease. Amino acid analysis of heat denatured ribonuclease showed a loss of lysine and histidine. According to the rather complex process of denaturation the enzymatic activity does not decay during the heating time in an exponential way with a single time constant. Nevertheless during the first two hours of inactivation and especially at higher temperature the survival curves follow this pattern rather closely. From the slope of the survival curve an effective activation enthalpy of 23 kcal/mole and an activation entropy of -22 cal/mole · degree were obtained. Comparison between denaturation induced by heat and by ionizing radiation shows that similar types of damage occur at the molecular level.

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