Thermal transitions of acylated ribonuclease.

The amino groups of ribonuclease have been specifically acylated using five N-acyl-succinimides in which the RCO-moieties varied in hydrocarbon chain length. The effects of these groups on the optical rotatory dispersion, circular dichroism, enzymic activity and stability to thermal denaturation have been determined. Thermal transitions, measured by changes in specific rotation at 365 nm, obeyed the criteria for a two-state process both before and after the acylation treatments. The transitions were fully reversible under the conditions of measurement and the observed standard enthalpies were constant over at least 80% of the transitions. The effect of acetylation was to decrease the transition temperature by 7°. Longer hydrocarbon chains decreased this further but only by a fraction of a degree per extra -CH2- group. The calculation of thermodynamic quantities is limited by the precision with which enthalpies can be measured but indicates that the standard free energy for the transition process decreases by about 270 cal per acetyl group and a further 20 cal per additional -CH2- group. The major source of destabilisation on acetylation appears to be the introduction of eleven hydrophobic CH3groups per protein molecule rather than the increase in net negative charge. The thermodynamic data permit inferences to be drawn about changes in the nature of the side chain environment on thermal denaturation.