Effect of temperature on the fluorescence quenching by N-bromosuccinamide of tryptophan residues in proteins

Abstract

The quenching of tryptophan fluorescence by N-bromosuccinamide was studied by the fluorescence stopped-flow technique over a temperature range of 5 to 35 °C in order to compare the reactivities of tryptophan residues in various molecules. The highest rates and lowest activation energies were found with the simple molecule N-acetyltryptophanamide. The hormone glucagon, which contains a single tryptophan residue, showed a somewhat lower rate and higher activation energy. α-Chymotrypsin, which has three groups of tryptophan residues in different environments, gave three rate constants which were lower than those with the other compounds, and the activation energies were correspondingly higher. There was a linear relationship between enthalpy of activation and the Gibbs energy of activation, the proportionality factor being 6.7. This indicates a ΔH≠ − ΔS≠ compensation, with an isokinetic temperature of 350 K. The compensation is discussed in the light of the steric effects.