Determination of the dioxygen quenching constant for protein and model indole triplets

Abstract

The decay of the indole triplet of single tryptophan-containing proteins and model compounds can be readily determined at room temperature in solution by monitoring the triplet absorption or emission following an exciting laser pulse. The dioxygen triplet quenching constants, can be measured for all these molecules and compared to the analogous singlet values determined by fluorescence methods. The dioxygen triplet quenching constant ( t k q) ranged from a high of 5.1·10 9 M −1·s −1 for the exposed indole of corticotropin to a low of 0.1·10 9 M −1·s −1 for the buried indole of asparaginase. The ratio of these values with their respective dioxygen singlet quenching constants ( s k q), t k q/ s k q, ranged from 0.3 to 0.6 for aqueous exposed polypeptide indoles. For globular proteins the t k q/ s k q value is observed to be 0.2 ± 0.1. This lower value for protein indoles is not attributable to ‘bulk’ environmental or hydrogen bonding effects, since the magnitude of t k q/ s k q( = 0.5 ± 0.1) for model indoles was independent of solvent dielectric constant, polarity, and proticity. Temperature-dependence studies were done to test whether t k q could be used to characterize the nature of the protein matrix. The activation energy ( E a for t k q was found to be 11 ± 2kcal/mol for most proteins. This E a was independent of whether the indole side-chain was solvent exposed or buried in the non-aqueous protein interior. Large E a values were also obtained for model indoles, naphtalene and nalidixic acid, dissolved in water, whereas the same compounds dissolved in 95% ethanol exhibited much smaller E a values. These data, in combination with the observation that the t k q of model indoles is insensitive to changes in solvent viscosity, indicate thet dioxygen quenching at the triplet level can not be easily used to characterize the dynamics of proteins.