REACTIVITY OF TRYPTOPHAN RESIDUES IN PROTEINS STOPPED-FLOW KINETICS OF FLUORESCENCE QUENCHING

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 ± 0.8) · 105 dm3 · mol-1 · s-1 at 23°C. The rate does not depend on the ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 ± 0.8) · 105 dm3 · mol-1 · s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 ± 0.3) · 105 dm3 · mol-1 s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in α-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 · 105, 0.56 · 105 and 0.11 · 105 dm3 · mol-1 · s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues. © 1979.