Single molecule electron paramagnetic resonance spectroscopy: hyperfine splitting owing to a single nucleus.

Individual pentacene-d(14) molecules doped into a p-terphenyl-d(14) host crystal have been studied by optically detected electron paramagnetic resonance spectroscopy. The magnetic resonance transitions between the triplet sublevels of the pentacene molecule and the splitting of the resonance lines for a molecule that contains a carbon-13 nucleus have been observed in an external magnetic field. This splitting is caused by the hyperfine interaction of the triplet electron spin with the single carbon-13 nuclear spin.

Investigation of diffusion-limited rates of chymotrypsin reactions by viscosity variation.

The possibility that the rates of acylation of chymotrypsin by certain highly reactive substrates approach the diffusion-controlled limits was investigated by measuring the values of kcat/Km for three substrates as a function of increasing viscosity with sucrose and ficoll as the viscosogenic reagents. The values of Kcat/Km (pH 8.0, 25 degrees C) representing the acylation rate constants are the following: N-(methoxycarbonyl)-L-tryptophan p-nitrophenyl ester, 3.5 x 10(7) M-1 s-1; N-acetyl-L-tryptophan methyl ester, 8 x 10(5) M-1 s-1; N-acetyl-L-tryptophan p-nitroanilide, 300 M-1 s-1. The rate constants decrease significantly with increasing viscosity for the first compound, decrease slightly for the second, and are insensitive to this perturbation for the third. The p-nitroanilide results taken together with the observation that the high concentrations of sucrose or ficoll used produce insignificant changes in kcat for the ester substrates argue against a general nonspecific perturbation in the enzyme structure effected by these reagents. The values of the association rate constants calculated from these results are 9 x 10(7) and 1 x 10(7) M-1 s-1 for the p-nitrophenyl and methyl esters, respectively. The values of kcat/Km divided by the association rate constants show that the rates of acylation by the p-nitrophenyl ester occur at ca. 40% and by the methyl ester at ca. 10% of the diffusion limits. Possibilities involving reorientation of a nonproductively bound substrate within the ES complex or desolvation of part of the active site of the enzyme are considered to account for the lower association rate constant for the methyl as compared to the p-nitrophenyl ester.