The inhibition of horse-liver esterase by rhodamine B

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Abstract
The inhibition of horse-liver esterase by the fluorescent dye rhodamine B was studied kinetically; and the interaction between enzyme and dye was investigated by measurements of the intensity and polarization of fluorescence. The inhibition is rapidly established and reversible, and competitive with respect to substrate. At constant substrate and dye concentrations, the percent inhibition is independent of enzyme and phosphate concentrations. The relation between the dye concentration and the apparent value of the Michaelis constant is not linear. It resembles the function entailed by the "apparent competitive" inhibition mechanism of Segal et al. (1952), but on analysis is found to deviate significantly from it. An inhibition mechanism is proposed which accounts for the experimental results, including the relation between the inhibitions of the hydrolyses of 2 different substrates. Distribution experiments and freezing-point measurements show that rhodamine B is monomeric in the aqueous solutions of pH 7.4 used for the enzyme studies. Measurements of the polarization of fluorescence demonstrate the reversible combination of rhodamine with large molecules in the enzyme preparation. The combination is accompanied by a change in absorption spectrum and an increase in fluorescent intensity. Displacement of the dye from combination by ethyl acetate gives evidence that the binding is specifically related to the esterase inhibition. The fluorescence data are analyzed quantitatively, and their relation to the inhibition results is discussed.