Measurement of catalytic reaction kinetics for adsorbed enzyme monolayers

We present a new assay based on total internal reflection fluorescence (TIR F) to quantify the catalytic activity of adsorbed enzyme monolayers on macr oscopically flat surfaces. The need for such an assay derives from a genera l shortage of assay methods that are sufficiently sensitive to measure reac tion kinetics for just a single monolayer of enzymes. The assay is based on the enzymatic conversion of a soluble, nonfluorescent fluorogenic substrat e reagent to a soluble, highly fluorescent product. The reaction occurs at the solid-liquid interface where the enzymes are adsorbed. Fluorogenic subs trates are introduced to the adsorbed layer by convective diffusion from so lutions undergoing steady laminar slit flow. The exponentially decaying eva nescent wave that is produced by total internal reflection serves as a "spe ctroscopic ruler" to resolve the spatial concentration profile of fluoresce nt products in solution near the interface. By measuring the steady-state f luorescence signal as a function of the Peclet number that characterizes ma ss transfer conditions in the experiment, it is possible to determine the e nzymatic reaction rate. Here we present the development of the method and i ts application to a test system of beta-galactosidase adsorbed to methylate d silica surfaces. Compared to the enzymatic rate constants for this enzyme in free solution, adsorption decreased the Michaelis-Menten rate constant k(cat) by a factor of 10 and increased the equilibrium binding constant K-m , by a factor of 4.5. Thus the intrinsic activity of the enzyme, as represe nted by the ratio k(cat)/K-m, decreased 45-fold due to adsorption, (C) 1999 Academic Press.