Determination of Ribonuclease H Surface Enzyme Kinetics by Surface Plasmon Resonance Imaging and Surface Plasmon Fluorescence Spectroscopy

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Abstract
The kinetics of the ribonuclease H (RNase H) surface hydrolysis of RNA−DNA heteroduplexes formed on DNA microarrays was studied using a combination of real-time surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS). Time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed using a simple model that couples diffusion, enzyme adsorption, and surface enzyme kinetics. This model is characterized by a set of three rate constants, enzyme adsorption (ka), enzyme desorption (kd), enzyme catalysis (kcat), and one dimensionless diffusion parameter (β). Values of ka = 3.15 (±0.20) × 106 M-1·s-1, kd = 0.10 (±0.05) s-1, and kcat = 0.95 (±0.10) s-1 were determined from fitting all of the SPRI and SPFS data sets. One of the most interesting kinetic parameters is the surface RNase H hydrolysis reaction rate constant (kcat), which was found to be ∼10 times slower than that observed in solution, but ∼100 times faster than that recently observed for the exonuclease III surface hydrolysis of double-stranded DNA microarrays (kcat = 0.009 s-1). Moreover, the surface coverage of the intermediate enzyme−substrate complex (ES) was found to be extremely small during the course of the reaction because kcat is much larger than the product of ka and the bulk enzyme concentration.

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