Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy

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 (k _a), enzyme desorption (k_d), enzyme catalysis (k _cat), and one dimensionless diffusion parameter (β). Values of k_a = 3.15 (±0.20) × 10⁶ M ^-1·s^-1, k_d = 0.10 (±0.05) s ^-1, and k_cat = 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 (k_cat), 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 (k_cat = 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 k_cat is much larger than the product of k_a and the bulk enzyme concentration.