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

Fang, S. and Lee, H.J. and Wark, A.W. and Kim, H.M. and Corn, R.M. (2005) Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonanace imaging and surface plasmon fluorescence spectroscopy. Analytical Chemistry, 77 (20). pp. 6528-6534. ISSN 0003-2700

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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.