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

S. Fang, H.J. Lee, A.W. Wark, H.M. Kim, R.M. Corn

Research output: Contribution to journalArticle

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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.
LanguageEnglish
Pages6528-6534
Number of pages7
JournalAnalytical Chemistry
Volume77
Issue number20
DOIs
Publication statusPublished - 15 Oct 2005

Fingerprint

Ribonuclease H
Enzyme kinetics
Fluorescence spectroscopy
Imaging techniques
Enzymes
Catalysis
Surface plasmon resonance
Hydrolysis
Microarrays
Rate constants
Desorption
Nucleic Acid Heteroduplexes
Adsorption
DNA
Kinetic parameters
Reaction rates

Keywords

  • enzymes
  • spectroscopy
  • ribonuclease
  • surface enzyme kinetics
  • surface plasmon
  • resonance imaging
  • fluorescence spectroscopy
  • protein-dna interactions
  • real-time
  • rnase-h
  • ultrasenstive detection
  • gold surfaces
  • label-free
  • microarrays
  • hybridization
  • adsorption
  • arrays

Cite this

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title = "Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonanace imaging and surface plasmon fluorescence spectroscopy",
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.",
keywords = "enzymes, spectroscopy , ribonuclease , surface enzyme kinetics , surface plasmon , resonance imaging, fluorescence spectroscopy, protein-dna interactions, real-time, rnase-h, ultrasenstive detection, gold surfaces, label-free, microarrays, hybridization, adsorption, arrays",
author = "S. Fang and H.J. Lee and A.W. Wark and H.M. Kim and R.M. Corn",
year = "2005",
month = "10",
day = "15",
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language = "English",
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pages = "6528--6534",
journal = "Analytical Chemistry",
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Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonanace imaging and surface plasmon fluorescence spectroscopy. / Fang, S.; Lee, H.J.; Wark, A.W.; Kim, H.M.; Corn, R.M.

In: Analytical Chemistry, Vol. 77, No. 20, 15.10.2005, p. 6528-6534.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonanace imaging and surface plasmon fluorescence spectroscopy

AU - Fang, S.

AU - Lee, H.J.

AU - Wark, A.W.

AU - Kim, H.M.

AU - Corn, R.M.

PY - 2005/10/15

Y1 - 2005/10/15

N2 - 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.

AB - 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.

KW - enzymes

KW - spectroscopy

KW - ribonuclease

KW - surface enzyme kinetics

KW - surface plasmon

KW - resonance imaging

KW - fluorescence spectroscopy

KW - protein-dna interactions

KW - real-time

KW - rnase-h

KW - ultrasenstive detection

KW - gold surfaces

KW - label-free

KW - microarrays

KW - hybridization

KW - adsorption

KW - arrays

U2 - 10.1021/ac051283m

DO - 10.1021/ac051283m

M3 - Article

VL - 77

SP - 6528

EP - 6534

JO - Analytical Chemistry

T2 - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 20

ER -