The origin of neocortical nitric oxide synthase-expressing ...
Validation of a Simple Assay for Nitric Oxide Synthase
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Validation of a Simple Assay for Nitric Oxide Synthase
Chelsea N. PeelerUniversity of Tennessee at
Martin
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NOSs = Nitric Oxide SynthasesGroup of enzymes that catalyze the production of nitric oxide from the amino acid L-arginine
*Dependent on the calcium ion
*Not dependent on the calcium
concentration
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iNOS i = inducible, immunity
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Nitric Oxide
Pre-1980 – atmospheric pollutant, bacterial
metabolite- readily reacts with atmospheric oxygen
to form nitrogen dioxidePost-1980 – implicated in a number of
biological processes 1992 “Molecule of the Year” Science
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Nitric Oxide Functions Primarily as a Signaling Molecule
Smooth muscle relaxantToo much is hazardous, just enough is crucial
for the body
Physiological processes regulated by NO signaling include:
VasodilationInhibition of platelet aggregationBronchodilationContractions of heart and skeletal muscleRegulator of ciliary beat frequencyNeurotransmissionMay assist in apoptosis
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NO AssaysExpensive, high powered, complex
Examples - oxyhemoglobin assay, mass spectrometry using 13N, chemiluminescence with luminal and hydrogen peroxide requiring a probe, and nitric oxide trapping reagents
Specific instrumentation required Trapping agents degenerate quickly, not thermo-
stable, susceptible to photolysis
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Basis of Assay MethodsMonitor the rate of conversion of NADPH to
NADP+
Monitor the amount of nitric oxide free radical produced- Consumption of DTNB- Electron Spin Resonance
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5, 5’-dithiobis-2-nitrobenzoic acid
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ESR for NO Determination Electron Spin Resonance detection of nitric oxide generation can be used to measure NO activity
- Transitions can be induced between spin states of the unpaired electron in NO by applying a magnetic field and then supplying electromagnetic energy, usually in the microwave range of frequencies - Resulting absorption spectra are described as ESR or EPR (electron paramagnetic resonance)
In our case, a high concentration of NO did not develop.
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Enzyme Kinetics
Where y-intercept = 1 / Vmax
x-intercept = -1 / KM
and slope = KM / Vmax
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OverviewNADPH/
iNOS (μL)Buffer (μL) L-arg. (μL) Initial
Absorbance
Absorbance after 30 minutes
700 0 1000 0.346 0.275
700 500 500 0.309 0.234
700 750 250 0.324 0.256
700 875 125 0.314 0.242
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Determination of the Michaelis Constant
Performed by varying the concentration of the substrate by one-half and one-fourth
Calculated by multiplying the slope of the line obtained by the maximum velocity
Compare value to: M
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Michaelis-Menten Plot Compare to typical assay:
-1KM
= -631.12 M-1
KM = 1.58 x 10-3 M
1Vmax
= 26.57 min
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Michaelis-Menten PlotCompare to typical assay:
1Vmax
= 84.32 min
-1KM
= -6292.39 M-1
KM = 1.59 x 10-4 M
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DTNB and NO reactionTest a typical iNOS-catalyzed reaction with DTNBAdded corresponding time-dependent iNOS reaction
to (1.244 x 10 -3 M) DTNB 0.002 decrease in absorbance over 8 h 40 min
interval (0.005 to 0.003)
No significant data obtained
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ConclusionsThrough the utilization of the paramagnetic properties of NO, the application of ESR on the NOS-catalyzed reaction was not successful, and this could be due to time restrictions on the production of NO. By observing the absorbance spectra of the NADPH molecule consumed in the NOS-catalyzed conversion of L-arginine to L-citrulline, the Michaelis constant was nearly identical to that of Cook’s.By observing the absorbance spectra of the product of the DTNB reaction with NO, there were no significant findings.
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AcknowledgementsDr. S.K. AireeDr. Misganaw GetanehJoe Cook
University of Tennessee at Martin College of Engineering and Natural Sciences (CENS)