Validation of a Simple Assay for Nitric Oxide Synthase  

Chelsea N. PeelerUniversity of Tennessee at

Martin

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

iNOS i = inducible, immunity

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

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

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

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

5, 5’-dithiobis-2-nitrobenzoic acid

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.

Enzyme Kinetics

Where y-intercept = 1 / Vmax

x-intercept = -1 / KM

and slope = KM / Vmax

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

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

Michaelis-Menten Plot Compare to typical assay:

-1KM

= -631.12 M-1

KM = 1.58 x 10-3 M

1Vmax

= 26.57 min

Michaelis-Menten PlotCompare to typical assay:

1Vmax

= 84.32 min

-1KM

= -6292.39 M-1

KM = 1.59 x 10-4 M

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

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.

AcknowledgementsDr. S.K. AireeDr. Misganaw GetanehJoe Cook

University of Tennessee at Martin College of Engineering and Natural Sciences (CENS)