Enzyme Kinetics of iNOS - University of Tennessee at Martin
Validation of a Simple Assay for Nitric Oxide Synthase Chelsea N. Peeler University of Tennessee at Martin NOSs = Nitric Oxide Synthases Group of enzymes that catalyze the production of nitric oxide from the amino acid L-arginine *Not dependent on the calcium concentration
*Dependent on the calcium ion iNOS i = inducible, immunity Nitric Oxide Pre-1980 atmospheric pollutant, bacterial metabolite
- readily reacts with atmospheric oxygen to form nitrogen dioxide Post-1980 implicated in a number of biological processes 1992 Molecule of the Year Science Nitric Oxide Functions Primarily as a Signaling Molecule
Smooth muscle relaxant Too much is hazardous, just enough is crucial for the body Physiological processes regulated by NO signaling include: Vasodilation Inhibition of platelet aggregation Bronchodilation Contractions of heart and skeletal muscle Regulator of ciliary beat frequency
Neurotransmission May assist in apoptosis NO Assays Expensive, 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 thermostable, susceptible to photolysis
Basis of Assay Methods Monitor 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-2nitrobenzoic 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 Overview NADPH/
iNOS (L)L) Buffer (L)L) L-arg. (L)L) Initial Absorbanc e Absorbanc e 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: 1 = 26.57 min Vmax
-1 = -631.12 MKM 1 KM = 1.58 x 10-3 M Michaelis-Menten Plot Compare to typical assay: 1 Vmax= 84.32 min -1 = -6292.39 M-1 KM KM = 1.59 x 10-4 M
DTNB and NO reaction Test a typical iNOS-catalyzed reaction with DTNB Added 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 Conclusions Through 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 Larginine to L-citrulline, the Michaelis constant was nearly identical to that of Cooks.
By observing the absorbance spectra of the product of the DTNB reaction with NO, there were no significant findings. Acknowledgements Dr. S.K. Airee Dr. Misganaw Getaneh Joe Cook University of Tennessee at Martin College of Engineering and Natural Sciences (CENS)
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