1. Validation of a Simple Assay for Nitric Oxide Synthase Chelsea N. Peeler University of Tennessee at Martin

2. NOSs = Nitric Oxide Synthases Group 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

3. iNOS i = inducible, immunity

4. 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

5. 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

6. NO Assays Expensive, high powered, complex  Examples - oxyhemoglobin assay, mass spectrometry using 13 N, 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

7. 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

9. 5, 5’-dithiobis-2- nitrobenzoic acid

10. 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.

11. Enzyme Kinetics Where y-intercept = 1 / V max x-intercept = -1 / K M and slope = K M / V max

12. Overview NADPH/ iNOS (μL) Buffer (μL)L-arg. (μL) Initial Absorbance Absorbance after 30 minutes 700010000.3460.275 700500 0.3090.234 7007502500.3240.256 7008751250.3140.242

13. Determination of the Michaelis Constant

14. Michaelis-Menten Plot Compare to typical assay: K M = -631.12 M -1 K M = 1.58 x 10 -3 M 1 V max = 26.57 min

15. Michaelis-Menten Plot Compare to typical assay: 1 V max = 84.32 min K M = -6292.39 M -1 K M = 1.59 x 10 -4 M

16. 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

17. 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 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.

18. Acknowledgements Dr. S.K. Airee Dr. Misganaw Getaneh Joe Cook University of Tennessee at Martin College of Engineering and Natural Sciences (CENS)