1 / 18

Validation of a Simple Assay for Nitric Oxide Synthase

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.

riona
Download Presentation

Validation of a Simple Assay for Nitric Oxide Synthase

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 *Not dependent on the calcium concentration *Dependent on the calcium ion

  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 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

  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

  8. 5, 5’-dithiobis-2-nitrobenzoic acid

  9. 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.

  10. Enzyme Kinetics Where y-intercept = 1 / Vmax x-intercept = -1 / KM and slope = KM / Vmax

  11. Overview

  12. 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

  13. Michaelis-Menten Plot Compare to typical assay: 1 Vmax = 26.57 min -1 KM = -631.12 M-1 KM = 1.58 x 10-3 M

  14. Michaelis-Menten Plot Compare to typical assay: 1 Vmax = 84.32 min -1 KM = -6292.39 M-1 KM = 1.59 x 10-4 M

  15. 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

  16. 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.

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

More Related