Transcript 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 thermo-stable,
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 (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:
𝐾𝑀 = 2.0 𝑥 10−3 M
Michaelis-Menten Plot
Compare to typical assay:
1
= 26.57 min
Vmax
-1 = -631.12 M-1
KM
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 Cook’s.
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)