Validation of a Simple Assay for Nitric Oxide Synthase

Validation of a Simple Assay for Nitric Oxide Synthase Validation of a Simple Assay for Nitric Oxide Synthase - Start

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Validation of a Simple Assay for Nitric Oxide Synthase - Description

 . 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. *Dependent on the calcium ion . ID: 698451 Download Presentation

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Validation of a Simple Assay for Nitric Oxide Synthase




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Slide1

Validation of a Simple Assay for Nitric Oxide Synthase  

Chelsea N. Peeler

University of Tennessee at Martin

Slide2

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

Slide3

iNOS

i = inducible, immunity

Slide4

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

Slide5

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

Slide6

NO AssaysExpensive, 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 requiredTrapping agents degenerate quickly, not thermo-stable, susceptible to photolysis

Slide7

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

Slide8

Slide9

5, 5’-dithiobis-2-nitrobenzoic acid

Slide10

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.

Slide11

Enzyme Kinetics

Where

y

-intercept = 1 /

V

max

x-intercept = -1 /

K

M

and slope = K

M

/ Vmax

Slide12

Overview

NADPH

/

iNOS

(μL)

Buffer

(μL)

L-arg. (μL)

Initial

Absorbance

Absorbance after 30 minutes

700

0

1000

0.346

0.275

700

5005000.3090.234700750250

0.324

0.256

700

875

125

0.314

0.242

Slide13

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

 

Slide14

Michaelis-Menten Plot

Compare to typical assay:

-1

K

M

= -631.12 M

-1

K

M

= 1.58

x

10

-3

M

1

V

max

= 26.57 min

Slide15

Michaelis-Menten Plot

Compare to typical assay:

1

V

max

= 84.32 min

-1

K

M

= -6292.39 M

-1

K

M

= 1.59

x

10

-4

M

Slide16

DTNB and NO reactionTest 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

Slide17

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.

Slide18

AcknowledgementsDr. S.K.

Airee

Dr.

Misganaw

Getaneh

Joe Cook

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

Slide19

Slide20


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