Validation of a Simple Assay for

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16 Νοε 2013 (πριν από 3 χρόνια και 6 μήνες)

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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



*Dependent on the
calcium ion

*Not dependent on the
calcium concentration

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
F
unctions 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
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


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
-
2
-
nitrobenzoic 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 /
V
max










x
-
intercept =
-
1 /
K
M




and slope = K
M
/
V
max


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:

𝐾
𝑀
=

2
.
0

𝑥

10

3

M




Michaelis
-
Menten

Plot

C
ompare to typical assay:



-
1

K
M


=
-
631.12 M
-
1

K
M

= 1.58
x

10
-
3

M




1

V
max

= 26.57 min

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




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 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.







Acknowledgements

Dr. S.K.
Airee

Dr.
Misganaw

Getaneh

Joe Cook

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