1. Nitric Oxide is Consumed, Rather thanNitric Oxide is Consumed, Rather than
Conserved, by Reaction withConserved, by Reaction with
Oxyhemoglobin Under PhysiologicOxyhemoglobin Under Physiologic
ConditionsConditions
Mahesh S. Joshi, PhD; T. Bruce Ferguson Jr. MD; Tae H. Han,
PhD; Daniel R. Hyduke, PhD; James C Liao, PhD; Tienush Rassaf,
PhD; Nathan Bryan, PhD; Martin Feelisch, PhD; Jack R. Lancaster,
Jr. PhD
Departments of Surgery, Physiology and Medicine, LSU Health
Sciences Center, New Orleans; Department of Chemical
Engineering, UCLA; Department of Physiology and Biophysics,
LSU Health Sciences Center, Shreveport

2. Q:Q:
How does NO produced by theHow does NO produced by the
endothelium escape being scavengedendothelium escape being scavenged
by blood (hemoglobin) and therebyby blood (hemoglobin) and thereby
effect the vasorelaxation of adjacenteffect the vasorelaxation of adjacent
smooth muscles?smooth muscles?

3. Hypothesis A: NO oxidation/inactivation
Free NO produced by the endothelium diffuses through the
blood vessels. A majority of NO gets consumed due to its
reaction with hemoglobin, but enough survives to reach smooth
muscle to initiate relaxation.
HbO2 + NO MetHb + NO3
-
In support of this hypothesis, we have previously demonstrated
that RBCs consume far less NO than an equivalent amount of
free hemoglobin (Liu et al JBC 1998)
Also, Jim Liao’s group has proposed that intravascular flow
together with existence of a RBC-free zone near the vessel wall
decreases the erythrocyte consumption of NO (Liao et al PNAS
1999)

4. Hypothesis B: NO conservation
The oxidation of NO to NO3
-
by oxyHb is of little significance.
Instead, NO preferentially binds to a minor population of deoxy
hemes in a cooperative manner and then through a transnitrosation
reaction nitrosates hemoglobin thiol to form SNO-Hb.
oxyHb 99%
deoxyHb 1% 30 µM
NO 10-100 nM
Hb(Fe+2
) + NO Hb(Fe+2
)NO (1)
Hb(Fe+2
)NO Hb-S-NO (2)
Thus, hemoglobin conserves, rather than, consumes NO
bioactivity.
?

5. 0
20
40
60
80
100
Hemoglobin RBCs
FeNO-Hb
SNO-Hb
Gow et al Proc.Natl.Acad.Sci. (1999)
PercentofTotalNitro
48 µM HbO2 + 1.2 µM NO 103 ± 38 nM SNO-Hb & 42 ± 15 nM HbNO

6. OxyHbOxyHb
mMmM NONO
A problem with bolus NO/OxyHb reactionA problem with bolus NO/OxyHb reaction
NO autooxidation ∝ [NO]2
2NO + O2 2 .
NO2
.
NO2 + NO N2O3

7. Working HypothesisWorking Hypothesis::
The reaction of hemoglobin with NO added as a
highly concentrated aliquot results in additional
reactions (to form SNO-Hb and HbNO) and these
conditions do not mimic the physiological milieu
where NO produced at a constant rate by the
endothelium reacts with blood hemoglobin oxidatively
to form mainly nitrate and methemoglobin.

8. Difference spectra of NO/OxyHb reaction
33 µM HbO2 + 2.2 µM NO
500 550 600 650 700
-0.02
-0.01
0.00
0.01
MAHMA/NO
NO bolus
Absorbance
Wavelength

9. 0
1
2
3
4
5
6
µM
Bolus Donor
NO3- NO2- MetHb
125 µM OxyHb + 5 µM NO

10. SNOHb assay: Chemiluminescence
SNOHb, nM
NO bolus 51 ± 1.9
NO donor 39.8 ± 5.9

11. Time course of spectral changes with NO donor

12. 0 20 40 60 80 100
0
20
40
60
80
100
0 20 40 60 80 100
0
20
40
60
80
100A
Bolus
10 mM Pi
Bolus
100 mM Pi
Donor
[MetHb]/([MetHb]+[NO2
-
])(%)
[Hb] (µM)
B
Bolus
10 mM Pi
Bolus
100 mM Pi
Donor
[Hb] (µM)
[NO2
-
]/([MetHb]+[NO2
-
])(%)
MetHb and Nitrite formation as a function
of [Hb]

13. Conclusions
1. Reaction of hemoglobin with NO uniformly distributed
throughout the solution results in almost complete
oxidation of NO to NO3
-
and methemoglobin.
2. Bolus addition of NO to a solution of hemoglobin
yields equal amounts of both NO2
-
and NO3
-
3. The isosbestic points in the spectral data clearly
indicate gradual conversion of OxyHb to only metHb
4. There is formation of equal amount of NO3
-
and
methemoglobin showing that all the NO3
-
results from
the oxidation of NO by hemoglobin.

14. Acknowledgements
LSU, New Orleans:
Bruce Ferguson, Jr.; Jack Lancaster, Jr.
UCLA, Los Angeles:
Jim Liao; Tae Han; Daniel Hyduke
LSU, Shreveport:
Martin Feelisch; Tienush Rassaf; Nathan Bryan

15. NO
eNOS
NO
NO
NO
NO
MetH
b+ NO3
-
SNOHb
XNO
XNO
XNO
NO
RBC
EC
SMC
Vascular movement of Free and bound NO

16. Evidence for the NO oxidative reaction:
During NO breathing in humans, NO predominantly reacts with the heme
moiety of hemoglobin either to form metHb and nitrate or to form
Hb(Fe+2
)NO and little SNO-Hb (Gladwin et al PNAS 2000).
The addition of a NO donor to a solution of aerated Mb formed low levels of
NO2
-
(2.4%), while bolus NO addition formed substantial (64%) NO2
-
(Zhang
& Hogg, 2002).
Reduction in RBC volume due to induced acute hemorrhage in rabbits
generates more than 3 fold increase in exhaled NO, showing that blood does in
fact scavenge NO (Carlin et al J. Surg. Res. 1997).