Duke Researchers Discover Central Role of Nitric Oxide in Hemoglobin Action
DURHAM, N.C. -- Duke University Medical Center researchers
have found that nitric oxide, combined with hemoglobin, is a
major regulator of gas exchange, as well as blood pressure, in
the circulatory system. The finding appears to have solved the
long-standing mystery of how blood carries oxygen to body
tissues and extracts waste carbon dioxide while keeping vessels
open and blood pressure steady.
Scientists say the discovery, detailed in the March 21 issue
of the British journal Nature, could quickly pay off in
developing the first effective blood substitute, and may
ultimately change the way many diseases are treated.
"We now know that nitric oxide is involved in the blood's
major functions," said cardiologist and pulmonologist Dr.
Jonathan Stamler in an interview. "Oxygen delivery is essential
to life and a deficiency in oxygen is associated with diseases
of every organ. The same picture is gradually emerging for
nitric oxide (NO). Understanding delivery of both in concert
could have profound therapeutic implications.
"The duet of hemoglobin and NO is fantastically symbiotic in
carrying out the machinery of life," he said. "Hemoglobin uses
a spritz of the NO it carries to help get oxygen into tissues.
And NO helps hemoglobin carry away the trash of carbon dioxide.
The work was funded by the National Institutes of Health and
the Pew Charitable Trusts. Working with Stamler was first
author, Duke research associate Li Jia, and Joseph Bonaventura
and Celia Bonaventura, from Duke's Nicholas School of the
Environment and the Marine Biomedical Center.
Nitric oxide, long known as a noxious gas in the atmosphere,
has been found over the past several years to play a major role
in numerous biological systems. For example, scientists
discovered that NO worked in the circulatory system to dilate
blood vessels. "Free" NO is released by endothelial cells on
the inside of vessel walls where it migrates to nearby muscle
cells and relaxes them, opening the vessel and lowering blood
At the same time, researchers observed that this free nitric
oxide was inactivated by hemoglobin, as the iron molecule in
hemoglobin essentially consumes NO.
Adding these bits of knowledge together -- that NO keeps
vessels open, but that hemoglobin destroys NO -- produced a
major paradox that no one could solve, Stamler said. How can
blood vessels maintain a constant pressure when the hemoglobin
that flows through them destroys NO on contact?
Stamler suspected that NO had to exist in some other form in
the blood, apart from the "free" NO that is made in the vessel
and destroyed by hemoglobin. So he and Jia worked with the
Bonaventuras, who are experts on hemoglobin, designing
investigations using blood from humans and rats.
After a series of experiments, the team discovered that a
NO-containing hemoglobin molecule is synthesized in the lungs
and that the NO attached to it differs from that produced in
vessel walls. Hemoglobin is a large protein complex containing
"heme" groups, which include a central iron molecule that
serves as a site on which to bind and carry oxygen. The same
site also destroys "free" NO. Stamler and his team found the
"new" NO attaches itself to the hemoglobin-oxygen complex on a
cysteine residue that keeps the NO away from the hemes.
Specifically, in its new form, this NO is attached to a
thiol and is called SNO (for S-nitrosothiols). SNO retains its
NO-like properties, but is "a souped-up cousin," Stamler said.
SNO is protected from inactivation by hemes, unlike NO produced
in vessel walls, and it has a wider range of functions than NO.
Not many SNOs have been found in the body to date, but the ones
that have been discovered are powerful forms of NO, he said.
For example, SNO can kill invading bacteria or microbes. Free
NO cannot, said Stamler.
"We always knew that the hemoglobin complex had two reactive
arms, a heme and a cysteine, to which other molecules could
attach, but no one knew what the cysteine's function was," he
said. "We now know that it serves to bind NO."
Further experimentation by the group uncovered the intricate
interplay between hemoglobin, oxygen, NO and carbon
* After hemoglobin loads up oxygen and SNO in the lung, the
hemoglobin-trio travels down arteries through the heart and
into the rest of the body to deliver its load of oxygen. That
process has to happen with open vessels and in a constant
pressure. So as the hemoglobin complex inevitably devours the
free NO gas that endothelial cells produced to dilate the
vessel, hemoglobin simultaneously releases the SNO molecule it
had been carrying. One "ineffective" NO is exchanged for
another "protected" NO. Blood pressure remains constant and
blood flow maintained in order to promote oxygen transport.
Hemoglobin detects how much NO has been removed from the
bloodstream and compensates with SNO, the researchers believe.
Still, the hemoglobin-SNO molecules are very abundant, so there
is still plenty of SNO still available as the hemoglobin
complex enters tissues.
* In tissue, the hemoglobin undergoes a major structural
change to release its load of oxygen. This same
"conformational" change also releases SNO, presumably to
increase the efficiency of oxygen utilization, Stamler said.
"Mitochondria in tissue make energy from oxygen, and the SNO
may help regulate the rate at which the mitochondria respire,
or use the oxygen." It is also possible that SNO regulates
capillary blood flow, he said.
* When the hemoglobin has released both its oxygen and SNO,
it can attract molecules of carbon dioxide. Carbon dioxide
(CO2) is the waste gas produced from oxygen respiration.
Hemoglobin binds CO2 and carries it to the lungs, where it is
exhaled. At that point, the "free" NO consumed by hemoglobin is
also released and exhaled. Then, the oxygenation process is
"Once, we thought the primary job of hemoglobin was to carry
oxygen," said co-author Joseph Bonaventura in an interview.
"Now we can show that nitric oxide delivery may be comparable
in importance. Here we have the lungs synthesizing a compound
and delivering it to tissue where it is metabolized, just as
In addition, the research shows that NO has a regulatory
"allosteric" function that has not been described before,
Stamler said. Allosteric regulation is when one molecule causes
a protein to change its shape and, thereby, its function.
Hemoglobin is "a classic allosteric protein" because its
function depends on whether or not oxygen is bound to
it,Stamler said. Now, the Duke researchers said, NO is also
involved in the allosteric transition of hemoglobin.
Moreover, the work draws attention to a new way of
regulating protein function through attachment of NO to
cysteine residues in proteins. "This mechanism, termed
S-nitrosylation, may be analogous to phosphorylation -- that
is, regulation by attaching a phosphate group. It is the major
signaling mechanism in cells. Here, regulation occurs by
attaching an NO group.
"This is the first demonstration that an NO group, attached
to a thiol within a cell, has regulatory function," Stamler
said. "There has been growing evidence that NO does its work in
the body, in part, by binding to a thiol, but no one had proven
it before. Thiol-related signaling has been disputed because no
one had found SNO proteins in cells.
"Defining how NO signals in biological systems is a major
step forward," he said.
Among the immediate benefits of the research, Jia said, is a
new way to design blood substitutes. A continuing problem in
designing blood replacements is that they have increased blood
pressure and imposed other constraints that result from
inactivation of NO, he said. "They have never been able to
regulate oxygen delivery properly because the synthetic
hemoglobin destroyed NO in the vessels, and without NO at all,
the vessels can't dilate," Jia said. Stamler added, "Now we
know that a substitute for hemoglobin may do well to have an
SNO attached, to replace NO being destroyed in the vessel
walls. Indeed, the whole idea of a blood substitute is to mimic
Furthermore, understanding hemoglobin's relationship with NO
could help in treating the damage caused when tissue is
deprived of oxygen, as in heart disease and stroke, and the
many diseases in which oxygen delivery is critical, Stamler