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October 5, 1999

Thanks to a 'Horrible Worm,' New Ideas on Hemoglobin

By solving the mystery of how a loathsome worm thrives inside the human gut, scientists have arrived at a sweeping new theory of how hemoglobin, the common protein that ferries oxygen to tissues, evolved.

That discovery, in turn, could have major implications in the treatments of certain diseases as diverse as hypertension and sickle cell anemia.

A much-scrutinized protein still holds surprises.

The mystery involved the worm called ascaris, which has enormous hemoglobin molecules but thrives in an oxygen-free environment. If not to carry oxygen, what is the hemoglobin for?

According to a report in the Sept. 30 issue of the journal Nature, the worm uses its hemoglobin and a simple gas called nitric oxide to seek out and destroy oxygen, not carry it.

This "oxygen eating" function of hemoglobin, researchers said, is a missing link in the two billion years of hemoglobin evolution.

Hemoglobin is commonly thought to specializes in oxygen transport, but the molecule started to do that only relatively recently, said Dr. Jonathan Stamler, a biochemist at Duke University and an author of the worm paper. Hemoglobin first evolved to destroy nitric oxide, a gas that poisoned early microbial life, he said.

Later it captured nitric oxide and put it to work destroying oxygen, a gas that poisoned primitive worms and other invertebrates.

Now, hemoglobin with the help of nitric oxide moves oxygen around the bodies of large animals with backbones.

All of hemoglobin evolution has been driven by the activity of nitric oxide, Dr. Stamler said, and this has enormous implications for medicine. While the role of oxygen in health and disease is widely appreciated, nitric oxide has not been given equal footing, and yet it is involved in almost everything the body does.

Every cell makes nitric oxide. Apart from helping to evolve circulatory systems that deliver oxygen to tissues, nitric oxide also carries, through complex and largely unexplained mechanisms, thoughts in the brain and sensations through the body, and it allows men to have erections.

Most diseases involve changes in how the body handles or metabolizes nitric oxide and oxygen, Dr. Stamler said, including heart disease and high blood pressure, stroke, asthma, many cancers, sickle cell anemia, tuberculosis, arthritis and other diseases.

On hearing about the discovery that worm hemoglobin destroys oxygen, Dr. Ross Hardison, a professor of biochemistry at Pennsylvania State University and a leading expert on hemoglobin, said: "It's like the scales have fallen from my eyes. It looks like hemoglobin goes very far back in evolution.

It was there before the kingdoms of life separated. There's no mistaking real hemoglobin. It's found in bacteria, yeast, protozoans, blue-green algae, plants, insects and now in these horrible worms."

Dr. Steven Gross, a biochemist at Cornell University Medical College in New York, called hemoglobin the most studied and best understood protein in the human body, and yet "it still holds surprises." The new story about hemoglobin evolution, he said, is "an elegant example of molecular evolution."

"When you think about it," he said, "it just makes you smile."

Two billion years ago "there were lots of crazy gases around, lots of nitric oxide and no oxygen," Dr. Stamler said. Therefore, the first microbial life forms evolved hemoglobin to capture nitric oxide, which was poisonous to those forms, and to break it down into harmless nitrate, which could be used as food.

All hemoglobins are composed of two parts: heme, a scaffold that holds an iron molecule in its center, and globin, a protein that assumes a characteristic folded shape and nestles the heme.

Five hundred million years after bacteria spawned hemoglobin, certain algae began producing oxygen, which slowly built up in the atmosphere, Dr. Stamler said. Plants appeared and produced more oxygen. But many creatures living at this time, including primitive worms, or nematodes, were poisoned by oxygen and needed to find ways to get rid of it. They preferred living in places without much oxygen. Many, like ascaris, still do.

Ascaris infects one in six people in the world, primarily in developing nations, said Dr. Daniel Goldberg, a microbiologist at Washington University School of Medicine in St. Louis and a lead author on the Nature article. To be infected, Dr. Goldberg said, people must have swallowed the worm's eggs, which are carried by human fecal matter. Once inside the gut, the parasites grow to the size of plump earthworms and begin crawling around. They can get into the lungs, pancreatic duct and appendix, or even crawl out the throat and be unconsciously reswallowed. Some 20,000 deaths a year worldwide are linked to the worm.

Remarkably, the ascaris's hemoglobin holds on to oxygen 20,000 times tighter than human hemoglobin holds on to oxygen, Dr. Goldberg said.

To find out why, the scientists went inside the worms -- as one put it, "where no man has gone before" -- using fiber optic probes and other high-tech approaches to measure the nitric oxide and oxygen content of the worm's gut.

There, the scientists discovered the new role for hemoglobin.

Instead of destroying nitric oxide, as bacteria do, the worm hemoglobin finds a way to use nitric oxide in what amounts to the first real respiratory cycle, Dr. Stamler said. The worm hemoglobin develops a special site near its center that can hold nitric oxide, now made by the worm itself, and then exposes it to oxygen molecules.

When the two gases combine, he said, a waste product is formed and oxygen is removed from the system. Through a change in hemoglobin structure, nitric oxide was now being used rather than eliminated.

This step paved the way for the further evolution of hemoglobin found in creatures that use energy-rich oxygen for respiration, Dr. Stamler said. Hemoglobin got packaged into red blood cells and evolved a way to change its shape so that it could carry oxygen or carbon dioxide through much larger circulatory systems. But nitric oxide did not go away. It remained at home in the hemoglobin molecule and took on the role of sentry, looking for tissues that were hungry for oxygen. When nitric oxide encounters such tissues, Dr. Stamler said, it hops off the hemoglobin molecule, enters the blood vessels, widens them and delivers oxygen. Hemoglobin changes its shape, goes through the lungs and picks up more oxygen and recycles nitric oxide.

Nitric oxide's role in delivering oxygen was not appreciated until very recently, Dr. Stamler said, and its role remains in dispute.

In fact, the dogma in the field of hemoglobin has been that oxygen and nitric oxide cannot exist side-by-side. But this belief stems from mixing molecules in test tubes at unnaturally high concentrations, he said.

Hemoglobins in the body need vanishingly small amounts of nitric oxide to work properly.

Only recently have tools been able to measure the gas, which moves at lightning speeds in living tissues.

This matters because many therapies are based on the assumption that hemoglobin automatically destroys nitric oxide, Dr. Stamler said. For example, when sickle cell anemia patients are given nitric oxide to open up their lungs, it is believed that none of the gas will get into other parts of the body.

This is not true, he said, and may explain systemic side effects from the therapy, including sudden drops in blood pressure.

Many experimental substitutes for blood have not worked, Dr. Stamler said, because they did not take nitric oxide into account.

A deeper understanding of hemoglobin, nitric oxide and oxygen biology should lead to new therapies, several experts said.

For example, worm hemoglobin might be used to kill cancerous tumors by starving the tissue of oxygen.

Nitric oxide enzymes might be used to treat high blood pressure because nearly everyone with the condition has below-normal levels of the simple gas.

And hemoglobin may contain clues for preserving oxygen in tissue damaged by stroke or heart disease.

In a still deeper mystery, hemoglobin exists in parts of the human body other than red blood cells, Dr. Stamler said.

Immune cells called macrophages make their own hemoglobin, which may release nitric oxide to help kill bacteria, fungi or other parasites.

Some cancer cells also produce their own hemoglobin. No one knows why.

The field is open to new discoveries.