Take a deep breath; the fresh air flows into your mouth ornose, down the airways into the lungs. There it dumps its cargoof oxygen (21 percent of air) into the hemoglobin-laden redblood cells (RBCs) of the circulation and picks up in return thewaste carbon dioxide. This you exhale along with the inertnitrogen (78 percent of air).

Meanwhile, pumped by the heart, the oxygen-rich, bright redarteries are distributing that incoming oxygen to every lasttissue and cell.

Now take a trip to Denver (elevation 5,280 feet) or Mexico City(7,350 feet). At these altitudes the air is thinner, the oxygensparser, the breathing harder. But after a night's rest, saidbiochemist Chien Ho of Carnegie-Mellon University, your bodybegins to acclimatize. The blood produces more 2,3-diphosphoglycerate, which helps inject the oxygen you inhaleinto the RBCs. To kick in this supercharger, nature has poweredthe hemoglobin (Hb) protein in these cells with potent allostericeffector molecules. These exert intricate inhibition/activationcontrols on oxygenation, something like a feedback speedgovernor.

"Normal human hemoglobin can't do that in vitro," Ho toldBioWorld. "To engineer Hb as a successful blood substitute, firstyou need to make its heme iron atom more stable againstoxidation. Second, you want low oxygen affinity. Hb grabsoxygen very tightly, doesn't want to give it up. We need toprovide the allosteric effector that helps unload it in ourbloodstream."

Ho described how he and his associates in Carnegie-Mellon'sbiology department are closing in on these two goals in today'sissue of the Proceedings of the National Academy of Science(PNAS). His report is cryptically titled "Production ofunmodified human adult hemoglobin in Escherichia coli."

Engineering human hemoglobin to replace donated blood is aglittering, tantalizing prize sought by many biotechnologycompanies and academic laboratories. But it's not simple.

For starters, Ho's team acquired two gift plasmids, one fromSomatogen of Boulder, Colo., the other from what was thenCetus of Emeryville, Calif. (which has since merged with ChironCorp.) The Somatogen expression vector carried genes encodingthe alpha and beta chains of human Hb's tetrameric structure.They melded the DNA in this cloning vehicle with the one fromCetus, which carried the gene that E. coli expresses to makemethionine aminopeptidase (Met-AP). That enzyme cleavesmethionine from an amino acid sequence.

Ho's game plan was to overcome one of the main stumblingblocks in synthesizing hemoglobin, being stuck with aredundant methionine residue at the protein's N-terminal."Many proteins don't care if you have one extra amino acid inthe N terminal," he observed. "Hemoglobin does care. Itsoxygenation property depends on the integrity of that terminalresidue."

Ideally, Hb, like other useful proteins, should be freeze-driedfor storage and shipment. No way -- yet. "In general," Hoexplained, "when you get a protein like Hb, you freeze-dry it ina vial at zero conditions. Then, when you need the thing, youinject some isotonic saline. But when you freeze-dry Hb, its ironatom goes from ferrous to ferric, Fe2 plus to Fe3 plus. Thatchanges the Hb molecule from oxyhemoglobin tomethemoglobin, which does not bind oxygen. "If you couldmake that iron atom more stable, lyophilization would be apossibility."

His paper in PNAS hints at ways to do that by converting theorientation of the heme molecule.

The Carnegie-Mellon group is making "all sorts of Hb mutants"by site-directed mutagenesis, based on computer modeling ofprotein structure and design. "Once we understand themechanism of oxygenation," Ho said, "we can design mutant Hbto alter oxygen affinity, which could have great implications fora blood substitute. In fact, the company that gave us theplasmids for the Hb, Somatogen, is very excited by our work."

"We distribute our basic expression plasmids to academic andbasic researchers just following normal rules of scientificcourtesy," Somatogen's vice president of R&D, Gary Stetler, toldBioWorld. "One of the standard practices for molecularbiologists for years has been, if you publish on something, youshould be able to provide the materials so that others canrepeat your observations."

He observed that "Chien Ho, with whom we have a veryinformal collaboration, has been a very courteous collaborator.Not everyone to whom we send our plasmids communicates tous the results they've gotten in a timely fashion."

Somatogen itself is far advanced toward clinical application ofits own recombinant human hemoglobin, using a yeastexpression system. "As far as I know," Stetler said, "we areexpected to be the first group to proceed into a surgical settingafter completing our initial safety studies."

He added that E. coli, Ho's host organism, "is generally a littlemore tractable and should increase accessibility to moreresearchers if Chien sends out his plasmid."

Ho's goal is to "get a completely unmodified human Hb in largequantity." At the moment, he has a "very good yield" of 100 to200 milligrams per liter of culture.

Because of the AIDS epidemic, he said, "a reliable source of theblood supply is very important medically. Our research is afirst step in that direction, working with recombinant DNAtechnology."

What about DNX Corp., which is harvesting recombinant humanHb from transgenic pigs? "Ultimately," replied Ho, "it comesdown to which expression system gives the cheapest productand the best one."

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.