2001 Lemieux Award Lecture Organic chemistry and hemoglobin: Benefits from controlled alteration

Abstract

Hemoglobin carries oxygen in circulation within red cells but does not function outside the cells because it fails not only to release oxygen but also dissociates into dimers that make up the tetrameric protein. Bifunctional anionic acylating agents that contain a structurally rigid bridge introduce cross-links that stabilize hemoglobin and alter its oxygen affinity so that it could be used to carry oxygen outside cells. Nitric oxide binds to hemoglobin and in circulation this causes undesirable increases in blood pressure. It had been reported that higher weight collections of hemoglobin do not cause vasoconstriction. Reagents with two pairs of reaction sites joined by a rigid link connect and cross-link two hemoglobins. The resulting bis-tetramers lack the cooperativity of the native protein and bind oxygen too tightly to be useful; occupation by oxygen blocks the sites from nitric oxide. Nitric oxide may be delivered from thionitrosyl groups, which occur in hemoglobin in the red cell. Cross-linked hemoglobin can be specifically nitrosylated. These species can then serve as circulating sources of nitric oxide resulting from an internal electron transfer.Résumé : Dans la circulation sanguine, l'hémoglobine transporte l'oxygène à l'intérieur des cellules rouges, mais à l'extérieur elle ne fonctionne pas parce qu'elle n'est pas en mesure de libérer l'oxygène et, de plus, elle se dissocie en dimères qui forment la protéine tétramère. Les agents acylants anioniques bifonctionnels qui contiennent un pont structuralement rigide permettent d'introduire des liens de réticulation qui stabilisent l'hémoglobine et qui altèrent son affinité pour l'oxygène de façon à la rendre utilisable pour le transport d'oxygène à l'extérieur des cellules. L'oxyde nitrique se lie à l'hémoglobine et dans la circulation ce qui provoque des augmentations indésirables de la pression sanguine. Il a été rapporté que des collections d'hémoglobines de masses moléculaires plus élevées ne provoquent pas de vasoconstriction. Des réactifs avec deux paires de sites réactionnels liés par une jonction rigide permettent de raccorder et de réticuler deux hémoglobines. Les bis-tétramères qui en résultent ne donnent pas lieu à la coopération qui existe dans la protéine naturelle et ils se lient trop fortement à l'oxygène pour être utiles, mais ils ne peuvent donc pas se lier à l'oxyde nitrique. Une extension des raccordements pourrait permettre de résoudre ces problèmes. L'oxyde nitrique peut être obtenu à partir de groupes thionitrosyles que l'on retrouve dans l'hémoglobine des cellules rouges. Il est possible d'effectuer une nitrosilylation spécifique de l'hémoglobine réticulée. Ces espèces servent de sources circulantes d'oxyde nitrique et elles résultent d'un transfert d'électron interne.

Cross-Linked Bis-hemoglobins: Connections and Oxygen Binding

“…The bis-tetramers are possible alternatives to nonspecifically linked oligomers in red cell substitutes. Research with PEG-Hb's has shown that an increase in the overall size of the species results in prolonged circulation life and minimizes increases in mean arterial blood pressure. , In addition, the bis-tetramers are good candidates for studying the relationship of structure to function in oxygen carriers. , The idea that cross-linked bis-tetramers might be less vasoactive in circulation than cross-linked tetramers derives from analyses of heterogeneously cross-linked hemoglobins that have been used in clinical trials. − The oxygen-binding properties of the individual species in such mixtures have not been determined, whereas those of the purified cross-linked tetramers in other trials have been measured . Our studies reveal that cross-linked bis-tetramers have higher oxygen affinities and decreased cooperativity compared to the clinically tested mono-tetramers.…”

“…In cases where oxygen-binding properties of red cell substitutes had been rationally produced, cooperative, low oxygen affinity species were sought to maximize oxygen delivery …”

“…18,19 In addition, the bis-tetramers are good candidates for studying the relationship of structure to function in oxygen carriers. 20,45 The idea that cross-linked bis-tetramers might be less vasoactive in circulation than crosslinked tetramers derives from analyses of heterogeneously crosslinked hemoglobins that have been used in clinical trials. [8][9][10][11][12] The oxygen-binding properties of the individual species in such mixtures have not been determined, whereas those of the purified cross-linked tetramers in other trials have been measured.…”

“…In cases where oxygen-binding properties of red cell substitutes had been rationally produced, cooperative, low oxygen affinity species were sought to maximize oxygen delivery. 45 However, such species would also be most able to acquire nitric oxide. Clinical trials have been conducted for situations where a small fraction of the total blood volume is replaced.…”

Modern cross‐linking strategies for synthesizing acellular hemoglobin‐based oxygen carriers

“…Before these results were known, cross‐linking reagents were engineered to target specific amino acid residues on the Hb molecule 31–33. In 2001, Kluger34 asked, “Can hemoglobin be chemically altered so that it can effectively deliver oxygen and remain in circulation outside the red cell?”. Kluger's group thus began their efforts to selectively introduce crosslinks in the cationic amino group regions of the Hb molecule.…”