Acetylated Hemoglobin Bis-Tetramers: From Chemical Designs to Physiological Impacts

Cross-linked human hemoglobins have been evaluated for clinical use as circulating oxygen carriers. However, their induction of vasoactivity was sufficiently problematic to lead to the cessation of clinical trials. The source of vasoactivity is likely to be endothelial extravasation causing the scavenging of endogenous nitric oxide. It was recently shown that species that consist of two coupled hemoglobin tetramers do not evoke vasoactivity in a sensitive murine model. Presumably these materials are too large to extravasate. In order to make this class of material more readily available, there is a need for improved methods that can form a cross-linked bis-tetramer without producing smaller species at the same time. A potentially efficient route to cross-linking and coupling two Hb tetramers is through phase-directed coppercatalyzed azide alkyne cycloaddition (PDCuAAC). However, introduction of the necessary azide-containing cross-link gives mixtures of tetrameric and bis-tetrameric proteins, as the PDCuAAC process appears to be limited to only those proteins where the cross-link containing the azide is exclusively within the β-subunits. In order to block formation of the azide cross-link within the α-subunits, subunit-specific introduction of the azide is necessary. This is achieved by blocking reaction at the reactive amino groups of the β-subunits in the site that binds the allosteric activator 2,3-diphosphoglycerate (DPG) with inositol hexaphosphate (IHP), permitting α-selective acetylation with acetyl 3,5-dibromosalicylate. After removal of IHP, reaction with an anionic crosslinker containing an azide group occurs within the β-subunits. The resulting α-acetylated β−β′-cross-linked hemoglobin azide (acHb>-N3) undergoes efficient PDCuAAC with bis-alkynes to produce cross-linked bis-tetramers. Analysis of circular dichroism spectra of the modified species shows that there is little change in the structure of the globin chains as a result of the chemical modifications. The oxygenation properties are consistent with those needed for effective oxygenation in circulation, while the bistetrameric structure is sufficiently large to avoid extravasation and depletion of nitric oxide. T potential utility of an acellular oxygen-carrier as an alternative to red cells in transfusions led to the design and production of various chemically altered derivatives of human hemoglobin (Hb). In acellular circulation, the native protein dissociates from a functional tetrameric (α2β2) state into nonfunctional αβ dimers. Introduction of chemical crosslinks within Hb permanently stabilizes the tetrameric form while producing desirable oxygenation properties. Reports of clinical evaluations of cross-linked tetramers in circulation revealed their tendency to induce significant hypertension. The observed vasoactivity is likely to be associated with extravasation of the tetramers into the endothelia and scavenging of nitric oxide, which signals relaxation of the blood vessels. Constricted vessels would be expected to lead to cardiac stress. The observation of these side effects led to termination of clinical trials for potential HBOCs. On the basis of this understanding, an acceptable design for an oxygen carrier from hemoglobin must overcome extravasation as well as transport oxygen. It is possible that increasing the size of the cross-linked Hb derivative should resist extravasation. It appears likely that connecting two hemoglobins would produce a species that would not extravasate. The initial route to a crosslinked bis-tetramer utilized tetrafunctional reagents that consist of linked pairs of conventional site-directed cross-linking reagents. The resulting bis-tetramers bind oxygen reversibly with appropriate affinity and cooperativity. Zapol and coworkers evaluated one example in circulation in mice whose condition is critically dependent on the availability of nitric oxide. They noted that in accord with expectations the materials did not evoke symptoms associated with vasoactivity. On the basis of those positive observations with a highly purified cross-linked bis-tetramer, the general approach seems promising, unlike alternatives that are mixtures from less specific approaches that can also contain unlinked hemoglobin αand β-subunits, αβ-dimers, and single tetramers, cross-linked or not. The approach used to produce the tested bis-tetramer from tetra-esters is affected by competing spontaneous hydrolysis, Received: August 21, 2014 Revised: October 2, 2014 Published: October 7, 2014 Article pubs.acs. org/biochemistry © 2014 American Chemical Society 6793 dx.doi. org/10.1021/bi501054b | Biochemistry 2014, 53, 6793−6799