Functional Cross-Linked Hemoglobin Bis-tetramers: Geometry and Cooperativity

Abstract

Hemoglobin-based oxygen carriers have been sought as stable, sterile alternatives to red cells in transfusions. Problems in clinical trials using cross-linked tetramers have led to proposals that larger assemblies of tetramers may alleviate some of the problems. A study of such assemblies requires materials with defined structures and physical properties. Evaluation of cross-linked bis-tetramers with inflexible linear links between the tetramers revealed that these have very low cooperativity in oxygen binding and would thus be inefficient as oxygen carriers. New, more flexible reagents were designed to cross-link and connect tetramers in two modes: with angular connectors that permit torsional movement (1-3) and with linear connectors that resemble previously studied systems (4-6). The resulting cross-linked bis-tetramers were produced in high yield and were isolated and characterized. Digest mapping showed that modifications were specifically introduced as expected at amino groups in the 2,3-bisphosphoglycerate binding sites within beta subunits. Circular dichroism showed that the secondary structure of the globin chains is maintained while the microenvironment of the hemes is altered. The bis-tetramers derived from 1-3 have oxygen affinity (P(50) = 3.6-4.7) and cooperativity (n(50) = 2.2-2.7) that appear to be suitable for efficient oxygen delivery to hypoxic regions along with increased mass that is expected to minimize extravasation.

Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives

“…This has previously been achieved in our lab (71)(72)(73). Tetrafunctional cross-linkers react selectively within hemoglobin tetramers while also creating inter-protein linkages between tetramers (71-73) to give cross-linked bis-tetramers of hemoglobin (BT-Hb) (Scheme 3.1).…”

“…The high purity of these compounds comes from the specific acylation reactions within the BPG binding site of the protein. The di-bromosalicyl (DBS) leaving groups of the cross-linker have negative charges that direct reaction specifically to the cationic BPG site, where the lysine-82 can react with electrophilic ester groups to form stable amide bonds (71,74). This site-specific cross-linking overcomes problems with heterogeneity and eliminates the presence of dissociated αβ-dimers (71,74).…”

“…The di-bromosalicyl (DBS) leaving groups of the cross-linker have negative charges that direct reaction specifically to the cationic BPG site, where the lysine-82 can react with electrophilic ester groups to form stable amide bonds (71,74). This site-specific cross-linking overcomes problems with heterogeneity and eliminates the presence of dissociated αβ-dimers (71,74). In addition, allosteric cooperativity is retained due to tetrameric stabilization and the inter-protein linkage effectively doubles the size of the hemoglobin molecule.…”

“…Homogeneous solutions of hemoglobin bis-tetramers (BT-Hb) can be produced efficiently to give materials with well-defined structural integrity (71). BT-Hb has an increased molecular size while retaining appropriate oxygen affinity and cooperativity.…”

“…Because of the extensive purification procedure to obtain pure bis-tetramers of hemoglobin, there were no unmodified tetrameric species present in the final product that was used in animal testing (Figure 4.2). The chemical reaction to produce bis-tetramers is subject to a competing hydrolysis reaction, and the reaction of the functional cross-linker with hemoglobin gives only 40 % BT-Hb (71,74). The rest was largely comprised of cross-linked hemoglobin that is not tethered through an inter-protein linkage.…”