Synthesis, characterization, and modeling of novel bovine hemoglobin-based oxygen carriers

Second generation hemoglobin-based O2 carriers (HBOCs) are being developed with high O2 affinity (low P50) in order to suppress vasoconstriction elicited by over-oxygenating tissues, a problem associated with low O2 affinity first generation HBOCs. Two novel reactive dialdehydes were synthesized by ring-opening 2-chloroethyl-Beta-D-fructopyranoside (2-CEFP) and 1-o-octyl-Beta-D-glucopyranoside (1-OGP) at the 1,2-diol position, respectively, to yield novel Hb polymerizing reagents. High-affinity polymerized HBOCs were synthesized by reacting R-state bovine hemoglobin (bHb) with ring-opened 2-CEFP and 1-OGP at cross-linker to bHb molar ratios ranging from 10:1 to 30:1. The resulting polymerized bovine HBOCs (bHBOCs) displayed P50s ranging from 7-18 mmHg, cooperativities ranging from 0.8-1.4, and methemoglobin (metHb) levels ranging from 3-10%. The cross-linking reaction also stabilized the third stepwise Adair coefficient for 1-OGP20, 1-OGP30, and 2-CEFP30. Additionally, the number-averaged molecular weight of each polymerized bHBOC was larger compared to bHb. bHb was purified from bovine red blood cells (bRBCs) via anion exchange chromatography preceded by dialysis. Q Sepharose XL, a strong anion exchange resin, was found to have twice the binding capacity for bHb compared to three other anion exchange resins that were studied in this work. MetHb levels remained below 2% with bHb concentrations between 0.7 and 1.7 mM. The effect that increasing the cross-linker to bHb molar ratio to 100:1 had on metHb levels and size distributions was further studied in detail. Purified bHb was found to elute from a size exclusion chromatography column as a dimer. bHBOCs synthesized with ring-opened 1-OGP and 2-CEFP each showed an increase in bHb species having both decreased and increased Mw distributions compared to bHb as the ring-opened 1-OGP to bHb molar ratio increased. However, for bHBOCs synthesized with 2-CEFP, the Mw distributions at cross-linker to bHb molar ratios between 50:1 and 70:1 remained constant. Furthermore, bHBOCs synthesized with 2-CEFP created Mw species centered at 63.6 and 96 kDa, correlating to intact tetrameric bHb and tetrameric bHb bound to a bHb dimer, respectively. Autoregulatory theory has been presented as a possible explanation for increased vasoconstriction observed in the transfusion of low-affinity HBOCs, where it is hypothesized that low-affinity HBOCs over-deliver O2 to tissues surrounding capillaries thereby eliciting vasoconstriction. An a priori model has been developed in which the performance of mixtures of acellular HBOCs (having P50s between 5 and 55 mmHg) and hRBCs has been simulated using a Krogh tissue cylinder model. At normoxic inlet pO2s, there was no correlation between O2 flux out of the capillary and the O2 affinity of the HBOC. However, a correlation was found between the average pO2 tension in the capillary and the O2 affinity of the HBOC.