High O₂ affinity hemoglobin‐based oxygen carriers synthesized via polymerization of hemoglobin with ring‐opened 2‐chloroethyl‐β‐D‐fructopyranoside and 1‐o‐octyl‐β‐D‐glucopyranoside

Abstract

Second generation hemoglobin-based O(2) carriers (HBOCs) are being developed with high O(2) affinity (low P(50)) in order to suppress vasoconstriction elicited by over-oxygenating tissues, a problem associated with low O(2) affinity first generation HBOCs. Our group has previously investigated the polymerization of hemoglobin (Hb) with dialdehydes as a strategy for engineering high O(2) affinity HBOCs. In this study, 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 P(50)s ranging from 7 to 18 mmHg, cooperativities ranging from 0.8 to 1.4, and methemoglobin (metHb) levels ranging from 3% to 10%. The cross-linking reaction also stabilized the third stepwise Adair coefficient for bHbs reacted with ring-opened 1-OGP at cross-linker to bHb molar ratios of 20:1 and 30:1 and for bHbs reacted with ring-opened 2-CEFP at molar ratios of 30:1. Additionally, the number-averaged molecular weight, M(n), of each polymerized bHBOC was larger compared to bHb. Molecular weight distributions leaning towards larger molecular weight bHBOCs were obtained by increasing the cross-linker to bHb molar ratio. Taken together, the results of this study have identified novel Hb polymerization reagents that are easy to synthesize, and that are capable of yielding bHBOCs with higher O(2) affinities and weight-averaged molecular weights compared to bHb.

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

“…Empty chromatography columns XK 26/40 (400 mm in length, 26 mm I.D., GE Healthcare, Piscataway, NJ) were packed with the anion exchange resin Q-sepharose XL (GE Healthcare). Tris(hydroxymethyl)aminomethane, NaCl, NaOH, EDTA, Triton X-100, HCl, KCN, KFe(CN) 6 , dialysis tubing with a molecular weight cutoff of 6-8 kDa and all other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).…”

“…A variety of HBOCs exist and include: recombinant Hbs [4], cross-linked Hbs [5], polymerized Hbs [6][7][8][9], PEGylated Hbs [10], liposome encapsulated Hbs [11][12][13], polymersome encapsulated Hbs [14] and other particle encapsulated Hbs [15][16][17]. In order to synthesize and/ or formulate these HBOCs, a pure source of Hb is needed.…”

Purification of bovine hemoglobin via fast performance liquid chromatography

“…There are many different types of HBOCs that are currently in development, including polymerized acellular Hbs [3][4][5][6][7], liposome encapsulated Hbs [8][9][10], polymersome encapsulated Hbs [11], and hydrogel-based O 2 carriers [12][13][14]. Since each of these HBOCs require purified stroma-free Hb as the starting material, it is essential to develop an efficient and streamlined purification method of Hb to ensure that the starting product is of high purity and high yield.…”

Purification of hemoglobin by tangential flow filtration with diafiltration

“…RBC substitutes currently in development include liposome-encapsulated Hb2–5 and cross-linked/polymerized Hb 6–9. Several of these hemoglobin-based oxygen carriers (HBOCs), such as Hemopure (Biopure, Cambridge, MA)10,11 and PolyHeme (Northfield Labs, Evanston, IL)12 have shown some promise in clinical applications.…”