Optimizing the Reaction Process of Cross-linking Ethylene Glycol Diglycidyl Ether with Hemoglobin

Ethylene glycol diglycidyl ether (EGDE) was used as across-linker for preparation of cross-linked hemoglobin (Hb) which is considered to be used as a candidate of red blood cell substitute. The products of cross-linking reaction were monitored and analyzed by SDS-PAGE, size exclusion HPLC and multi-angle laser light scattering (MALLS). Results demonstrate that 90% of the product could be intramolecularly cross-linked Hb when the reaction is carried out at pH 8.7 and Hb 20 mg?mL?1, molar ratio of EGDE to Hb being 100:1, and reaction for 4 h. Adjusting pH to 7.5,increasing Hb concentration up to 200 mg·mL-1 while decreasing the reactant ratio (EGDE to Hb)to 25:1, prolonging the reactive time to 12 h, intermolecularly cross-linked product is formed. The molecular weight distribution of intermolecular cross-linked Hb is between 145 kD and 375 kD. The mixture contains 74.7% of dimer, 18.9% of trimer and 6.4% of oligomer respectively. Both intramolecularly cross-linked Hb and intermolecularly cross-linked Hb demonstrate oxygen carrying abilities. Animal experiments with rats show that the cross-linked Hb prepared has no toxicity effect.