Crosslinking Hemoglobin With Ethylene Glycol Diglycidyl Ether For Preparation Of A Red Blood Cell Substitute

Ethylene glycol diglycidyl ether (EGDE) was developed as a protein crosslinker to prepare crosslinked bovine hemoglobin(bHb), which had the potential to be a red blood cell substitute. Reaction conditions such as temperature, pH, the molar ratio of EGDE to bHb, bHb concentration, reaction time had significant influence on bHb crosslinking. To identify reactive groups on bHb, we developed a method to distinguish different reactive groups. The –SH of bHb mainly reacted with EGDE under pH7.5. The –NH2 of bHb mainly reacted with EGDE under pH8.7-9.5. The product was determined by SDS-PAGE and multi-angle laser light scattering (MALLS). It showed that EGDE could be used either as an intramolecular crosslinker or as an intermolecular crosslinker depending on the reactive conditions. When the bHb concentration was less than 20mg/ml, 35℃,pH8.7,and the reactive time was no more than 4 hours, 90% of the product was intramolecular crosslinked hemoglobin under the reactant ratio of 100:1(EGDE to bHb). Increasing bHb concentration up to 200mg/ml and prolonging the reactive time to 12h while decreasing the reactant ratio to 25:1 and adjusting pH to 7.5, intermolecular crosslinked product was formed. Intermolecular crosslinked bHb had a narrow distribution between 140kD and 380kD. The mixture contained 74.7% of dimer, 18.9% of trimer and 6.4% of oligomerPDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.comrespectively. Animal experiments with rats showed their biological safety. The characterizations of intramolecular crosslinked bHb and intermolecular crosslinked bHb, including oxygen carrying ability, PI, the stability , UV-VIS scan analysis , the secondary structure analysis by FTIR spectroscopy were researched.