乙二醇二缩水甘油醚交联血红蛋白的反应优化

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

将乙二醇二缩水甘油醚（EGDE）作为血红蛋白（Hb）的交联剂制备红细胞代用品.采用SDS-PAGE电泳、高效液相色谱和多角度激光散射检测器联用鉴定交联产物.实验表明,在pH 8.7,血红蛋白浓度为20 mg·mL^-1,修饰剂EGDE与血红蛋白的摩尔比达100：1时反应4 h,交联产物中90%为分子内交联的血红蛋白.在pH 7.5、血红蛋白浓度为200mg·mL^-1,修饰剂EGDE与血红蛋白的摩尔比为25：1时反应12 h,交联产物主要为寡聚血红蛋白,分子量范围在145-375 kD之间;其中二聚体血红蛋白占总蛋白量的74.7%,三聚体血红蛋白18.9%,四到六聚血红蛋白6.4%,几乎无高聚物生成.采用血氧分析仪对交联产物进行活性鉴定,制备的分子内交联和寡聚血红蛋白均具有携氧能力,小鼠的生物学实验表明产品无异常毒性,具备了作为红细胞代用品的可能性.

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 intramoleeularly cross-linked Hb when the reaction is carried out at pH 8.7 and Hb 20mg·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.mLl 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 intermoleeular 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 intrarnoleeularly cross-linked Hb and intermoleeularly cross-linked Hb demonstrate oxygen carrying abilities. Animal experiments with rats show that the cross-linked Hb prepared has no toxicity effect.