Preparation Of High-P50 Hemoglobin Oxygen Carriers And Preliminary Study Of Its Biological Effects Activity

Objectives:To study develop a simple method of preparation of high-P₅₀ hemoglobin oxygen carriers by oxidized raffinose,and to preliminary study its biological effects activity.Methods:Procine blood and bovine blood were used as the raw materials and ultra-pure hemoglobin was obtained through multiple purification steps;raffinose was oxidized by sodium-m-periodate,and then o-raffinose as cross-linker was directly modified without any further purification;by optimizing the reaction conditions such as oxidation time of Raf-finose,the ratio of raffinose to hemoglobin,the final concentration of hemoglobin,reaction temperature,the stability of reaction,choose the most convenient and optimized reaction conditions.The physical properties of the polymerized hemoglobin,such as the average molecular weight,oxygen affinity,and oxidation tendency of the polymerized hemoglobin,were detected by SEC-HPLC,UV detection,and oxygen dissociation curve analysis.The efficiency of oxygen release of two different types of heamoglobin oxygen carriers and the survival time when endogenous hemoglobin is less than 0.05g/dl(that is, more than 90%exchange transfusion with HBOCs),were obtained through the rat exchange transfusion model.Results:The simple method of raftinose cross-linked hemoglobin can be developed to prepare the HBOCs.Compared with classical methods,the main advantage is as follows:raffinose oxidation time may be shortened within 60min;oxidation of hemoglobin can be controlled when the reaction temperature is 20℃;the proportion of MetHb can be controlled less than 5%;the ratio of raffinose to hemoglobin and the final concentration were dependent on the average molecular weight of HBOCs;at the same time,we can obtain HBOCs of different molecular weights.The results of the rat change transfusion experiment showed that the efficiency of HBOCs' oxygen release in vivo is directly related to P₅₀,the efficiency of HBOCs with high P₅₀ is higher;the rat survival time after transfusion with high-P₅₀ HBOCs is up to 490 min when the endogenous hemoglobin is less than 0.05g/dl.Conclusion:The simplified method for developing raffinose polymerization hemoglobin is similar to the classic method,and with good reproducibility.Oxygen affinity of HBOCs by this method is similar to that of Hemosol's.And its efficiency of oxygen release is high in rat change transfusion.