Study Of Bovine Hemoglobin Modified By PEG As A Red Blood Cell Substitute

As the result of the rapid growth of blood demand, insufficiency of origin naturalblood, moreover the serious viral infection, the development of artificial red bloodcell substitutes that have oxygen-carrying ability has become an international hot spotof scientific research. Conjugating bovine Hemoglobin (bHb) with PEG can lengthenhemoglobin's half-life in vivo and reduce the kidney toxicity and immunogenicity ofbHb. Moreover, considering that there is abundant bovine blood, PEG-bHb is onepotential method for preparing red blood cell substitutes .This thesis which concerns about the surface-conjugating of bHb with PEG ismainly divided into three parts:First, two kinds of methods for the purification of bHb, method of CarbonMonoxide saturation and centrifuging method combining heating treatment, are testedto prepare hemoglobin from bovine blood. Results show that the method ofcentrifuging combining heating is more applicable to prepare hemoglobin. The bHbprepared in the lab has a higher purity than the standard Sigma bHb with 96% to 90%.Meanwhile, the yield rate is about 75%. The bHb prepared in the lab is digested insulphuric acid, and the lipids content is determined to be 0.606mg/ml by complexingwith Ammonium Molybdate and measuring the absorbance。In addition, colorimetryis used to determine the concentration of MetHb in routine purification of bHb.Second, a new route is used to synthesize SC-mPEG of which the structure isverified through NMR test .Third the yield rates of PEG-bHb under dissimilar conditions are studied andmore important , a series of methods used to characterize the PEG-bHb mixtures arebuilt. It is known from the experiment result that the yield rate is higher in pH8-10than other circumstance. There are quite some differences between the products ofPEG-bHb under different ratio of SC-mPEG to bHb(20-100:1). The higher the ratio ofSC-mPEG to hemoglobin, the more high-molecular PEG-bHb in the mixture ofproduct. Compared with reaction ratio and pH of reaction system, the reactiontemperature and time are not so important to the reaction.In view of the fact that it is not easy to get clear information about PEG-bHbthrough routine measurements, the method of TNBS is used to determine what theaverage conjugation rate of PEG-bHb is. Meanwhile GPC, which can separatesdifferent things according to their molecular weights, is used to analyze the products.From the results of GPC the detailed information of PEG-bHb in different molecularweight is acquired. In addition, it is found that the Hemoglobin tetramer keeps theirforms in the PBS system according to the GPC results. The Native-PAGE experimentprovides more "true" information about the PEG-bHb and enhances thecharacterization of PEG-bHb.The work of this thesis is meant to establish a research route and a series ofanalysis methods for the research of modification of hemoglobin with PEG and makea basis for further research on improving the PEG-bHb.