Study On Polyethylene Glycol(PEG)-Conjugation Of Various Hemoglobin As Novel Blood Substitutes

Synthesizing conjugations of polyethylene glycol (PEG) to bovine homeglobin (bHb) including porcine and human homeglobin , which can reduce antigenicity and prolong vascular retention time in circulation is a promising way to manufacture blood substitute. In this research, the approach of theoretical analysis of protein structure and computer-assisted design was firstly applied to select the potential conjugating site and judge the possibility of PEG-conjugation, and then the physicochemical properties of conjugates were examined, the molecular mechanism and function of conjugates, were studied finally.Our results demonstrated that twelve and thirteen lysine residues located on surface of αβ dimer of bovine Hb and porcine Hb respectlivey. were potential conjugating sites which had higher activities ,and that PEG whose molecular weight is 5000 doltans by proper linker could specifically bind to ε-amino group of lysines, which resulted in the conjugates whose molecular weight of is over 64KD. Moreover , PEG5000 with linker covalently binding to lysine residues of bHb could not influence the structure of oxygen binding in bHb/pHb.A simple and efficient procedure for purifying bHb from bovine blood has been established. The purification steps include: removing red blood cells(RBC) by centrifuge; disruption of RBC by phosphate buffer; anion-exchange chromalography on Q-sepharose. The electrophotogram of bHb revealed a single band on PAGE being in different concentration and PG-PAGE. That the basic structure of purified Hb was not changed was demonstrated by spectroscopy methods , namely Raman spectrum and scanning spectrum.The fractionation of PEG-bllb conjugates was preferably carried out by DEAE-Spherodex, an strongly anion exchange medium which is capable of selectively binding those conjugates having a molecular weight of less than 64KD, namely a degree of substitute of less than six PEG-conjugates per ap dimer of bllb because the conjugated lib molecular of various degrees of substitution will have different isoelectric point.The toxicity and ability of carrying oxygen of conjugates were compared by using rat models of hemorrhagic. The results showed that the conjugates of PEG-bovine hemoglobin had not obviously mortal toxicity simultaneously ameliorated MAP together with heart rate of hypovolemic rats to certain extent.The studies under development showed that the structure-based,computer-assisted design of conjugation of hemoglobin same conjugation may represent prospective leads for the development of blood substitutes,and that eventually prove to have important role in transfusion medicine.