开环棉籽糖分子内交联血红蛋白及其戊二醛的二次交联

用高碘酸钠氧化制备开环棉籽糖(O-Raffinose),将其作为交联剂对脱氧态无基质血红蛋白(Stroma-freehemoglobinSFHb)进行修饰.十二烷基磺酸钠-聚丙烯酰胺不连续电泳(SDS-PAGE)结果显示,开环棉籽糖交联血红蛋白(O-Ra-Hb)与SFHb相比抗解聚性有明显提高;琼脂糖(Sepharose)凝胶层析结果显示,SFHb和O-Ra-Hb出峰时间和峰形大小基本相同,说明交联反应主要发生在分子内的亚基间,基本没有分子间交联产物.用戊二醛(GDA)在优化条件下对O-Ra-Hb进行分子间二次交联,SDS-PAGE结果显示,二次交联产物稳定性比O-Ra-Hb有了进一步提高;Sepharose凝胶层析结果表明,产物中分子量为65000的未聚合的O-Ra-Hb组分为22.6%,分子量为130000～600000的组分为76.9%,分子量为600000以上的组分仅为0.5%,达到了预期目的.     

Preparation and in vitro characteristics of a blood substitute based on pyridoxylated polyhemoglobin

Stroma-free hemoglobin (SFHb) is prepared from outdated blood. Red cells are washed, lysed in hypotonic phosphate buffer, and stromal lipid is then removed by toluene extraction and high-speed centrifugation. Pyridoxal-phosphate (PP) added in a 4∶1 molar ratio to deoxygenated SFHb, is covelently linked across the polyphosphate binding site of the Hb tetramer by reduction with NaBH₄ under N₂ for 18 h. Excess reagents are removed by dialysis. Subsequent crosslinking using 5.0% glutaraldehyde in the presence of lysine for 12–36 h yields soluble, macromolecular polyhemoglobin (PolyHb). Progress of the reaction is monitored by gel chromatography. In vitro work shows that different molecular weight (MW) PolyHb can be produced, ranging in weight from 130,000 to over 1,000,000. Intermediate MW PolyHb (<600,000) remains stable for months at 4°C, withstands prolonged incubation at 37°C, and has a viscosity similar to blood. SFHb has a lowP ₅₀=15 torr, which has been raised to 26 torr by pyridoxylation. When PP-SFHb is crosslinked into PP-PolyHb, it can reversibly carry oxygen with a lower affinity (P ₅₀=16 torr) than nonpyridoxylated PolyHb (P ₅₀=11 torr). These favorable physicochemical properties of PP-PolyHb suggest that further progress toward a potential artificial blood substitute has been made.