Disuccinimidyl suberate(DSS)intramolecularly cross-linked hemoglobin(Hb)was developed as a novel red blood cell substitute.A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to ...Disuccinimidyl suberate(DSS)intramolecularly cross-linked hemoglobin(Hb)was developed as a novel red blood cell substitute.A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb.SDS-PAGE was also used as a complement.It was proved that 83.8%of the product was intramolecularly cross-linked Hb with weight-average molecular weights(Mw)of 67.5 kD,12%was dimeric Hb with Mw of 146.6 kD,and 4.2%was trimeric Hb with Mw of 306.4 kD.The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCI2.Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the two a subunits within a Hb molecule,resulting in stabilization of the tetramer structure.However,the cross-linking was not site-specific.The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg,and the Hill coefficient decreased from 2.22 to 1.41.Result of isoelectric focusing showed that the pi of DSS cross-linked Hb was in the range of 4.6-5.2,similar to that of serum albumin.The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs.Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects.The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.展开更多
基金supported by the National Natural Science Foundation ofChina(Grant No.20136020)Key Project of Innovation Program by the ChineseAcademy of Sciences.
文摘Disuccinimidyl suberate(DSS)intramolecularly cross-linked hemoglobin(Hb)was developed as a novel red blood cell substitute.A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb.SDS-PAGE was also used as a complement.It was proved that 83.8%of the product was intramolecularly cross-linked Hb with weight-average molecular weights(Mw)of 67.5 kD,12%was dimeric Hb with Mw of 146.6 kD,and 4.2%was trimeric Hb with Mw of 306.4 kD.The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCI2.Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the two a subunits within a Hb molecule,resulting in stabilization of the tetramer structure.However,the cross-linking was not site-specific.The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg,and the Hill coefficient decreased from 2.22 to 1.41.Result of isoelectric focusing showed that the pi of DSS cross-linked Hb was in the range of 4.6-5.2,similar to that of serum albumin.The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs.Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects.The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.
