Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute.
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 MgCl2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the two alpha 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 pl 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.
Lu
Xiuling
X
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, China.
Zheng
Chunyang
C
Xu
Yuhong
Y
Su
Zhiguo
Z
D000818:Animals; D001802:Blood Substitutes / Q000737:chemistry*; D001803:Blood Transfusion; D002417:Cattle; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry*; D006168:Guinea Pigs; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008027:Light; D008297:Male; D013058:Mass Spectrometry; D008956:Models, Chemical; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012542:Scattering, Radiation; D021241:Spectrometry, Mass, Electrospray Ionization; D013388:Succinimides / Q000494:pharmacology*; D013997:Time Factors
D001802:Blood Substitutes; D003432:Cross-Linking Reagents; D006454:Hemoglobins; D013388:Succinimides; C019358:disuccinimidyl suberate