TY - JOUR
T1 - Intramolecular cross-linking of oxy hemoglobin by bis sulfosuccinimidyl suberate and sebacate
T2 - Generation of cross-linked hemoglobin with reduced oxygen affinity
AU - Manjula, Belur N.
AU - Smith, Paul K.
AU - Malavalli, Ashok
AU - Seetharama Acharya, A.
N1 - Funding Information:
This work was supported by National Institutes of Health Grant HL-38665 and a Grant-in-Ad from American Heart Association, National. The encouragement By Dr. R. L. Nagel to pursue these studies and the facilities extended to us is very much appreciated.
PY - 1995/1/1
Y1 - 1995/1/1
N2 - The sulfosuccinimidyl esters of suberic and sebacic acids readily introduce intramolecular crosslinks into oxy HbA at pH 7.4, the relative efficiency of crosslinking by the suberate ester being slightly higher than that of sebacate. Nearly quantitative intramolecular crosslinking of HbA (0.5 mM) is achieved at pH 7.4 and 4C by using 5 and 10 fold molar excess of the suberic and sebacic acid, respectively. In contrast to the facile crosslinking reaction seen with the bis sulfosuccinimidyl sebacate, bis sulfosuccinimidyl sebacate and bis (3:5 dibromo salicyl) sebacate did not introduce any crosslinking into HbA despite the fact that the ‘crosslinking arm’ of the two bifunctional reagents is the same. The discrepant reactivity of the two reagents demonstrates the ‘steering’ influence of the negative charge of the leaving group of the reagent, namely sulfo succinimidyl moiety to specific domains of HbA rich in positively charged groups. A second advantage is also anticipated in the use of the sulfosuccinimidyl esters of aliphatic dicarboxylic acids. We speculate that the intermediate in the crosslinking reaction mimics the structural aspects of the low oxygen affinity ‘psuedo crosslinked Hb’. Conversion of the low oxygen affinity ‘psuedocrosslinked Hb’ into crosslinked Hb by the formation of the second isopeptide bond may lead to the ‘freezing in’ of the elements of low oxygen affinity structure. Consistent with this speculation, the suberate crosslinked Hb indeed exhibited low oxygen affinity even though the crosslinking reaction was carried out in the oxy state. The need of only oxy conformation for nearly quantitative intramolecular crosslinking of HbA makes this crosslinking approach, an attractive tetramer stabilization procedure for the large scale production of hemoglobin based blood substitute.
AB - The sulfosuccinimidyl esters of suberic and sebacic acids readily introduce intramolecular crosslinks into oxy HbA at pH 7.4, the relative efficiency of crosslinking by the suberate ester being slightly higher than that of sebacate. Nearly quantitative intramolecular crosslinking of HbA (0.5 mM) is achieved at pH 7.4 and 4C by using 5 and 10 fold molar excess of the suberic and sebacic acid, respectively. In contrast to the facile crosslinking reaction seen with the bis sulfosuccinimidyl sebacate, bis sulfosuccinimidyl sebacate and bis (3:5 dibromo salicyl) sebacate did not introduce any crosslinking into HbA despite the fact that the ‘crosslinking arm’ of the two bifunctional reagents is the same. The discrepant reactivity of the two reagents demonstrates the ‘steering’ influence of the negative charge of the leaving group of the reagent, namely sulfo succinimidyl moiety to specific domains of HbA rich in positively charged groups. A second advantage is also anticipated in the use of the sulfosuccinimidyl esters of aliphatic dicarboxylic acids. We speculate that the intermediate in the crosslinking reaction mimics the structural aspects of the low oxygen affinity ‘psuedo crosslinked Hb’. Conversion of the low oxygen affinity ‘psuedocrosslinked Hb’ into crosslinked Hb by the formation of the second isopeptide bond may lead to the ‘freezing in’ of the elements of low oxygen affinity structure. Consistent with this speculation, the suberate crosslinked Hb indeed exhibited low oxygen affinity even though the crosslinking reaction was carried out in the oxy state. The need of only oxy conformation for nearly quantitative intramolecular crosslinking of HbA makes this crosslinking approach, an attractive tetramer stabilization procedure for the large scale production of hemoglobin based blood substitute.
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U2 - 10.3109/10731199509117947
DO - 10.3109/10731199509117947
M3 - Article
C2 - 7493052
AN - SCOPUS:0029044107
SN - 1073-1199
VL - 23
SP - 311
EP - 318
JO - Artificial Cells, Blood Substitutes, and Biotechnology
JF - Artificial Cells, Blood Substitutes, and Biotechnology
IS - 3
ER -