Intramolecular cross-linking of oxy hemoglobin by bis sulfosuccinimidyl suberate and sebacate

Generation of cross-linked hemoglobin with reduced oxygen affinity

B. N. Manjula, P. K. Smith, A. Malavalli, A. S. Acharya

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 4°C 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 'psuedocrosslinked 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.

Original languageEnglish (US)
Pages (from-to)311-318
Number of pages8
JournalArtificial Cells, Blood Substitutes, and Immobilization Biotechnology
Volume23
Issue number3
StatePublished - 1995

Fingerprint

Hemoglobin
Crosslinking
Hemoglobins
Oxygen
Esters
Cross-Linking Reagents
Blood Substitutes
Dicarboxylic Acids
Freezing
Fatty Acids
Acids
bis(sulfosuccinimidyl)suberate
bis(sulfosuccinimidyl) sebacate
Chemical elements
Conformations
Stabilization
sebacic acid
suberic acid

ASJC Scopus subject areas

  • Biotechnology
  • Biomedical Engineering
  • Biomaterials
  • Hematology

Cite this

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title = "Intramolecular cross-linking of oxy hemoglobin by bis sulfosuccinimidyl suberate and sebacate: Generation of cross-linked hemoglobin with reduced oxygen affinity",
abstract = "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 4°C 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 'psuedocrosslinked 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.",
author = "Manjula, {B. N.} and Smith, {P. K.} and A. Malavalli and Acharya, {A. S.}",
year = "1995",
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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, B. N.

AU - Smith, P. K.

AU - Malavalli, A.

AU - Acharya, A. S.

PY - 1995

Y1 - 1995

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 4°C 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 'psuedocrosslinked 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 4°C 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 'psuedocrosslinked 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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VL - 23

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EP - 318

JO - Artificial Cells, Nanomedicine and Biotechnology

JF - Artificial Cells, Nanomedicine and Biotechnology

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