Intramolecular Cross-Linking of Oxy Hemoglobin by<i>BIS</i>Sulfosuccinimidyl Suberate and Sebacate: Generation of Cross-Linked Hemoglobin with Reduced Oxygen Affinity

Manjula, Belur N.; Smith, Paul K.; Malavalli, Ashok; Acharya, Asha

doi:10.3109/10731199509117947

Cited by 5 publications

(1 citation statement)

References 3 publications

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“…All these reactions have been carried out in the deoxy state. Manjula et al (1995) introduced the use of sulfosuccinimidyl suberate and sulfosuccinimidyl sebasate as affinity directed intramolecular crosslinkers for generating an intramolecular crosslink within the bb cleft of Hb in the oxy state. The negatively charges of sulfo groups in these crosslinkers facilitates the affinity directed noncovalent interactions of the crosslinkers at the bb-cleft in the oxy conformation of Hb prior to the formation of the crosslink.…”

Section: Zero Length Intermolecular Crosslinkingmentioning

confidence: 99%

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

Acharya

Intaglietta

Tsai

et al. 2013

Hemoglobin-Based Oxygen Carriers as Red Cell Substitutes and Oxygen Therapeutics

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Section: Zero Length Intermolecular Crosslinkingmentioning

confidence: 99%

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

Acharya

Intaglietta

Tsai

et al. 2013

Hemoglobin-Based Oxygen Carriers as Red Cell Substitutes and Oxygen Therapeutics

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Perturbation of the Intermolecular Contact Regions (Molecular Surface) of Hemoglobin S by Intramolecular, Low-O2-Affinity-Inducing Central Cavity Cross-Bridges

Malavalli

et al. 2000

J Protein Chem

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The general assumption among researchers on hemoglobin is that the intramolecular central cavity cross-bridging of Hb does not result in any generalized perturbations at the protein surface. A corollary of this is that central cavity cross-bridges are unlikely to influence the polymerization of deoxy HbS, since polymerization is a protein surface phenomenon involving the participation of multiple protein surface amino acid residues. In an attempt to evaluate this experimentally, we have introduced two low-O2-affinity-inducing central cavity cross-bridges into HbS, beta(beta)-sebacyl [between the two Lys-82(beta) residues] and alpha(alpha)-fumaryl [between the two Lys-99(alpha) residues], and investigated their influence on the polymerization of the deoxy protein. The O2 affinities of the cross-bridged HbS exhibited sensitivity toward the buffer ions and pH in a cross-link-specific fashion. The modulation of the O2 affinity of these cross-bridged HbS in the presence of allosteric effectors, DPG and L-35, is also very distinct, reflecting the differences in the conformational features these two cross-bridges induce within the central cavity at the respective effector-binding domains. In addition, the alpha(alpha)-fumaryl cross bridge inhibited the polymerization, reflecting the perturbation of the microenvironment of one or more intermolecular contact residues, protein surface residues, as a consequence of the central cavity cross-bridge. On the other hand, the beta(beta)-sebacyl cross-bridge exerted a slight potentiating effect on the polymerization of HbS. This reflects the fact that the perturbations at the protein surface are limited and favor polymerization. The results presented demonstrate that the structural changes induced by the central cavity cross-bridges are very specific and not simply restricted to the sites of modification, but are propagated to distant sites/domains, both within and outside the central cavity. It is conceivable that other surface regions that are not involved in the polymerization could also experience similar structural/conformational consequences. These results should be taken into consideration in designing intramolecularly cross-bridged asymmetric hybrid HbS for mapping the contribution of the intermolecular contact residues in the cis and trans dimers of deoxy HbS during polymerization.

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Probing the Conformation of Hemoglobin Presbyterian in the R-State

et al. 2003

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The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108(beta)->Lys mutation-induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the alpha1beta1 and alpha1beta1 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(alpha) of Tg-HbP, a residue of the alpha1beta1 interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule. First, it facilitates the generation of deoxy (T)-state marker at 14.2 ppm (from 2,2-dimethyl-s-silapentane-5-sulfonate) on the interaction of oxy Hb with inositol hexa-phosphate without changing the ligation state. Second, it increases the geminate yield of the 10 ns photoproduct of CO-Hb. Third, it enhances the propensity of phosphate to increase the geminate yield. Fourth, it potentiates the ability of phosphate to induce deoxy-like features at the heme environment in the R-state. Fifth, it induces T-state-like signatures at the switch and hinge regions of the alpha1beta2 interface. Finally, molecular modeling studies have indicated an increased affinity for the four anion binding sites mapped in the midcentral cavity of Hb caused by the presence of Lys-108(beta). In short, Lys-108(beta) in HbP induces a propensity for oxy Hb to access T-like conformational features in different regions of the oxy Hb molecule and also enhances the T-like signatures in the oxy state on interaction with allosteric effectors without changing its ligation. Interestingly, the intrinsic T-like conformational features of the R-state of HbP, in addition to those induced by the addition of allosteric effectors to liganded HbP, appear to be reminiscent of features of the B-state conformation of Hb found in rHb 1.1 (recombinant hemoglobin). We propose that the lowered oxygen affinity of Tg-HbP in the presence of allosteric effectors is a consequence of an altered R-state conformation of Hb, which reflects the facilitation of switching the R-state of HbP to the T-state compared with the normal R-state of HbA, thereby reducing HbA's affinity to oxygen.

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Intramolecular Cross-Linking of Oxy Hemoglobin byBISSulfosuccinimidyl Suberate and Sebacate: Generation of Cross-Linked Hemoglobin with Reduced Oxygen Affinity

Cited by 5 publications

References 3 publications

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

Perturbation of the Intermolecular Contact Regions (Molecular Surface) of Hemoglobin S by Intramolecular, Low-O2-Affinity-Inducing Central Cavity Cross-Bridges

Probing the Conformation of Hemoglobin Presbyterian in the R-State

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