Michele Perrella

PEGylation Promotes Hemoglobin Tetramer Dissociation

Hemoglobin conjugated with poly(ethylene glycol) (PEG) acts as an oxygen carrier free in plasma, substituting red blood cells in supplementing oxygen in hypo-oxygenation pathologies. Given the complexity of oxygen delivery controls, subtle structural and functional differences in PEGylated hemoglobins might be associated with distinct physiological responses and, potentially, adverse effects. We have compared hemoglobin PEGylated under anaerobic conditions, called PEG-Hb(deoxy), with hemoglobin PEGylated under aerobic conditions, called PEG-Hb(oxy), a product that mimics Hemospan, produced by Sangart, Inc. SDS PAGE and MALDI-TOF analyses demonstrated that PEG conjugation yields products characterized by a broad distribution of PEG/hemoglobin ratios. The elution profiles in size-exclusion chromatography indicate that both products exhibit a more homogeneous distribution of molecular weight/hydrodynamic volume under deoxy conditions and at higher concentrations. PEG-Hb(oxy) shows high oxygen affinity, low modulation of allosteric effectors, almost no cooperativity, a fast and monophasic CO binding, and a limited dependence of functional properties on concentration, whereas PEG-Hb(deoxy) exhibits oxygen binding curves that significantly depend on protein concentration, and a slow CO binding, similar to native hemoglobin. PEGylated CO-hemoglobins, probed by flash photolysis, exhibited a lower amplitude for the geminate rebinding phase with respect to native hemoglobin and a negligible T state bimolecular CO rebinding phase. These findings are explained by an increased dissociation of PEGylated hemoglobins into dimers and perturbed T and R states with decreased quaternary transition rates. These features are more pronounced for PEG-Hb(oxy) than PEG-Hb(deoxy). The detected heterogeneity might be a source of adverse effects when PEGylated Hbs are used as blood substitutes.

Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration

Blood transfusion is still a critical therapy in many diseases, traumatic events and war battlefields. However, blood cross-matching and storage may limit its applicability, especially in Third World countries. Moreover, haemoglobin, which in red blood cells is the key player in the oxygen transport from lung to tissues, when free in the plasma causes hypertension and renal failure. This investigation was aimed at the development of a novel haemoglobin-based oxygen carrier with low vasoactivity and renal filtration properties. Human haemoglobin was chemically conjugated with polyethylene glycol (PEG) under either aerobic or anaerobic conditions, following different chemical procedures. The resulting PEGylated haemoglobin products were characterized in terms of oxygen affinity, cooperativity, effects of protons and carbon dioxide concentration, and oxidation stability, and were transfused into rats to evaluate vasoactivity and renal filtration. A deoxyhaemoglobin, conjugated with seven PEG and seven propionyl groups, which we called Euro-PEG-Hb, did not produce profound hypertension, was 99% retained within 6 h, and exhibited oxygen binding properties and allosteric effects more similar to human haemoglobin A than the other tested PEGylated haemoglobin derivatives, thus appearing a very promising candidate as blood substitute.

Isoelectric focusing and electrophoresis at subzero temperatures

Abstract Electrophoretic and isoelectric focusing separations have been achieved at −20 to −30°C, i.e., at temperatures considerably lower than previously reported by using as supporting media gels of acrylamide-methylacrylate copolymers and dimenthylsulfoxide-water mixtures. Hybrids of human and sickle cell hemoglobin and partially oxidized human carboxyhemoglobin have been separated in the temperature range −20 to −30°C, both by a discontinuous buffer gel electrophoresis and by isoelectric focusing.

[12] Detection of hemoglobin hybrid formation at subzero temperature

Publisher Summary This chapter discusses the detection of hemoglobin hybrid formation at subzero temperature. The study of hybrid species (asymmetric hybrids) in mixtures of unlike tetrameric hemoglobins yields significant information on the structure–function relationship of hemoglobin. The formation of hybrid molecules between hemoglobin S and minor hemoglobin components present in the red cells of sickle cell blood—such as hemoglobin A, hemoglobin A2, and hemoglobin F—is recognized as an important factor in the inhibition of hemoglobin S gelling promoted by these minor components. Intermediates in the reactions of hemoglobin with ligands or with oxidants can also be considered hybrid molecules. In this case, the dimers forming the tetrameric hybrid belong to the same hemoglobin species but exist in different liganded or valency states (ligand and valency hybrids). Hybridization reactions that occur via the dimerization of tetramers and subsequent reassociation of dimers are one possible source of instability of the asymmetric hybrid molecules. Standard chromatographic and electrophoretic methods are used for the isolation of stable valency and asymmetric hybrids. The ability to isolate hybrids by these methods depends on a favorable ratio between the rate of tetramer dissociation and the rate of protein separation.

The functional properties of sickle cell blood

It is known that whole blood from patients with sickle cell anemia has a decreased oxygen affinity [ 1,2]. Purified hemoglovin S solutions, however, are normal [3,4]. A systematic study of the effect of 2,3-DPG and of other known allosteric regulators of oxygen affinity of HbS in whole blood seems desirable, indeed essential, to understand the reason for the altered oxygen affinity of the hemoglobin S molecule in the intact red cell environment. A more complete understanding of the functional properties of SSblood would also be important to assess the physiological significance of such an alteration, and to evaluate the therapeutic effect of cyanate, administration.

Determination of the equilibrium constants for oxygen-linked CO2 binding to human hemoglobin

The binding of CO2 to the a-amino groups of hemoglobin as carbamate [l] contributes significantly to CO2 transport and is partly responsible for lowering the oxygen affinity of hemoglobin to its physiologic value [2] . Direct measurements at pH 7.4 of the CO* binding constant (X) of hemoglobin specifically modified by cyanate at the a-amino groups [3] (i.e., I@

The Bohr Effect of Hemoglobin Intermediates and the Role of Salt Bridges in the Tertiary/Quaternary Transitions

‘, a& and c&3

Understanding mechanisms in a cooperative protein: the CO ligation intermediates of hemoglobin

) have shown that the P-chains have a higher affinity than the a-chains for CO2 in Hb. In HbCO, however, both (Yand P-chains have been found to have the same CO2 affinity, although lower’than in Hb [4]. The pH-dependent h is related to [H’J as follows [2,5] :

[21] Low-temperature electrophoresis methods

Understanding mechanisms in cooperative proteins requires the analysis of the intermediate ligation states. The release of hydrogen ions at the intermediate states of native and chemically modified hemoglobin, known as the Bohr effect, is an indicator of the protein tertiary/quaternary transitions, useful for testing models of cooperativity. The Bohr effects due to ligation of one subunit of a dimer and two subunits across the dimer interface are not additive. The reductions of the Bohr effect due to the chemical modification of a Bohr group of one and two α or β subunits are additive. The Bohr effects of monoliganded chemically modified hemoglobins indicate the additivity of the effects of ligation and chemical modification with the possible exception of ligation and chemical modification of the α subunits. These observations suggest that ligation of a subunit brings about a tertiary structure change of hemoglobin in the T quaternary structure, which breaks some salt bridges, releases hydrogen ions, and is signaled across the dimer interface in such a way that ligation of a second subunit in the adjacent dimer promotes the switch from the T to the R quaternary structure. The rupture of the salt bridges per se does not drive the transition.

Subzero temperature quenching and electrophoretic methods for the isolation of protein reaction intermediates.

Hemoglobin is a regulatory component of the oxygen transport to the tissues, and for decades has been a prototype to develop new strategies for the study of the structure/function relationships in proteins. One of the most difficult, and so far, unattained objectives of hemoglobin research has been the study of the hemoglobin molecules in a state of partial ligation with oxygen, or intermediates, as a means of testing theories of cooperativity. A cryogenic technique has been developed for the isolation, identification and quantification of the reaction intermediates of hemoglobin and CO, which in many aspects is a close approximation to the physiological ligand. The technical features that are crucial for the evaluation of the significance of the experimental data obtained using this technique and various approaches to the analysis of the data are reported. The discussion points out the importance of accessing direct information on the nature and concentrations of the intermediates in solution to clarify mechanisms of cooperativity as opposed to the less informative studies of the bulk properties of the solution.