Photolyses of mammalian oxy-hemoglobin studied by nanosecond photoacoustic calorimetry.

Enthalpy and conformational volume changes in photolyses of oxy-hemoglobin (HbO(2)) of human, bovine, pig, horse and rabbit are investigated by photoacoustic calorimetry. In the experiment, a pulsed Nd:YAG laser is used as an exciting source, and a PVDF film transducer and a PZT transducer are used to detect the photoacoustic signals. Based on the time scales of the excitation and detection systems as well as the photolysis processes of HbO(2), it can be indicated that the measured enthalpy and conformational volume changes are related to slow geminate recombination and tertiary relaxation in photolyses of HbO(2), which are with the time scale of 30-40 ns and 100-150 ns, respectively. The results show that the enthalpy and conformational volume changes are different for both photolysis processes of HbO(2) and also for various mammals. The different results among the five mammals are analyzed and discussed briefly.

Enthalpy and conformational volume changes of mammalian oxy-hemoglobins investigated by pulsed photoacoustic calorimetry.

Enthalpy and conformational volume changes induced by laser photo-dissociation reactions of mammalian oxy-hemoglobin, such as human, bovine, pig, horse and rabbit oxy-hemoglobins, are investigated by pulsed photoacoustic calorimetry. Generally, the response time of the photoacoustic calorimetry is restricted by the width of the laser pulse and the bandwidth of the acoustic detector. Considering the time window of the experimental system, the enthalpy and conformational volume changes detected should be caused by the tertiary relaxation of the heme proteins. In order to calculate the enthalpy and conformational volume changes, the quantum yields of the photo-dissociation products of oxy-hemoglobins must be measured and taken into account. Finally, the enthalpy and conformational volume changes of the oxy-hemoglobins connected with the tertiary relaxation are obtained, which show that for all measured mammalian oxy-hemoglobins the values of enthalpy changes are in the range of 30.0-46.8 kcal/mol and volume changes are of 2.3-7.8 ml/mol although the quantum yields for the different species may have much bigger differences. A possible explanation of the results is presented.

Differential pathways in oxy and deoxy HbC aggregation/crystallization.

CC individuals, homozygous for the expression of beta(C)-globin, and SC individuals expressing both beta(S) and beta(C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype. To date, the question remains as to why HbC forms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal formation induced under conditions of concentrated phosphate buffer (2g% Hb, 1. 8M potassium phosphate buffer) and viewed by differential interference contrast microscopy. Oxy HbC formed isotropic amorphous aggregates with subsequent tetragonal crystal formation. Also observed, but less numerous, were twisted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in the oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored the formation of a greater morphologic variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed flat, hexagonal crystals. These results suggest: (1) the Lys substitution at beta6 evokes a crystallization process dependent upon ligand state conformation [i. e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) the oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of HbC that energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.

Glutaraldehyde effect on hemoglobin: evidence for an ion environment modification based on electron paramagnetic resonance and Mossbauer spectroscopies.

Glutaraldehyde is a widely used reagent for hemoglobin cross-linking in blood substitutes research. However, hemoglobin polymerization by glutaraldehyde involves modifications of its functional properties, such as oxygen affinity, redox potentials, and autoxidation kinetics. The aim of this article is to investigate, by electron paramagnetic resonance and Mossbauer spectroscopies, the changes that occur in the iron environment after glutaraldehyde cross-linking. Spectrometric studies were performed with native hemoglobin and hemoglobin cross-linked as soluble and insoluble polymers. Spectrometry data comparison with glutaraldehyde-modified hemoglobin functional properties allows to interpret from a structural point of view that glutaraldehyde action occurs as a decrease of the O--N(F8His) distance, an increase of the Fe--N(F8His) bond length, and the decrease of the distal-side steric hindrance.

Spin label probes of the environment of cysteine beta-93 in hemoglobin.

The environment of cysteine beta-93 is altered during the oxygenation of hemoglobin. Electron spin resonance was used to probe the hemoglobin conformation in this crucial region on the proximal side of the heme. Spin-labeled hemoglobins in both the R-liganded state [methemoglobin and oxyhemoglobin] and the T-unliganded state [deoxyhemoglobin as well as Ni(II) and Cu(II) substituted hemoglobins] were investigated. Included in this study are iodoacetamide and maleimide labels with different constraints at the point of reaction with the SH-group, as well as a series of pyrrolidinyloxyl maleimide labels of different chain length. From differences in the correlation time of the spin labels it was possible to identify two distinct strongly immobilized configurations in addition to the relatively mobile configuration with the label on the surface of the protein. By dipolar interactions between the spin labels and paramagnetic Cu(II) at the heme center, the relative position of the three orientations for the spin label are defined. Differences are observed between the two hemoglobin conformations with respect to the relative population of the various orientations and with respect to the potential barrier associated with the reorientation of the spin labels.

Pseudo cross-link of human hemoglobin with mono-(3,5-dibromosalicyl)fumarate.

The reaction of human oxyhemoglobin with mono(3,5-dibromosalicyl)fumarate, produces a derivative specifically acylated at the two lysines beta 82, which can be purified with a 70% yield. The oxygen affinity of this derivative at 37 degrees C at pH 7.4, 0.1 M Cl- is of 12 mm Hg, and is not affected by organic phosphate. In the presence of 5% CO2, the oxygen affinity decreases to 25 mm Hg. In all cases the cooperativity is lowered, with a value of n in the Hill plots near 2. Sedimentation velocity measurements indicate that, contrary to normal hemoglobin, this derivative fails to dissociate into dimers upon exposure to pH 5.5. The stability of the tetrameric structure is probably due to a modification of the beta-beta interface, resulting from electrostatic and hydrophobic interactions introduced in the beta cleft by the fumaryl residues. These new interactions are probably the origin of a new reverse Bohr effect group at alkaline pH. Consistent with the stabilization of the tetrameric structure, the half-time of retention of this compound in the rat is increased 4-fold with respect to that of normal hemoglobin. These characteristics cast a favorable light on the usage of this compound as an oxygen carrier in transfusional and perfusional fluids.