Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry.

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe3+), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that 1H2O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T1 shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T2 linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R2) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future.

The effects of conductivity and electrochemical doping on the reduction of methemoglobin immobilized in nanoparticulate TiO2 films.

Methemoglobin (bovine) is immobilized from aqueous phosphate buffer (pH 5.5) solution into thin porous TiO(2) (anatase) films at ITO electrode surfaces. Films of TiO(2) are produced in a deposition process employing 40 nm diameter TiO(2) nanoparticles suspended in dry methanol followed by calcination. The pore size in these films is sufficient for methemoglobin (ca. 6 nm diameter) to diffuse into the porous structure (over several hours) and to remain immobilized in electrochemically active form. The electrochemical reduction of methemoglobin immobilized in TiO(2) and immersed in aqueous phosphate buffer at pH 5.5 is observed in two steps with (i) a small quasi-reversible voltammetric response at -0.16 V vs. SCE (Process 1) and (ii) an irreversible reduction peak at ca. -0.5 V vs. SCE (Process 2). The irreversible response is recovered only after slow chemical re-oxidation of hemoglobin to methemoglobin. At sufficiently negative applied potential "electrochemical doping" of the TiO(2) host is observed to lead to a considerably enhanced reduction Process 1. TiO(2) can be temporarily switched from a non-conducting (irreversible electron transfer) into a conducting (reversible electron transfer) state.

Conformational transition of aquomethemoglobin: intramolecular histidine E7 binding reaction to the heme iron in the temperature range between 220 K and 295 K as seen by EPR and temperature-jump measurements.

Temperature-dependent EPR and temperature-jump measurements have been carried out, in order to examine the high-spin to low-spin transition of aquomethemogobin (pH 6.0). Relaxation rates and equilibrium constants could be determined as a function of temperature. As a reaction mechanism for the high-spin to low-spin transition, the binding of N epsilon of His E7 to the heme iron had been proposed; the same mechanism had been suggested for the ms-effect, found in temperature-jump experiments on aquomethemoglobin. A comparison of the thermodynamic quantities, deduced form the measurements in this paper, gives evidence that indeed the same reaction is investigated in both cases. Our results and most of the findings of earlier studies on the spin-state transitions of aquomethemoglobin, using susceptibility, optical, or EPR measurements, can be explained by the transition of methemoglobin with H2O as ligand (with high-spin state at all temperatures) and methemoglobin with ligand N epsilon of His E7 (with a low-spin ground state). Thermal fluctuations of large amplitude have to be postulated for the reaction to take place, so this reaction may be understood as a probe for the study of protein dynamics.

Allosteric transition in triply met-haemoglobin.

Methaemoglobin undergoes a transition to a T-like form at acid pH in the presence of strong effectors such as inositol hexakisphosphate (IHP), as evidenced by spectroscopic and oxidation potential measurements. Since oxygen and CO do not bind to the ferric haems, it is difficult to compare the properties of the R-met and T-met forms with those of ferrous haemoglobin. We have therefore prepared 90% oxidized samples, where the dominant signal for ligand (oxygen or CO) binding is due to tetramers with three met haems. Measurements were made of the oxygen equilibrium curves and CO rebinding kinetics after photodissociation. Without effectors, the partially oxidized samples show mainly R-state properties. Addition of IHP at acid pH induces an increase in T-state behaviour, as indicated by a lower oxygen affinity and a higher fraction of the slow bimolecular component for CO rebinding.

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.

Modeling the hemoglobin switchpoint with cyanomet valency hybrids: Raman spectroscopic probes of tertiary and quaternary structure.

Hybrid hemoglobins with cyanomet hemes in the alpha or in the beta chains have been investigated by resonance Raman (RR) spectroscopy, using ultraviolet (230 nm) and visible (441.6 nm) excitation. For the CO adducts, the UVRR spectra are identical with that of native HbCO, showing the tyrosine and tryptophan signals to be insensitive to ligand substitution within the R state. In the absence of CO, the doubly ligated hybrids show differences in the UVRR spectra, relative to the CO adducts, which are the superposition of two difference spectra: (1) the T-R difference spectrum obtained by subtracting the spectrum of HbCO from that of deoxyHb and (2) a perturbed R state spectrum, characteristic of deligated chains within the R state. These spectra arise from alterations, respectively, in the quaternary contacts of interface aromatic residues and in the tertiary contacts of interior aromatic residues. From the amplitudes of the difference spectra, the T state population was determined to be 30% for (alpha FeII beta FeIIICN)2 and 43% for (alpha FeIIICN beta FeII)2, in good agreement with the kinetic analysis of Cassoly and Gibson [Cassoly, R., & Gibson, Q. H. (1972) J. Biol. Chem. 247, 7332]. Addition of inositol hexaphosphate (IHP) increased the T state population, but only by a modest amount, to 40 and 53%, respectively, in contrast to the frequent assumption that the T state conversion is quantitative in the presence of IHP. Since current understanding of the quaternary state dependence of the Fe-histidine stretching frequency is based on that assumption, the RR band envelope for this vibration was reexamined.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubility of fluoromethemoglobin S: effect of phosphate and temperature on polymerization.

The polymerization properties of the fully liganded fluoromet derivative of hemoglobin S (FmetHb S) were investigated by electron microscopy and absorption spectroscopy. Polymerization progress curves, as measured by increasing sample turbidity at 700 nm, exhibit a delay time (t(d)) consistent with the double nucleation mechanism. The pattern of fiber growth, as monitored by electron microscopy, is also indicative of a heterogeneous nucleation process, and dimensions of the fibers were found to be comparable to that of deoxyHb S. The polymerization rate constant (1/t(d)) depends exponentially on Hb S concentration, and the size of the homogeneous and heterogeneous nuclei also depend on FmetHb S concentration. As for deoxyHb S, higher concentrations of protein and phosphate favor fiber formation, while lower temperatures inhibit polymerization. Solubility experiments reveal, however, that eight times more FmetHb S is required for polymerization. The current studies further show that reaction order is independent of phosphate concentration if Hb S activity and not concentration is considered. The allosteric effector, inositol hexaphosphate (IHP), promotes fiber formation, and temperature-dependent reaggregation of FmetHb S suggests that IHP stabilizes pregelation aggregates. These studies show that FmetHb S resembles deoxyHb S in many of its polymerization properties; however, IHP-bound FmetHb S potentially provides a unique avenue for future studies of the early stages of Hb S polymerization and the effect of tertiary and quaternary protein structure on the polymerization process.