The facilitated diffusion of oxygen by hemoglobin and myoglobin.

We have clarified the use of Wyman's differential equation for the facilitated oxygen flux through a slab of solution of myoglobin or hemoglobin by showing that there is a unique choice of boundary condition on the carrier concentration to be employed in conjunction with it. The singular perturbation solution of Wyman's equation, due to Murrayand Mitchell and Murray, has been extended. By means of it, the paradox of Wittenberg, that the facilitated oxygen flux per mole of heme is apparently independent of the protein carrier, has been resolved.     

Changes in conformation and function of hemoglobin and myoglobin induced by adsorption to silica.

Adsorption of myoglobin (Mb) or hemoglobin (Hb) to silica (Cab-O-Sil) causes marked alterations in protein hydrogen exchange kinetics. The exchange is slower for cyanometMb and faster for both cyanometHb and oxyHb in adsorbed state than for the corresponding species in the free state. For Hb, adsorption increases oxygen affinity (P₅₀ = 12.9 mmHg vs. 16.7 for free) and decreases cooperativity (n = 2.05 vs. 2.87 for free). Myoglobin has the same oxygen affinity in both the free and adsorbed states.     

Dielectric properties of hemoglobin and myoglobin. II. Dipole moment of sperm whale myoglobin

This paper is concerned with the molecular origin of the dipole moment of sperm whale myoglobin as it can be calculated from the dielectric dispersion at 1 Mcps on the basis of a mechanism of orientational polarization. It was possible to compare the dielectric increment of native myoglobin and its change during the reaction with bromo acetate with dipole moments calculated according to the known coordinates of the charged groups of the molecule. The agreement between the two shows that in myoglobin only the permanent dipole moment due to these charged groups is important, and that contributions from other possible sources remain within the limits of experimental error.     

Stereochemistry of carbon monoxide binding to myoglobin and hemoglobin

The binding of carbon monoxide to myoglobin and hemoglobin is examined to determine the origin of the deviation of the FeCO geometry from that found in model systems. Possible distortions due to protein-ligand interactions are analyzed with special attention to protein relaxation. It is estimated that the protein can support a strain of less than 10 kcal per mole; this may be sufficient to produce a displacement of a linear FeCO unit from the heme normal.     

Resonant recognition model and protein topography. Model studies with myoglobin, hemoglobin and lysozyme

This study describes the further extension of the resonant recognition model for the analysis and prediction of protein--protein and protein--DNA structure/function dependencies. The model is based on the significant correlation between spectra of numerical presentations of the amino acid or nucleotide sequences of proteins and their coded biological activity. According to this physico-mathematical method, it is possible to define amino acids in the sequence which are predicted to be the most critical for protein function. Using sperm whale myoglobin, human hemoglobin and hen egg white lysozyme as model protein examples, sets of predicted amino acids, or so-called 'hot spots', have been identified within the tertiary structure. It was found for each protein that the predicted 'hot spots', which are distributed along the primary sequence, are spatially grouped in a dome-like arrangement over the active site. The identified amino acids did not correspond to the amino acid residues which are involved in the chemical reaction site of these proteins. It is thus proposed that the resonant recognition model helps to identify amino acid residues which are important for the creation of the molecular structure around the catalytic active site and also the associated physical field conditions required for biorecognition, docking of the specific substrate and full biological activity.     

Picosecond phase grating spectroscopy of hemoglobin and myoglobin: energetics and dynamics of global protein motion.

Phase grating spectroscopy has been used to follow the optically triggered tertiary structural changes of carboxymyoglobin (MbCO) and carboxyhemoglobin (HbCO). Probe wavelength and temperature dependencies have shown that the grating signal arises from nonthermal density changes induced by the protein structural changes. The material displaced through the protein structural changes leads to the excitation of coherent acoustic modes of the surrounding water. The coupling of the structural changes to the fluid hydrodynamics demonstrates that a global change in the protein structure is occurring in less than 30 ps. The global relaxation is on the same time scale as the local changes in structure in the vicinity of the heme pocket. The observed dynamics for global relaxation and correspondence between the local and global structural changes provides evidence for the involvement of collective modes in the propagation of the initial tertiary conformational changes. The energetics can also be derived from the acoustic signal. For MbCO, the photodissociation process is endothermic by 21 +/- 2 kcal/mol, which corresponds closely to the expected Fe-CO bond enthalpy. In contrast, HbCO dissipates approximately 10 kcal/mol more energy relative to myoglobin during its initial tertiary structural relaxation. The difference in energetics indicates that significantly more energy is stored in the hemoglobin structure and is believed to be related to the quaternary structure of hemoglobin not present in the monomeric form of myoglobin. These findings provide new insight into the biomechanics of conformational changes in proteins and lend support to theoretical models invoking stored strain energy as the driving force for large amplitude correlated motions.     

Thin-layer microcalorimetric studies of oxygen and carbon monoxide binding to hemoglobin and myoglobin.

A thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand binding macromolecules. We have measured the enthalpy of binding oxygen and carbon monoxide to horse myoglobin, human hemoglobin A0 and sperm whale myoglobin in phosphate buffer at pH 7.6, with the enzyme reducing system of Hayashi. Reactions of human hemoglobin were also done under various buffer conditions in order to elucidate the Bohr effect. These binding reactions were found not to exhibit a detectable enthalpy change over the temperature range of 10 degrees C to 25 degrees C. The enzyme reducing system was shown to react with oxygen in a manner that releases a substantial amount of heat. This problem was corrected by using a minimum amount and by placing the buffer and enzyme system in the reference cell effectively cancelling the oxygen enzyme reaction heat as well as the heat of gas dissolution. It was also demonstrated that glucose-6-phosphate, one of the reducing system components, in 50 mM concentrations can influence the heat of binding oxygen and carbon monoxide to hemoglobin. This effect was shown to be absent in the myoglobins and also with hemoglobin at glucose-6-phosphate concentrations less than 5 mM.     

The Nernst equation applied to oxidation-reduction reactions in myoglobin and hemoglobin. Evaluation of the parameters

Analyses of the binding of oxygen to monomers such as myoglobin employ the Mass Action equation. The Mass Action equation, as such, is not directly applicable for the analysis of the binding of oxygen to oligomers such as hemoglobin. When the binding of oxygen to hemoglobin is analyzed, models incorporating extensions of mass action are employed. Oxidation-reduction reactions of the heme group in myoglobin and hemoglobin involve the binding and dissociation of electrons. This reaction is described with the Nernst equation. The Nernst equation is applicable only to a monomeric species even if the number of electrons involved is greater than unity. To analyze the oxidation-reduction reaction in a molecule such as hemoglobin a model is required which incorporates extensions of the Nernst equation. This communication develops models employing the Nernst equation for oxidation-reduction reactions analogous to those employed for hemoglobin in the analysis of the oxygenation (binding of oxygen) reaction.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase

At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangement of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regeneration.     

Picosecond transient absorption study of photodissociated carboxy hemoglobin and myoglobin.

The optical transient absorption spectra at 30 ps and 6.5 ns after photolysis are compared for both carboxy hemoglobin (HbCO) and carboxy myoglobin (MbCO). Both 355- and 532-nm excitation pulses were used. In all cases the shapes of the optical difference spectra thus generated are stationary over the complete time-scale studied. The photolysis spectra for MbCO are not significantly different from the equilibrium difference spectra generated on the same picosecond spectrometer when measured to an accuracy of +/- 0.5 nm. In addition, spectral parameters for delegated HbCO generated on the same spectrometer but detected by two different techniques, either by a Vidicon detector or point by point with photomultiplier tubes, are reported; the results are different from some of the previously reported picosecond experiments.     

Low temperature photodissociation studies of ferrous hemoglobin and myoglobin complexes by M?ssbauer spectroscopy.

57Fe-enriched complexes of hemoglobin and myoglobin with CO and O2 were photodissociated at 4.2 degrees K, and the resulting spectra were compared with those of the deoxy forms. Differences in both quadrupole splitting and isomer shift were noted for each protein, the photoproducts having smaller isomer shift and larger quadrupole splitting than the deoxy forms. The photoproducts of HbCO and HbO2 had narrow absorption lines, indicating a well-defined iron environment. The corresponding myoglobin species had broader absorption lines, as did both deoxy forms. The weak absorption lines of photodissociated NO complexes appeared to be wide, possibly indicating magnetic interaction with the unpaired electron of the nearby NO.     

Soret circular dichroism of cobalt-substituted myoglobin and hemoglobin derivatives

Circular dichroic spectra in the Soret region were obtained for the following cobalt-substituted hemoproteins: ^CoMb³, ^CoMbO₂, ^CoMbNO, ^CoMb⁺, ^CoHb, ^CoHbO₂, ^CoHbNO and ^CoHb⁺ and compared with the corresponding spectra of the native species to delineate the sensitivity of Soret circular dichroism to ligation, quaternary structures, metal ion substitution and its magnetic moment. Soret rotational strengths, R, were calculated, and dissymmetry ratios were used to reveal hidden transitions. The results indicate that Soret circular dichroism is sensitive to the metal ion, its oxidation state, ligation and local environment but neither to quaternary structural changes as proposed by Ferrone &; Topp (1975), nor to the magnetic moment of the metal ion as suggested by Li &; Johnson (1969).     

The aerobic reduction of Fe(III) complexes by hemoglobin and myoglobin

The ability of hemoglobin (myoglobin) to reduce directly low-molecular-weight complexes of Fe(III) to form methemoglobin (metmyoglobin) and the Fe(II)-tris(2,2'-bipyridine) complex under aerobic conditions is described. The reduction is not mediated by superoxide, O-.2, as shown by increased rates under anaerobic conditions and lack of inhibition by superoxide dismutase. The chemical nature of the Fe(III) complex presented influences the rate of reduction; one of the most effective chelating agents of cellular origin is Fe(III) X ATP. This mechanism may be of fundamental importance in the mobilization and utilization of iron in biological systems.     

Spectrophotometric determination of myoglobin in cardiac and skeletal muscle: separation from hemoglobin by subunit-exchange chromatography.

Myoglobin is extracted from muscle and separated from blood hemoglobin by subunit-exchange chromatography on a column of Sepharose 4B to which hemoglobin α-β subunits are linked covalently. Hemoglobin is retained on the column. Myoglobin in the effluent is determined spectrophotometrically as ferrous myoglobin or as carbon monoxide ferrous myoglobin. The method is applicable to cardiac, smooth, or skeletal muscle from mammals, reptiles, birds, and teleost fish, but failed with the one amphibian and the one shark tested.