Masao Kotani

Studies on the function of abnormal hemoglobins I. An improved method for automatic measurement of the oxygen equilibrium curve of hemoglobin

Abstract An apparatus with which the continuous oxygen equilibrium curve of hemo globin can be recorded automatically was constructed and its performance was examined. The oxygen pressure in the hemoglobin solution and the degree of saturation of the hemoglobin with oxygen are determined by means of a Clarks oxygen electrode and by spectrophotometry, respectively. The deoxygenation of oxyhemoglobin is attained by introducing nitrogen gas into the reaction cell after about 90 min and the reoxygenation of deoxyhemoglobin by introducing air. The deoxygenation curve coincided well with the successive oxygenation curve of the same sample. The curves measured using monochromatic lights of various wavelengths in the visible range coincided well with each other, in so far as the measurements were done under the same conditions. This method is applicable to hemoglobin solutions of concentrations of 0.01–2%. The curve is reproduced very well if the experiments are carried out under the same conditions and within several days. This reproducibility, however, becomes worse when the experiments are carried out over a longer period. The standard error of the fractional saturation for the curves which have been measured over the past one year depends on the position on the curve and is a maximum, about 2%, near the point of half saturation. This apparatus can furnish us with easy, speedy and accurate means to measure the oxygen equilibrium curve and can facilitate the observation of fine structures of the curve. This method is suitable for studies on the function of abnormal hemoglobins.

Analysis of thermal equilibrium between high-spin and low-spin states in ferrihemoglobin complexes

Abstract Paramagnetic susceptibilities and absorption spectra of ferrihemoglobin complexes (Hb(Fe 3+ ) · CN − , ·N 3 − , · OH − , · OCN − and · H 2 O) have been measured between liquid N 2 and room temperatures. It has been found that temperature-dependent transitions exist in squares of effective Bohr magneton numbers for these complexes except ferrihemoglobin cyanide, as seen in the case of ferrimyoglobin complexes, These transitions are explained on the basis of thermal equilibria between high-spin and low-spin states of Fe 3+ in hemes. In the case of acid ferrihemoglobin (pH 5.9) a strange behavior has been observed near the freezing point of solution. From the analysis of temperature dependence of the equilibrium constant in high spin ⇌ low spin states, energy and entropy differences between the two states are estimated. Thermodynamic quantities ΔH ° and ΔS ° are calculated by use of e and γ values for these complexes. Compensation phenomena between ΔH ° and ΔS ° are also described together with the results of ferric myoglobin and cytochrome c peroxidase complexes. The data of three hemoproteins are compared. This report is the fourth in our series of reports on thermal equilibrium between high-spin and low-spin states.

Magnetic susceptibility measurements on hemoproteins down to 4.2°K

Abstract To obtain information on the electronic state of iron ion in various hemoproteins (myoglobin, hemoglobin, Rhodospirillum rubrum hemoprotein (RHP) and cytochrome c (Cyt. c)), the magnetic susceptibilities of these substances were measured from room temperature down to 4.2° K. After plotting the experimental susceptibility data against the reciprocal of temperature, the diamagnetic part of the magnetic susceptibility could be easily determined by extrapolating the χ-1/T curve to the point where 1/T was zero. The temperature dependence of the effective number of Bohr magnetons was calculated from the paramagnetic part, which was obtained by substracting the diamagnetic part from the total magnetic susceptibility. The following compounds were of high-spin type: Mb(Fe3+) pH = 6,Mb(Fe 2+ ) pH = 6, Mb(Fe 3+ )F, Hb(Fe 3+ ) pH = 6, Hb(Fe 2+ ) pH = 6, RHP(Fe 3+ ) pH = 6, RHP(Fe 3+ ) pH = 11, RHP(Fe 2+ ) pH = 6, RHP(Fe 2+ ) pH = II , catalase (Fe3+) pH = 6 and catalase (Fe3+)F. From the temperature dependence of neff, the value of D (where H=DSz2) of Mb(Fe3+) pH = 6 was estimated to be 10 cm−1 which was in good agreement with the value obtained from the anisotropy of the susceptibility. Mb(Fe3+)CN, Cyt. c (Fe3+) pH = 6, Cyt c (Fe3+) pH = 11, Cyt. c (Fe2+) pH = 6, Cyt. c (Fe2+) pH = 11, catalase (Fe3+)CN and catalase (Fe3+)N3 were of low-spin type and neff for Mb(Fe3+) pH = 9 had a medium value, between the high- and low-spin types.

Theoretical Study on the Effective Magnetic Moments of Some Hemeproteins

It is pointed out that the measurement of the static magnetic susceptibilities over a wide temperature range will be able to the detailed information about the electronic structure of the paramagnetic heme derivatives. On the basis of the data given by the electron paramagnetic resonance experiment, formulas for the temperature dependence of the effective magnetic moment (ne,rr) of the ferrihemoglobin fluoride and the ferrihemoglobin azide are derived; further the temperature dependence of nett of the ferrohemoglobin is studied theoretically and it is shown from the observed value of nett at room temperature that the non-cubic ligand field is weaker in the case of ferrohemoglobin compared with the ~ase of ferrihemoglobin azide. The structure of molecules in biological substances, particularly of proteins, can be studied by means of chemical, optical, electrical and rheological experiments, but in the case of paramagnetic molecules such as hemoglobin magnetic properties can be utilized as important sources of information. In this line we have two methods-the electron paramagnetic resonance (EPR) and the measurement of static paramagnetic susceptibilities. The EPR method, when it can be applied at all, is capable of giving fairly detailed information concerning the behavior of magnetic electrons in the molecule, particularly concerning wave-function and ligand fields to which 3d electrons in heme iron are subjected; the measurement of principal g values in ferrihemoglobin azide by Gibson and Ingram/> and the determination of orbital energies of 3d electrons from analysis of these g-values by Griffith> are fruitful examples of such studies. The study of static magnetic susceptibilities is regarded very often as inferior to EPR in the accuracy and the varieties of information obtainable from experiments. The former method has, however, merits which are not always shared by EPR. One of the merits is its wide applicability to various cases. For example in the case of hemes with reduced iron (Fe++) the number of unpaired electrons is even, so that the degeneracy is completely lifted as long as the symmetry of the ligand field is not particularly high. In such * On leave of absence from the University of Tokyo. Present address: Department of Physics. Faculty of Science, The University of Tokyo. Theoretical Study on the Effective Magnetic Moments of Some Hemeproteins 5 cases the method of EPR is not usually applicable. Even in the case of odd number of electrons, EPR is difficult to apply in the presence of large magnetic anisotropy unless a single crystal of reasonable size can be produced and the spin-lattice relaxation time is sufficiently long. Contrary to this, the measurement of the susceptibility can be carried out even in the case of even number of electrons as well as for samples not in the state of single crystals. In this sense the measurement of the susceptibility is not always superseded by EPR, but these two methods are complementary to each other. The study of the behavior of the susceptibility at low temperatures is expected to provide valuable information, although practically all measurements have been hitherto made at room temperatures. In this paper the author will show, on some examples, how one can predict the temperature dependence of the static susceptibility, based on the results of EPR experiments carried out on the same molecule or closely related molecules. The discussion in the following is based on the so-called ligand field theory. In its most naive form the ligand field theory was introduced as a model in which electrons on the central metallic ion (Fe) are subjected to inhomogeneous electrostatic field due to the ligands, but it is now generally known that the formalism of the theory remains true even when there is a partial covalency in the bonds between Fe and the ligands. Particularly when the five d orbitals are classified into two groups, de(d11.z, dza:, dx11) and dr(dz2, dx2_112), dr itself is non-magnetic, so that the precise forms of dr orbitals do not matter so much. This is fortunate because dr orbitals may participate in forming a bonds with the ligands and are often much deformed from their atomic d shape. de orbitals may also form weak covalent rc bonds with ligands, but these rc bonds are usually weak, and this phenomenon can be taken into account in the theory by reducing the magnitude of the spin-orbit interaction parameter a. The results of measurement of paramagnetic susceptibility . are usually represented by Curies formula

Paramagnetic anisotropy measurements on acid ferrimyoglobin and ferrimyoglobin fluoride

Abstract The anisotropy of the magnetic susceptibility of type A ferrimyoglobin single crystals was measured by a highly sensitive torque meter from liquid-N2 temperature down to the temperature of pumped liquid He. The torque meter covered the torque range of about 10−4 dyne·cm. The magnetic anisotropy of a haemoprotein single crystal containing more than 5·10−8 moles of ferric haem in a high-spin state could be measured with this apparatus. Fe3+ in the crystals investigated was in the high-spin state. The zero-field splitting of this state can be expressed by the spin-Hamiltonian D√Sz2 − 1/3S(S+1)√, (with the z axis perpendicular to the haem plane). The temperature dependence of the measured torque was best fitted to the theoretical curve by taking D ≈ 10.5 for acid ferrimyoglobin and D ≈ 6.5 for ferrimyoglobin fluoride. These values are in good agreement with those obtained from the mean magnetic susceptibility measurements. The possible presence of an FSz4 term in the spin-Hamiltonian is discussed.

Paramagnetic Properties and Electronic Structure of Iron in Heme Proteins

Publisher Summary This chapter discusses in-plane magnetic anisotropy of heme, both in the high-spin state and in the low-spin state. If the magnetic field is applied parallel to the heme plane and is rotated in the same plane, g values of electron paramagnetic resonance absorption (EPR) signal vary sinusoidally between maximum and minimum values. The absolute directions of these principal axes of in-plane anisotropy, in reference to the geometrical structure of heme, are not certainly known. Studies show that for ferri-MbF– (high-spin) and ferri-Mb.N3– (low-spin) these principal axes do not coincide, but those for fluoride and azide make an angle of about 45°. To show this, a single crystal of ferri-Mb was prepared, in which hydrogen fluoride and sodium azide was added simultaneously to the crystal. Then some of the hemes catch F– and some N3–, and signals from high-spin hemes and those from low-spin hemes could be observed on the same crystal. On the assumption that the attachment of F– and N3– does not affect the geometry of hemes, it was possible to compare the directions of principal axes of anisotropy of two derivatives. By constructing a Lissajous-like plot, it has been shown that the principal axes of high-spin heme and those of low-spin hemes make an angle of about π/4.

Studies on the function of abnormal hemoglobins II. Oxygen equilibrium of abnormal hemoglobins: Shimonoseki, Ube II, Hikari, Gifu, and Agenogi

Abstract Oxygen equilibrium functions of abnormal hemoglobins discovered from Japanese families were studied. Hb Shimonoseki (E3α, Gln → Arg), Hb Ube II (E17α, Asn → Asp), Hb Hikari (E5β, Lys → Asn) and Hb Gifu (EF4β, Asn → Lys) have the same function as that of Hb A. These results further confirm the widely known fact that external residues generally do not have a very important role in the function of hemoglobin. Hb Agenogi (F6β, Glu → Lys), however, has a different function from that of Hb A:it has a slightly but obviously lower oxygen affinity than that of Hb A while the shape of the curve of the Bohr effect and the heme heme interaction are normal. A plausible mechanism for the lower oxygen affinity is proposed from the viewpoint of altered surroundings of oxygen-linked acid groups which are brought about by an interaction between the groups and the newly introduced residue, lysine.

ELECTRONIC STRUCTURE OF IRON IN PORPHYRIN COMPLEXES

In the present paper I should l i e to discuss the electronic structure of the iron atom in heme, which is the well-known prosthetic group of hemoproteins. Since heme is a molecule consisting of a porphyrin and an Fe atom, the problem of the electronic structure of the Fe atom in heme is a part of the problem of the electronic structure of hemes. Many experimental results, however, particularly those of magnetic measuremWs, can be explained and discussed from the atomic viewpoint, in which the Fe atom or ion is considered to be subjected to electrostatic and other influences due to surrounding atoms such as the pyrrole and imidazole nitrogens. We can use ligand field theory, which has proved very effective in theoretical interpretations of energy levels and other electronic properties of coordination compounds of metallic ions. The heme found in Hb and Mb has the structure shown in FIGURE 1 ;the porphyrin of this heme is called protoporphyrin, and has two vinyl groups and two propionic acid groups, as well as methyl groups, attached to peripheral carbon atoms. Pi electrons of vinyl groups are conjugated with those of the main porphyrin ring. If we disregard these “tails,” the porphyrin and the heme possess tetragonal symmetry. The porphyrin is essentially planar, but there are evidences from x-ray analysis of hemes and myoglobin tha: the Fe atom is sometimes located above the porphyrin plane by as much as 1/2 A. The geometry of hemin chloride, separated from protein, has been measured in detail by Konig’ by the x-ray diffraction method, and the result is shown in FIGURE 2. This shows that the Fe atom is actually out of the porphyrin plane, and the symmetry of the molecule viewed from the Fe atom is approximately C In the following, a Cartesian coordinate system is introduced, with its origin at Fe nucleus, z axis perpendicular to the porphyrin plane, and x, y axes parallel to lines connecting pyrrole nitrogens, which are located diagonally opposite. These four pyrrole nitrogen atoms constitute the four ligands of the Fe atom under consideration. In the case of the native hemes in which we are interested, the Fe atom is further coordinated by another nitrogen atom of the imidazole of histidine. This nitrogen atom is the fifth ligand, and is situated approximately on the negative z axis. The plane of the imidazole ring is roughly perpendicular to the porphyrin plane, and the tetragonal symmetry is reduced, if we take the imidazole into account. Furthermore, at the sixth coordination point on the positive z axis, different kinds of small molecules and atomic ions can be coordinated, at least in myoglobin and hemoglobin. The reversible attachment of oxygen molecule at the sixth position is important in the physiological function of these molecules. Now, hemes can include the reduced and oxydized forms. The Fe atom exists in ferrous states in the reduced form and in ferric states in the oxidized form. The following abreviations are used for hemoglobins and myoglobins: Hb(FeZ+)X, for the hemoglobin whose hemes are in reduced form, Hb(FeS+)X for the oxidized form, and similar designations for myoglobins. Here X denotes the ligand which