Hiroshi Watari

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.

Properties of hemoglobin M, milwaukee-1 variant and its unique characteristic

Abstract The physicochemical properties of Hb M Milwaukee-1 (α 2 β 2 67Glu ) were studied in comparison with other Hb M variants and Hb Sydney (α 2 β 2 67Ala ). Using the patients blood containing Hb M Milwaukee-1. EPR spectra with high-spin-type signals were revealed which resembled those of Hb M Saskatoon indicating that the glutamic acid residue at E 11 has an effect on the heme iron analogous to the tyrosine residue at E7. In an O 2 equilibrium study, Hb M Milwaukee-1 showed a diminished heme-heme interaction, 1.4–1.6 in terms of n value, and this value seemed to be dependent on pH or temperature. Although the affinity for O 2 was as low as about 60 mm Hg of P 50 at pH 6.5, the Bohr effect was distinctly present in this Hb M, and its magnitude was larger than that of Hb A. The effect of temperature on the O 2 affinity, however, was significantly smaller in this Hb M than that observed in Hb A. In contrast to Hb Sydney, Hb M Milwaukee-1 was as stable as Hb A in an heat denaturation experiment, reflecting the stabilizing effect of the intramolecular bonding between the ferric heme iron and the substituted glutamic acid residue on the whole molecule.

Dependence of EPR Transition Probability on Magnetic Field

Bleanys theory of the g -value dependence of EPR transition probability is extended to a general case of rhombic ligand field by assuming that the system has a fictitious spin of 1/2. Given three principal g -values and a line shape function, changes in EPR spectra of polycrystalline aggregates with static magnetic field are calculated and a general equation of spectral curve are obtained. In the limiting case of a zero line width, the equation is reduced to an elliptic integral. This approach is applied to the simulation of the EPR spectra of normal and abnormal haemoglobins and pyrocatechase, and the theoretically and experimentally obtained spectra exhibit a good fit.

Hemoglobins M: identification of Iwate, Boston, and Saskatoon variants.

Hemoglobin M variants, M Iwate, M Boston, M Saskatoon are easily and accurately identified by electron spin resonance with small amounts of patients blood. In hemoglobin M Iwate and M Boston the electron spin resonance of both fresh blood (unprocessed) and isolated pure ferrihemoglobin M revealed similar signal shapes; whereas that of hemoglobin M Saskatoon was doublet in fresh blood and triplet in pure ferrihemoglobin M.

Changes in intracellular pH during repeated exercise

SummaryThe rates of change in intracellular pH during repeated exercise sessions with rest periods was determined by 31 phosphorus-nuclear magnetic resonance spectroscopy (31P-MRS). Five long-distance runners and six healthy male subjects as controls performed a 2-min femoral flexion at 20 kg · m · min−1 in a 2.1 T superconducting magnet with a 67-cm bore and repeated this exercise four times with 2-min rest periods intervening. In all cases during exercise the inorganic phosphate (Pi) peak split into two, the earlier increased rapidly (high-pH Pi) and the later (low-pH Pi) increased more slowly. The Pi peaks were separated by a fitting procedure using the least square mean method. The high-pH Pi area during exercise decreased as the number of repeated exercise periods increased, while the low-pH Pi area gradually increased. Although the total Pi area decreased exponentially during the recovery period, the high-pH Pi area decreased first and then the low-pH Pi area reduced gradually. The pH values were estimated from the chemical shift between the phosphocreatine peak and each split peak in the Pi. The high-pH in pooled data ranged from 6.6 to 7.0 during exercise and recovery, while the low pH decreased to 6.2 during exercise. As the number of exercise periods increased, each pH value gradually became less acidic, although there was a tendency to more acidity in the control subjects than in the long-distance runners. In conclusion, it was possible to obtain by non-invasive, continuous31P-MRS, a split pattern of Pi peaks during exercise and there were at least tow different intracellular pH values during exercise, suggesting that each Pi peak might be attributed to the types of muscle fibre recruited.

Electron spin resonance studies of ferrihaemoglobin MOsaka

Abstract 1. 1. As reported previously, the electron spin resonance spectrum of ferrihaemoglobin MOsaka showed a doublet signal in the g = 6 region. This is considered to be due to the haem iron of the αM-chain on the basis of measurements of electron spin resonance at each stage in the purification of ferrihaemoglobin MOsaka. 2. 2. The doublet signal is considered to occur as a result of a splitting of the degenerate g-value of 6.o. A theoretical absorption curve due to electron spin resonance for ferrihaemoglobin MOsaka, in an amorphous state, is given and is compared with the integrated experimental curve of electron spin resonance. 3. 3. When the E-term in a general spin Hamiltonian was treated as a second-order perturbation, split g-values were calculated as: gξ = 6 − 24E/D, gy = 6+24E/D and gz = 2. From the experimental g-values, the ratio of the E-term to the D-term was estimated as 0.01. A lowering of the symmetry of the ligand field on the haem iron in the αM-chain of ferrihaemoglobin MOsaka is discussed.

Semiquinone formation of d-amino-acid oxidase by irradiation

Abstract It has been found that oxidized d -amino-acid oxidase ( d -amino-acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) is transformed to a semiquinone by irradiation with visible light. The semiquinone formation was detected by measurements of the optical absorption spectrum and electron spin resonance. A fairly stable semiquinone was obtained under anaerobic conditions but not under aerobic conditions since it easily reacted with molecular oxygen. After a cycle of semiquinone formation and reoxidation, the d -amino-acid oxidase activity was found to be completely restored. Although no evidence was obtained about an electron donor for the semiquinone formation, it did not appear likely, from the measurement of the activation energy of 5 kcal per mole, that water donated electrons. The electron spin resonance signal of the semiquinone exhibited an asymmetrical shape. Possible interpretations for the phenomena are discussed.

Effect of Passive and Active Recovery on PCr Kinetics

Recently, 31phosphorus-nuclear magnetic resonance spectroscopy (31P-MRS) has been used as a noninvasive technique to measure changes in the concentrations of adenosine 5’-triphosphate (ATP), phosphocreatine (PCr) and inorganic phosphate (Pi), as well as the intramuscular pH, both during and after exercise. Splitting of the Pi peak into two has been observed during exercise and is attributable to two different pH distributions in exercising muscle (high pH and low pH) (1, 3, 5, 8, 11, 15). Previously, we reported that the two split Pi peaks showed different time courses at the onset of exercise and during recovery (16, 19, 22); high-pH Pi increased promptly at the onset of exercise and disappeared rapidly after exercise, while the low-pH Pi peak increased gradually after a delay of approximately 60 sec at the onset of exercise and decreased over a longer period after exercise was stopped.

Sur la Résonance Paramagnétique Electronique et la Structure Hyperfine des Flavines et des Flavine-Enzymes

Le developpement de lappareil nous permete dobtenir le spectre plus precis de la resonance paramagnetique electronique.La structure hyperfine de riboflavine, flavine mononucleotide et flavine adenine dinucleotide est composee de 32 raies dans le milieu acide.Les derivees simples de lisoalloxazine montrent la shf de 18 raies et chaque raie est separee egalement. Labsorption hyperfine est symmetrique et son rapport est estime comme 1: 3: 5: 7: 9: 12: 13: 14: 15. La densite du spin delectron apparie autour des noyaux N (10) et N (1) est determinee comme etant dans la relation, ρN (10): ρN (1) =2: 1.Labsorption de RPE de la solution de loxydase D-acide aminee vient fort proportionnellement a la duree de lillumination. Labsorption de FADH liee a lenzyme proteique atteind a la valeur de mill six cent fois superieure a celle de la FADH libre.On observe la RPE en ajoutant a la xanthine oxydase quelque substrat, xanthine, hypoxanthine, adenine ou DPNH.II y a trois agents effectifs a labsorption dans le systeme de la xanthine oxydase, cest-a-dire, FADH, Mo5+ et Fe3+ en negligant la possibilite de la formation de quelque intermede RPE actif a cote du substrat.