Taku Nakano

Spectrophotometric determination of hydrogen peroxide: catalase activity and rates of hydrogen peroxide removal by erythrocytes.

A new method of hydrogen peroxide determination for the measurement of catalase activity and rates of hydrogen peroxide removal by erythrocytes was described. Hydrogen peroxide was determined by converting it to the indamine dye with a water-soluble ironporphyrin and measuring the absorbance at 590 nm. This method was applied to the assay of catalase in hemolysates from human, rat and mouse blood. The activities obtained were in agreement with those obtained by other methods including UV method. The present method was also applied to the determination of rates of hydrogen peroxide removal by intact erythrocytes from human subjects, rats and mice. Data suggested that normal erythrocytes have substantial capacity to remove extracellular hydrogen peroxide. From the measurement of catalase activity in erythrocytes treated with 3-amino-1,2,4-triazole and rates of hydrogen peroxide removal by the erythrocytes, it is deduced that rate constants related to the hemoglobin content (k/g Hb) for hydrogen peroxide removal by catalase in normal and acatalasemic erythrocytes are 42.0 +/- 6.0 and 8.0 +/- 3.0, respectively.

Synthesis of Diazirinyl Photoprobe Carrying a Novel Cleavable Biotin

Photoaffinity labeling is a powerful tool for the identification of receptor proteins and their binding sites. The major drawback of this method, however, is the reliability of the labeling and the purity of labeled peptides after purification from a large number of unlabeled fragments. The recent development of diazirine-based biotinylated probes provides an efficient solution to this problem. In these probes, the diazirine photophore contributes to the formation of a stable cross-link that greatly improves the efficiency. The application of an N-acetylglucosamine photoprobe carrying biotinylated diazirine provided the first information regarding acceptor-site peptides of b-1,4-galactosyltransferase. We have also developed a novel method for the one-step introduction of a biotinylated diazirine photophore into unprotected carbohydrate ligands. Since biotin–avidin binding is essentially irreversible (Kd = 10 15 m), several approaches have been investigated to achieve efficient recovery of biotinylated products from an immobilized avidin matrix. Although the use of monomeric avidin contributes to the isolation of biotinylated products because of the lower affinity to biotin (Kd = 10 8 m), it requires a high

Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides

Hydrogen peroxide removal rates by hemoglobin were enhanced in the presence of reduced pyridine nucleotides. The species which had the activity to oxidize pyridine nucleotides was purified from human blood and identified as hemoglobin A. Hydrogen peroxide removal rates by hemoglobin A without reduced pyridine nucleotides at 0.2 mM hydrogen peroxide were 0.87+/-0.11 micromol/s/g hemoglobin, and the removal rates using 0.2 mM NADH and NADPH were 2.02+/-0.20 and 1.96+/-0.31 micromol/s/g hemoglobin, respectively. We deduced that the removal reaction by hemoglobin included formations of methemoglobin and the ferryl radical and reduction of the latter with pyridine nucleotides. The hydrogen peroxide removal ability by hemoglobin was less than that by catalase but was larger than that by glutathione peroxidase-glutathione reductase system at 0.2 mM hydrogen peroxide. Under acatalasemic conditions, it was suggested that NAD(P)H were important factors to prevent the oxidative degradation of hemoglobin.

V-51 and Mn-55 NMR studies of metal carbonyls.

The metal chemical NMR shifts of metal carbonyls are reported and discussed in relation to the bonding nature characteristic of carbonyl compounds of the M(CO)6- and Mn(CO)5X-types, where M is V or Mn, and X is a halogen or SCN. Theoretically evaluated chemical shift data based upon Ramsey’s expression agree well with those observed for the M(CO)6-type carbonyls. Regarding Mn(CO)5X, the variation of the shifts for halogens is elucidated theoretically and revealed to be dependent upon the π-bonding nature between the metal and the halogen atom.

Characterization of Acatalasemic Erythrocytes Treated with Low and High Dose Hydrogen Peroxide HEMOLYSIS AND AGGREGATION

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mm hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mm, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (·Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.

Laser photolysis of pyrenesulfonate and pyrenetetrasulfonate via two-photon ionization in aqueous and reverse micellar solutions

Abstract We investigated the laser photolysis of tetrasodium 1,3,6,8-pyrene-tetrasulfonate (Na 4 PS 4 ) and sodium 1-pyrenesulfonate (NaPS) in aqueous and reverse micellar solutions. The photoproducts as well as their yields were found to strongly depend on the reaction parameters such as pH, dissolved gases and the size of water pool. The primary reaction in aqueous solution was commonly presented by laser-induced formation of the cation radicals followed by hydroxylation of them. In the case of PS 4 4− , pyranine was efficiently and highly selectively formed, possibly via desulfonation of the cation radical, P + S 4 4− , followed by the hydroxylation. On the other hand, from PS − , hydroxypyrenesulfonate (PSOH) was initially formed in alkaline solution and desulfonation occurred secondarily, leading to the formation of hydoxypyrene. These photoreactions were markedly suppressed within a small water pool of the reverse micelle. As the size of water pool increased similar photochemical reactions occurred depending on the dissolved gases. The observed micellar effects could be explained based on the pH of water pool and the nature of the cation radicals in it.

(1Z,2Z)-1,2-Bis(3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene)hydrazine

The title compound, C16H14N4S2, crystallizes in symmetry group C2. The molecule is planar with C2h symmetry, with the inversion centre at the mid-point of the hydrazine N-N bond, and it has an N-N s-trans conformation and a Z,Z configuration. The particular crystal examined was a racemic twin, as suggested by the Flack parameter of 0.41 (2) [Flack (1983). Acta Cryst. A39, 876-881].