Kunihiko Tajima

One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells.

BackgroundIsolation of a concentrated, living preparation of malarial parasite-infected red blood cells (PRBCs) that have low contamination of white blood cells (WBCs) facilitates research on the molecular, biochemical and immunological aspects of malarial parasites. This is currently carried out by a two-step method, including the concentration of PRBCs using density gradient centrifugation through Percoll or Nycodenz, followed by the removal of host WBCs using a cellulose powder column or a commercially available filtration unit. These two-step methods can help isolate sufficient PRBCs, but they are laborious. In this study, a simplified one-step procedure that takes advantage of the difference between diamagnetic low-spin oxyhaemoglobin and paramagnetic haemozoin (haem polymer) was described. The paramagnetic polymer is deposited in the food vacuoles of the parasite, allowing the use of magnetic separation to efficiently and rapidly concentrate PRBCs while removing contaminating host WBCs.MethodsThe magnetic removal of WBCs using a commercial LD column (MACS) was evaluated as a new method for concentrating and purifying PRBCs. To compare this method with the two density gradient centrifugation methods using Percoll or Nycodenz, we analysed the quantities of enriched PRBCs and contaminating host WBCs as well as the viability of malarial parasites in the final preparations.ResultsThe quantity of PRBCs and the viability of malarial parasites in the isolated PRBCs were similar between magnetic and centrifugation methods. However, 90–99% of the contaminating WBCs were removed from the starting material using a magnetic column, whereas WBC content did not change using the Percoll or Nycodenz methods.ConclusionThe use of a commercially available magnetic LD column is effective, safe and easy for the one-step purification of PRBCs. This simple method does not affect the viability of malarial parasites.

Identification and characterization of L-arabonate dehydratase, l-2-keto-3-deoxyarabonate dehydratase and L-arabinolactonase involved in an alternative pathway of l-arabinose metabolism: Novel evolutionary insight into sugar metabolism

Azospirillum brasiliense possesses an alternative pathway of l-arabinose metabolism, different from the known bacterial and fungal pathways. In the preceding articles, we identified and characterized l-arabinose-1-dehydrogenase and α-ketoglutaric semialdehyde dehydrogenase, which catalyzes the first and final reaction steps in this pathway, respectively (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623 and Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888). We here report the remaining three enzymes, l-arabonate dehydratase, l-2-keto-3-deoxyarabonate (l-KDA) dehydratase, and l-arabinolactonase. N-terminal amino acid sequences of l-arabonate dehydratase and l-KDA dehydratase purified from A. brasiliense cells corresponded to those of AraC and AraD genes, which form a single transcriptional unit together with the l-arabinose-1-dehydrogenase gene. Furthermore, the l-arabinolactonase gene (AraB) was also identified as a component of the gene cluster. Genetic characterization of the alternative l-arabinose pathway suggested a significant evolutional relationship with the known sugar metabolic pathways, including the Entner-Doudoroff (ED) pathway and the several modified versions. l-Arabonate dehydratase belongs to the ILVD/EDD family and spectrophotometric and electron paramagnetic resonance analysis revealed it to contain a [4Fe-4S]2+ cluster. Site-directed mutagenesis identified three cysteine ligands essential for cluster coordination. l-KDA dehydratase was sequentially similar to DHDPS/NAL family proteins. d-2-Keto-3-deoxygluconate aldolase, a member of the DHDPS/NAL family, catalyzes the equivalent reaction to l-KDA aldolase involved in another alternative l-arabinose pathway, probably associating a unique evolutional event between the two alternative l-arabinose pathways by mutation(s) of a common ancestral enzyme. Site-directed mutagenesis revealed a unique catalytic amino acid residue in l-KDA dehydratase, which may be a candidate for such a natural mutation.

Reaction of biological phenolic antioxidants with superoxide generated by cytochrome P-450 model system.

Scavenging of a superoxide and simultaneous formation of free radicals with phenolic antioxidants were investigated with cobalt and iron tetraphenylporphyrin-thiolate complexes as models of P-450 enzymes. The kinetics of decay of the superoxide and development of free radical ESR signal intensities were studied. Based on a molecular orbital calculation of the hyperfine splitting, the radical species generated were confirmed to be those of phenoxyl radicals. The biological implication of superoxide, including active oxygen forms, for the reaction was discussed.

Creation of a type 1 blue copper site within a de novo coiled-coil protein scaffold.

Type 1 blue copper proteins uniquely coordinate Cu(2+) in a trigonal planar geometry, formed by three strong equatorial ligands, His, His, and Cys, in the protein. We designed a stable Cu(2+) coordination scaffold composed of a four-stranded α-helical coiled-coil structure. Two His residues and one Cys residue were situated to form the trigonal planar geometry and to coordinate the Cu(2+) in the hydrophobic core of the scaffold. The protein bound Cu(2+), displayed a blue color, and exhibited UV-vis spectra with a maximum of 602-616 nm, arising from the thiolate-Cu(2+) ligand to metal charge transfer, depending on the exogenous axial ligand, Cl(-) or HPO(4)(2-). The protein-Cu(2+) complex also showed unresolved small A(∥) values in the electron paramagnetic resonance (EPR) spectral analysis and a 328 mV (vs normal hydrogen electrode, NHE) redox potential with a fast electron reaction rate. The X-ray absorption spectrum revealed that the Cu(2+) coordination environment was identical to that found in natural type 1 blue copper proteins. The extended X-ray absorption fine structure (EXAFS) analysis of the protein showed two typical Cu-N(His) at around 1.9-2.0 Å, Cu-S(Cys) at 2.3 Å, and a long Cu-Cl at a 2.66 Å, which are also characteristic of the natural type 1 blue copper proteins.

Rotation and orientation of axially coordinated imidazoles in low-spin ferric porphyrin complexes

Abstract The variable temperature 1H NMR spectra of a series of low-spin bis(imidazole)tetrakis(2, 4, 6-trialkylphenyl)porphinatoiron(III) chlorides, (R-TPP)Fe(L)2Cl, showed that the rotation of axially coordinated 2-alkyl- and benzimidazoles slowed down on the NMR time scale at low temperature to give four pyrrole signals. The 13C NMR spectra of the 13C enriched (Me-TPP)Fe(2- methylimidazole)2Cl at the meso positions gave two meso signals with equal intensity at the temperature range below −25 °C. These results indicate that each of the axial ligands is perpendicularly aligned over a diagonal CmesoFeCmeso axis. The shift range of the pyrrole protons reached as much as 12 ppm at −56 °C. The apparent unfavorable orientation of the ligands was explained in terms of the S4 deformed structure of the porphinatoiron core in solution. The relatively small slopes in Curie plots of the pyrrole-H and the meso 13C signals were also ascribed to the deformed structure of the core. In the mixed ligand complexes having a hindered 2-isopropylimidazole and an unhindered 1-methylimidazole ligand, it was suggested from the 1H and 13C NMR splitting patterns that the rotation of only one of the ligands, 2-isopropylimidazole, was frozen. In these complexes the spread of the pyrrole signals increased to nearly 20 ppm. The activation free energies for rotation were determined by the dynamic NMR technique. They changed in the range of 11.3 to 13.6 kcal mol−1 depending upon the bulkiness of the axial ligands and o-alkyl substituents. Close examination of the dynamic process using the saturation transfer technique revealed that the dissociation of the axial ligands occurred concomitantly during the rotation process in the case of the bis(2-isopropylimidazole) complexes. In contrast, pure rotation process was observed in the complexes with 2-methyl-, 2-ethyl-, 1, 2-dimethyl- and benzimidazole. X band ESR spectra of these complexes were taken at 4.2 K in CH2Cl2 glass. Although complexes with perpendicularly aligned planar ligands tend to exhibit so called ‘strong gmax’ type signals, the complexes studied here showed signals with smaller gz values.

A possible model of hemoprotein-peroxide complexes formed in an iron-tetraphenylporphyrin system

A ferric low-spin species with an anomalously small g3-g1 separation generated by the reaction of Fe(III)tetraphenylporphyrin with t-butylhydroperoxide in the presence of tatramethylammonium hydroxide was characterized by ESR spectroscopy. The reaction kinetics, investigated by monitoring ESR intensity, indicated that this low-spin complex is highly reactive and easily changed to non-heme type iron complexes. The rhombic parameters of this complex are very similar to those of heme-peroxide adducts such as [Fe(II)-hemoglobin-O2]- and [Fe(II)-horseradish peroxidase-O2]-.

Creation of a binuclear purple copper site within a de novo coiled-coil protein.

Although various kinds of metal binding proteins have been constructed by de novo design, the creation of a binuclear metal binding site remains especially challenging. The purple copper site in subunit II of COX, referred to as the Cu(A) site, has two copper ions bridged by two Cys residues. We constructed the Cu(A) site consisting of two Cys and two His residues in a de novo designed four-helical coiled-coil protein. The protein bound two copper ions and exhibited a purple color, with relatively intense absorption bands at 488 and 530 nm in the UV-vis spectrum. The EPR spectrum displayed unresolved hyperfine splittings in the g(∥) region, which was similar to the native or engineered Cu(A) site with an A(∼480)/A(∼530) > 1. The extended X-ray absorption structure analyses of the protein revealed the presence of the Cu(2)S(2) core structure, with two typical N(His)-Cu bonds per core at 1.90 Å, two S (Cys)-Cu bonds at 2.21 Å, and the Cu-Cu bond at 2.51 Å, which are also characteristic structures of a purple copper site.

Generation of FeIIIOEP-hydrogen peroxide complex (OEP = octaethylporphyrinato) by reduction of FeIIOEP–O2 with ascorbic acid sodium salt

ESR and optical absorption spectra have been recorded for a frozen dimethylformamide (DMF) solution of the complex FeIIIOEP(-OH)(-OOH)(OEP = octaethylporphyrinato) prepared by mixing FeIIOEP(pyridine)–O2 with ascorbic acid sodium salt.

Spectroscopic characterization of coaxially and π—π stacked binuclear copper(II) complexes of ω,ω′-bis(pyridine-2-carboxamido)alkanes in acidic solution

Abstract Using electron spin resonance (ESR) and visible spectroscopies, coaxially stacked binuclear copper(II) complexes of ω,ω′-bis(pyridine-2-carboxamido)alkanes in acidic aqueous solution (pH 3.8–5.0), H2peda (alkane = ethane), H2ppda (alkane = propane), and H2pbda (alkane = butane), were characterized. trans-1,2-Bis(pyridine-2-carboxamido)cyclohexane or trans-H2pcyhda (alkane = trans-1,2-cyclohexane) did not provide coaxially stacked binuclear complex, because of the lower conformational freedom at the HNCH-CHNH bond. The coaxially stacked structure causes π—π stacking between the pyridyl rings. The conformation of the propanediyl chain is gauche—gauche, while the ethanediyl and butanediyl chains contain an eclipsed conformation. The driving force for the formation of these binuclear complexes in acidic solution is intramolecular π—π stacking interaction between the pyridyl rings in concert with hydrophobic interaction. Although H2ppda gave crystals of a binuclear complex with many lattice water molecules, H2peda and H2pbda ligands provided crystals of copper(II) polymer complexes from the respective acidic solution. The trans-H2pcyhda ligand forms a copper(II) binuclear complex with open-chain structure in acidic solution. Crystal structures of the polymer complexes are also presented.

Crystal structure and ESR study of a nitrodochromium(V) complex

Abstract The crystal structure of the nitridochromium(V) complex [CrN(salen)] (H2salen = N,N′-bis(salicylidene)ethylenediamine) has been determined. This complex crystallizes in the monoclinic system, space group P21/c, a = 9.575(1), b = 12.322(3), c = 12.999(2) A, β = 103.57(1)°, V = 1490.8(5) A 3 , Z = 4; final R2 = 0.059 (R = 0.054), wR2 = 0.082 (wR = 0.044) for 1935 observed reflections (I⩾1.5σ(l)). The coordination geometry is square pyramidal with an N2O2 donor set (LN202) in the basal plane and the nitride ion at the apex. The UV-Vis spectra of the complex have been re-measured in several solvents and exhibited a d-d band maximum around 540 nm (ϵ ≈200 M−1cm−1). This is the first example of an observed d-d band among the nitridochromium(V) complexes isolated so far. The ESR spectra have been also re-measured in more detail. The ESR parameters have been compared with those of the [CrN(LN4)] complexes and a remarkable nephelauxetic effect of the 3d sub-shell in comparison with the 3s sub-shell is indicated for the nitridochromium(V) complexes.