Atsushi Taga

Determination of the association constant of monovalent mode protein—sugar interaction by capillary zone electrophoresis

Protein-sugar interaction was observed by capillary zone electrophoresis, using a few beta-galactose-specific lectins and lactobionic acid as protein and sugar models, respectively. The lectin peaks were retarded in a concentration-dependent manner by addition of lactobionic acid in a carrier, and association constants of monovalent mode interactions could be obtained from t1 (migration time of protein), t2 (migration time of complex, obtainable as the migration time at the plateau) and the slope of the (t-t1)-1 vs. [S]-1 plots, where t and [S] are the migration of protein in the presence of lactobionic acid and the concentration of lactobionic acid, respectively. The values for Ricinus communis agglutinin, peanut agglutinin and soy bean agglutinin at pH 6.8 were 3.3 . 10(3), 9.1 . 10(2) and 1.1 . 10(2)1 mol-1, respectively. This method required only small amounts of protein samples and was reproducible. The amount of the sugar could be minimized under the conditions that the carrier was a buffer containing the sugar whereas the electrode solutions consisted only of the buffer.

Derivatization at capillary inlet in high-performance capillary electrophoresis Its reliability in quantification

Abstract The possibility of using derivatization at the capillary inlet for quantitative analysis by capillary electrophoresis was explored using amino acids as model compounds. It was shown that by using an appropriate introduction reagent and sample introduction program, i.e. reagent, then sample and once again reagent (so-called sandwich mode), selected amino acids were derivatized in high yields with o-phthaladehyde as in pre-capillary derivatization. Analyses of a number of amino acids by this at-inlet derivatization technique, followed by separation in a low pH phosphate buffer containing CHAPS, demonstrated the efficacy of this technique in automated amino acid analysis.

Analysis of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone derivatives by capillary/microchip electrophoresis and capillary electrochromatography

The 1-phenyl-3-methyl-5-pyrazolone (PMP) method has many advantages over hitherto reported methods based on reductive amination and hydrazone formation. This short review summarizes the various aspects of the PMP method, including the principle of derivatization, the simplicity of derivatization procedure, the high sensitivities to UV monitoring and ESI-MS, and the diversity of separation modes in capillary electrophoresis, and presents a number of application data for carbohydrate analysis in biological samples by this method. It also describes successful automation of carbohydrate analysis by in-capillary derivatization with PMP and miniaturization to microchip electrophoresis with whole channel UV detection allowing rapid (within 1 min) analysis of small amounts of PMP derivatives of carbohydrates. Furthermore, it discusses the possibility of capillary electrochromatography in carbohydrate analysis as PMP derivatives, and proposes an in-capillary modification strategy for improving column efficiency and elution time reproducibility.

Derivatization of amino acids in a moving zone of o-phthalaldehyde in the middle of a capillary for amino acid analysis by capillary electrophoresis

Problems in in-situ derivatization in a narrow zone in the middle of a capillary was investigated using a model system composed of amino acids and o-phthalaldehyde (OPA). Hydrostatic introduction of a solution of amino acid(s),the running buffer and the OPA solution for specified periods of time in this order from the anodic end of a capillary, followed by application of an appropriate voltage between both ends, resulted in formation of the OPA derivatives of amino acids, which were immediately separated by zone electrophoresis while moving through the rest of the capillary and detected at the detector window. Peak intensity was varied among amino acids, due to the variation of zone-overlapping period, accordingly reaction time. This technique of in-capillary derivatization was liable to be affected by various factors, including sample/reagent introduction times and applied voltage, giving rather lower reproducibility of determination than the derivatization at the inlet. However, it was useful for kinetic studies of such rapid reactions as those adopted in this work as a model system.

Characteristic features of the throughout-capillary technique of in-capillary derivatization in capillary electrophoresis

Abstract Characteristic features of the throughout-capillary technique of in-capillary derivatization for capillary electrophoretic analysis were explored using a rapid derivatization model. Selected amino acids (phenylalanine, glycine and glutamic acid) were converted to their OPA derivatives, while they were moving through an electrophoretic solution containing OPA in the electric field, and the resultant OPA–amino acids were concurrently analyzed by zone electrophoresis with UV detection. A deep trough appeared based on sample–reagent displacement, and the base line was noisy and drifty, especially at high reagent concentrations. Peaks were fronting and peak width varied among amino acid species, presumably due to the variation of the difference in velocity between an amino acid and its OPA derivative. However, the throughout-capillary technique is the simplest of all techniques of pre-capillary and in-capillary derivatizations, and there was good linearity between relative peak area and amino acid concentration. The quantification was reproducible with R.S.D.∼3.5%. The rate constant of the derivatization reaction could be roughly estimated by plotting logarithm of amino acid concentration vs. reaction time. The obtained values were approximately identical with the values obtained by the zone-passing technique. This paper also compares this technique with other techniques of in-capillary derivatization with respect to peak area, signal-to-noise ratio and column efficiency.

High-performance capillary electrophoresis of O-glycosidically linked sialic acid-containing oligosaccharides in glycoproteins as their alditol derivatives with low-wavelength UV monitoring

Several O-glycosidically linked monosialooligosaccharides from glycoproteins were separated as their alditol derivatives in ca. 10 min in borate buffer (pH 9.6) containing sodium dodecyl sulfate (SDS), and sensitively detected at the 10(-4) M level by measuring absorption at 185 nm. Oligosaccharides having higher degree of polymerizations migrated faster, and N-acetyl- and N-glycolylneuraminic acid-containing oligosaccharide analogues could be resolved from each other under the conditions employed. Good linearity was demonstrated between 0.9 and 20 mM concentrations for relative response of N-acetylneuraminyllactose as a model compound to lactobionic acid as an internal standard. The detection limit was 0.2 mM, which corresponded to 0.80 pmol as the injected amount. The relative standard deviation of relative response at 9 mM was 1.97% (n = 7). The established system was successfully applied to microanalysis of sialooligosaccharides in bovine submaxillary mucin and swallow nest material.

Unusual separation of 1-phenyl-3-methyl-5-pyrazolone derivatives of aldoses by capillary zone electrophoresis

The 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of aldoses showed unusual behavior in capillary zone electrophoresis in neutral buffers, giving excellent separation. Examination of the migration behavior of the PMP derivatives of aldopentoses and aldohexoses indicated that the derivatives of epimers having the 2,3-trans hydroxyl groups migrated faster than those having the 2,3-cis hydroxyl groups. This phenomenon can be explained by intramolecular ring formation by hydrogen bonding between the carbonyl group in the pyrazolone ring and the hydroxyl groups at C-2 and C-3 in the carbohydrate moiety. The 2,3-trans disposition will be favorable for ring formation and thereby cause reduction of the negative charge due to suppressed enolization.

Determination of cefixime and its metabolises by high-performance capillary electrophoresis

Cefixime (CX), an oral cephalosporin antibiotic, and its metabolites in human digestive organs were separated by various modes of high-performance capillary electrophoresis. The zone electrophoresis mode in phosphate buffer (pH 6.8) containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate gave the best separation, permitting the complete resolution of CX and all of five metabolites. On the other hand, the plain zone electrophoresis mode in phosphate buffer (pH 6.8) offered a simple procedure for the direct determination of urinary CX concentration using intact urine samples.

Mangiferin induces apoptosis by suppressing Bcl-xL and XIAP expressions and nuclear entry of NF-κB in HL-60 cells

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-d-glucoside (C-glucosylxanthone), is a xanthone derivative that is widely distributed in higher plants. Recently, mangiferin was found to exhibit potential antitumor effects. However, the molecular mechanisms of this effect have not been elucidated. In the present study, we attempt to clarify the mechanism of mangiferin-induced apoptosis in the human acute myeloid leukemia cell line HL-60; mangiferin was found to induce apoptosis. We also observed a concurrent increase in caspase-3 activity and DNA fragmentation. Furthermore, on examining the survival signals expressed during apoptotic induction, we observed that mangiferin caused a remarkable decrease in the nuclear entry of NF-κB p65. However, there were no changes in the expression of other survival signals, such as extracellular signal-regulated kinase 1/2, protein kinase B, and p38 mitogenactivated protein kinase. In addition, mangiferin suppressed the expressions of Bcl-xL and XIAP; however, we did not note any changes in the levels of Bcl-2, Bax, and Bim. These results indicate that mangiferin induces apoptosis by suppressing NF-κB activation and expressions of Bcl-xL and XAIP. These findings suggest that mangiferin may be useful as an anticancer agent and can be used in combination therapy with other anticancer drugs for the treatment of acute myeloid leukemia.

Simultaneous determination of the association constants of oligosaccharides to a lectin by capillary electrophoresis

Abstract The association constants ( K a values) of oligosaccharides as 8-amino-1,3,6-naphthalenetrisulfonate (ANTS) or 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives to lectins were determined in the reverse (an oligosaccharide derivative as sample and a lectin as additive) system by electrophoresis in a capillary coated with linear polyacrylamide. The determination was as reliable as in the direct (a lectin as sample and an oligosaccharide derivative as additive) system by electrophoresis in a capillary base fused silica. The reverse system had the advantage that the K a values of multiple oligosaccharides could be determined simultaneously. Thus, the K a values of lactose and melibiose (as PMP derivatives) to M r 60 000 Ricinus communis agglutinin could simultaneously be determined by this method. The K a values of a number of isomaltooligosaccharides (as ANTS derivatives) having various degrees of polymerization (DPs) to M r 46 000 Lens culinaris agglutinin could also be simultaneously determined. The K a values gradually increased with DP, implying the participation of not only the non-reducing end but also the interior portion of the oligosaccharide chain. The K a value of an oligosaccharide obtained in the multiple solute reverse system was consistent with that obtained by the single solute reverse system, demonstrating the reliability of this simultaneous determination.