Kenzaburoh Yao

Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction

A simple and rapid method for sialic acid determination in sialoglycoproteins by acidic ninhydrin reaction is described. The method is based on the reaction of sialic acids with an acidic ninhydrin reagent (K. Yao and T. Ubuka (1987) Acta Med. Okayama 41, 237-241). By heating a sample solution containing sialoglycoprotein with the reagent at 100 degrees C for 10 min, a stable color with an absorption maximum at 470 nm was produced. The standard curve was linear in the range of 20 micrograms to 3 mg of fetuin, a sialoglycoprotein, per 3.0 ml of the reaction mixture. The reaction is specific only for sialoglycoproteins among various proteins examined. The acidic ninhydrin method was applied to the determination of sialic acids in sialoglycoproteins in ascites fluids of Ehrlich ascites tumor-bearing mice.

High-performance liquid chromatographic determination of taurine and hypotaurine using 3,5-dinitrobenzoyl chloride as derivatizing reagent

A method for the determination of taurine and hypotaurine in biological samples involving the preparation of their 3,5-dinitrobenzoyl derivatives followed by HPLC was established. Taurine and hypotaurine in aqueous media were reacted with 3,5-dinitrobenzoyl chloride in the presence of triethylamine to prepare 3,5-dinitrobenzoyl derivatives. These derivatives were separated on a C18 reversed-phase column and detected by recording the absorbance at 254 nm. Derivatives of taurine and hypotaurine were obtained in yields of 91.4 +/- 3.3 and 85.6 +/- 2.6%, respectively. The calibration graphs for taurine and hypotaurine were linear between 2.5 and 500 microM with correlation coefficients of 0.999. The method was applied to the determination of taurine and hypotaurine in human and rat urine and blood and in rat liver and heart.

Preparation and characterization of S-[2-carboxy-1-1H-imidazol-4-yl)ethyl]glutathione and its derivatives as proposed precursors of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine, a compound found in human urine

Formation of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid (I) and S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (II), compounds found in human urine, has been demonstrated by enzymatic degradation of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (III). Compound (III) was chemically synthesized in 72% yield by incubating the reaction mixture of trans-urocanic acid and 3-fold excess GSH at 65 degrees C for 1 wk, which was accompanied by formation of N-(S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteinyl)glycine (IV) in 15% yield. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-N-gamma-glutamylcysteine (V) was produced by partial hydrolysis of compound (III) in HCl. The synthesized compounds were characterized mainly by fast-atom bombardment mass spectrometry and high-voltage paper electrophoresis as well as chemical degradation. Incubation of compound (III) with rat kidney homogenate in a Tris buffer (pH 8), formed compound (II) in 80% yield possibly via compound (IV). Yield of compound (II) was increased by adding glycylglycine to the reaction mixture. However, little degradation of compound (III) occurred in the use of rat liver, brain, heart or spleen homogenate as the enzyme source. Compound (II) was further metabolized to compound (I) by incubation with rat kidney homogenate in a phosphate buffer of pH 7.4. From these results, we suggest that the urinary compounds are products of enzymatic degradation of compound (III) and that GSH may participate in the metabolism of urocanic acid, the first catabolite of L-histidine.

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine in normal human urine.

SummaryA compound, which had the same mobility on a high-voltage paper electrophoretogram and the sameRF value on a thin-layer chromatogram as those ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (I), was partially purified from human urine by ion-exchange column chromatography. The compound gave a signal at m/z 260 on its FAB mass spectrum, which was assigned as MH+ of compound I. These results suggest that the urinary compound is compound I and it is a physiological precursor of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid [Kinuta et al., (1991) Biochem J 275: 617–621].

Oxidative deamination of L-cystine by L-amino acid oxidase from snake venom: Formation of S-(2-oxo-2-carboxyethylthio)cysteine and S-(carboxymethylthio)cysteine

Summary Oxidative deamination of L-cystine by snake venom L-amino acid oxidase from Crotalus terrificus terrificus was studied. Formation of S-(2-oxo-2-carboxyethylthio)cysteine (OCETC) was shown by ion-exchange chromatography and by its reduction to S-(2-hydroxy-2-carboxyethylthio)cysteine by sodium borohydride at pH 3. In the absence of catalase in the reaction mixture, S-(carboxymethylthio)cysteine was formed through decarboxylation of OCETC. OCETC was shown to be further deaminated to give a ninhydrin-negative disulfide.

Assay of sialidase activity using ion-exchange chromatography and acidic ninhydrin reaction

A new assay method for sialidase (EC 3.2.1.18) activity using ion-exchange chromatography and acidic ninhydrin reaction has been developed. Fetuin, 4-methylumbelliferyl-N-acetylneuraminic acid (MUB-NANA), gangliosides and N-acetylneuramin-lactose were examined as substrates. Free sialic acid liberated from these substrates by sialidase reaction was isolated with a Dowex 1-X8 column (trifluoroacetate form, 1.5 cm x 0.5 cm I.D.) and determined by acidic ninhydrin reaction. Among the substrates tested, MUB-NANA was the best in the present method, N-Acetylneuramin-lactose could not be used as the substrate, because it was not separated from liberated sialic acid under the conditions used. The recovery of N-acetylneuraminic acid was above 88%, and the sensitivity of the method was 20 nmol in 300 microliters of the reaction mixture. The method was applied to the sialidase assay during its purification from rat skeletal muscle, and a Michaelis constant of 1.15 mM was obtained with MUB-NANA as the substrate. The method using the acidic ninhydrin reaction was simple and exhibited good reproducibility.

Increase in cystathionine content in rat liver mitochondria after D,L-propargylglycine administration

SummaryIntraperitoneal administration of D,L-propargylglycine to rats resulted in an increase in the cystathionine content of whole liver and liver mitochondria. Cystathionine in mitochondria was identified by amino acid analysis, thin layer chromatography, high-voltage paper electrophoresis and liquid chromatography-mass spectrometry. The cystathionine content of whole liver was 5.37 ± 1.59µmol per g of fresh liver at 14 h after the administration of 50 mg of D,L-propargylglycine per kg of body weight, while 0.07 ± 0.02µmol of cystathionine per g of fresh liver was detected in the control rats. The cystathionine content of liver mitochondria from both groups of rats was 9.40 ± 1.20 and 0.19 ± 0.04 nmol of cystathionine per mg of protein, respectively. The mitochondrial cystathionine increased dose-dependently with the increase of D,L-propargylglycine administered. The increase was proportional to the time after the administration up to 12 h, and then decreased. The increase of cystathionine in the liver mitochondria was linearly proportional to that in the whole liver. These results suggest that cystathionine in liver mitochondria is in an equilibrium with that in the cytosol.

Preparation and analysis of a volatile derivative of cysteic acid

SummaryWe have reported preparations and gas chromatographic analyses of volatile derivatives of sulfuric acid and taurine (Masuoka et al., 1988; 1989). By extending these studies, we have developed a method for the gas chromatographic determination of cysteic acid. Cysteic acid was converted to the N-isobutoxycarbonyl derivative by the reaction with isobutyl chloroformate in the presence of sodium hydroxide. After desalting with a cation-exchange column, the derivative was converted to the silver salt by reacting with silver oxide. The resulting silver salt was quantitatively esterified with methyl iodide in the presence of dimethyl sulfate and silver oxide. Dimethyl N-isobutoxy-carbonylcysteate [methyl 2-(N-isobutoxycarbonylamino)-3-(methoxysulfonyl) propanoate] formed was analyzed by gas chromatography. The calibration curve was linear up to 5.0µmol per ml of cysteic acid and the recovery was more than 95%.