Determination of Chitin Based on the Colorimetric Assay of Glucosamine in Acidic Hydrolysate.

A colorimetric method for the glucosamine (GlcN) assay was applied for the determination of chitin, which can be hydrolyzed to produce GlcN. A 10-mg sample was mixed with 10 mL of a 5 mol/L HCl aqueous solution, and the mixture was kept at 100°C for 12 h. Under these conditions, chitin was completely depolymerized and deacetylated to produce GlcN, even when the sample was a crab shell. A 20-µL aliquot of the hydrolysate was mixed with 20 µL of a 5 mol/L NaOH aqueous solution and 200 µL of a 50 mmol/L Na2SiO3, 600 mmol/L Na2MoO4, 1.5 mol/L CH3COOH and 30% (v/v) dimethyl sulfoxide solution. The mixture was kept at 70°C for 30 min. In the mixture, GlcN reduced the Mo(VI) species to form a blue molybdosilicate anion, which gave an absorbance maximum at around 750 nm. Since N-acetylglucosamine and chitin oligosaccharides could not render the reaction mixture blue, GlcN in the hydrolysate could be assayed colorimetrically with high selectivity. When a standard chitin sample was examined, the GlcN concentration in the hydrolysate was determined to be 0.97 ± 0.02 g/L (as hydrochloride salt), indicating that the sample contained 10.0 ± 0.2 mg chitin (as an N-acetylglucosamine homopolymer). Calcium cation, amino acids, and proteins did not interfere with the GlcN assay. Thus, the proposed method was successfully applied to determine chitin in a crab shell sample.

Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes consisting of catalytic domains. The substrate specificities of adenylation (A) domains determine the amino-acid building blocks to be incorporated during nonribosomal peptide biosynthesis. The A-domains mediate ATP-dependent activation of amino-acid substrates as aminoacyl-O-AMP with pyrophosphate (PPi) release. Traditionally, the enzymatic activity of the A-domains has been measured by radioactive ATP-[(32)P]-PPi exchange assays with the detection of (32)P-labeled ATP. Recently, we developed a colorimetric assay for the direct detection of PPi as a yellow 18-molybdopyrophosphate anion ([(P2O7)Mo18O54](4-)). [(P2O7)Mo18O54](4-) was further reduced by ascorbic acid to give a more readily distinguishable blue coloration. Here we demonstrate the lab protocols for the colorimetric assay of PPi released in A-domain reactions.

Microplate Assay of α-Glucosidase and Its Inhibitors Based on the Direct Reduction of Molybdosilicate by Glucose.

A colorimetric method for monosaccharide determination (Anal. Sci., 2013, 29, 1021) was optimized for the high-throughput screening of α-glucosidase, which hydrolyzes an α-1,4-glycosidic bond of starch and related oligo- and polysaccharides, followed by the release of D-glucose from the non-reducing ends. In a microplate, 40 µL of a sample solution was mixed with 160 µL of a 50 mM Na2SiO3, 600 mM Na2MoO4, 1.5 M CH3COOH, and 20% (v/v) dimethyl sulfoxide solution, which was yellowish due to the formation of a yellow molybdosilicate. The mixture was kept at 80°C for 60 min. In the mixture, glucose reduced the Mo(VI) species directly to form a blue heteropolymolybdate(V/VI). Thus, 0.1 mM level glucose can be determined by the color change from yellow to blue. Since maltose cannot render the mixture blue as strongly as glucose, the present method has been successfully applied to a microtiter plate assay of α-glucosidase with the disaccharide. Also, the method has been applied to an assay of α-glucosidase inhibitors, acarbose and quercetin.

Simple and rapid separation of soyasaponin Bb from a soy extract.

A simple method to separate soyasaponin Bb from a soy extract is presented. This method is based on the difference in the solubility of soyasaponin Bb and Ba and other components into 3:7 and 1:1 (v/v) acetone-water mixed solvents. The crude soyasaponin consisting of soyasaponins Aa, Ab, Ba, and Bb at the 10 wt% level and other components was examined as the soy extract. A 10 mg quantity of the crude soyasaponin was mixed with 1 mL of the 3:7 acetone-water containing 0.1 mol/L HCl, and the supernatant was removed to obtain a precipitate, which was found to contain mainly soyasaponins Bb and Ba. The precipitate was mixed with 0.4 mL of the 1:1 acetone-water containing 0.1 mol/L HCl; the supernatant was transferred, and was mixed with 0.6 mL of water to obtain a precipitate, which was found to contain mainly soyasaponin Bb. The yield was ca. 30%, which may be much higher than that by the conventional preparative chromatographic approach. The separation method is rapid and easy to carry out, and is useful for the preparation of a soyasaponin Bb sample.

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase.

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na2SiO3, 600 mM Na2MoO4, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.

Colorimetric determination of pyrophosphate anion and its application to adenylation enzyme assay.

A colorimetric pyrophosphate assay based on the formation and reduction of the 18-molybdopyrophosphate ([(P2O7)Mo18O54](4-)) anion in an acetonitrile-water mixed solvent was modified and improved. The [(P2O7)Mo18O54](4-) anion is precipitated from the acetonitrile-water solution containing MoO4(2-) and HCl, and is re-dissolved in neat acetonitrile or propylene carbonate. This separation process decreases the interference by ATP, and prevents a yellow coloration of the reducing agent, ascorbic acid, due to excess Mo(VI) species. In the organic solvent, the [(P2O7)Mo18O54](4-) anion is reduced to a more intense blue molybdopyrophosphate species. The application of the colorimetry to the assay of adenylation enzymes is also described in this note.