Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Yuta Mutaguchi, Taketo Ohmori, Haruhiko Sakuraba, Kazunari Yoneda, Katsumi Doi, Toshihisa Ohshima

研究成果: ジャーナルへの寄稿記事

10 引用 (Scopus)

抄録

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes - aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO) - to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD+)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD+-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.

元の言語英語
ページ(範囲)1-6
ページ数6
ジャーナルAnalytical Biochemistry
409
発行部数1
DOI
出版物ステータス出版済み - 2 1 2011

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Aspartic Acid
Assays
Wavelength
Enzymes
aspartate racemase
NAD
Oxidoreductases
Formazans
Food
High performance liquid chromatography
Cell Extracts
Oxidation-Reduction
Reaction rates
High Pressure Liquid Chromatography
Cells

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

これを引用

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems. / Mutaguchi, Yuta; Ohmori, Taketo; Sakuraba, Haruhiko; Yoneda, Kazunari; Doi, Katsumi; Ohshima, Toshihisa.

:: Analytical Biochemistry, 巻 409, 番号 1, 01.02.2011, p. 1-6.

研究成果: ジャーナルへの寄稿記事

Mutaguchi, Yuta ; Ohmori, Taketo ; Sakuraba, Haruhiko ; Yoneda, Kazunari ; Doi, Katsumi ; Ohshima, Toshihisa. / Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems. :: Analytical Biochemistry. 2011 ; 巻 409, 番号 1. pp. 1-6.
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