TY - JOUR
T1 - Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems
AU - Mutaguchi, Yuta
AU - Ohmori, Taketo
AU - Sakuraba, Haruhiko
AU - Yoneda, Kazunari
AU - Doi, Katsumi
AU - Ohshima, Toshihisa
N1 - Funding Information:
This work was supported by a grant for Promotion of Basic Research Activities for Innovate Bioscience from the Bio-oriented Technology Research Advancement Institution (BRAIN) . We thank M. Yohda for preparation of the hybrid plasmid pPH0670E containing the P. horikoshii AspR gene, S. Furuya for preparation of testes from male C57BL/6J mice, and H. Akano for his kind gift of Chinese vinegar.
PY - 2011/2/1
Y1 - 2011/2/1
N2 - 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.
AB - 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.
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U2 - 10.1016/j.ab.2010.10.016
DO - 10.1016/j.ab.2010.10.016
M3 - Article
C2 - 20951671
AN - SCOPUS:78650297006
SN - 0003-2697
VL - 409
SP - 1
EP - 6
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -