Visible wavelength spectrophotometric assays of L-aspartate and D-aspartate using hyperthermophilic enzyme systems.
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.
Mutaguchi
Yuta
Y
Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Ohmori
Taketo
T
Sakuraba
Haruhiko
H
Yoneda
Kazunari
K
Doi
Katsumi
K
Ohshima
Toshihisa
T
D019342:Acetic Acid / Q000737:chemistry; D000591:Amino Acid Isomerases / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000032:analysis*; D026603:D-Aspartic Acid / Q000032:analysis*; D029968:Escherichia coli Proteins; D007536:Isomerism; D008099:Liver / Q000737:chemistry; D008773:Methylphenazonium Methosulfate / Q000031:analogs & derivatives / Q000737:chemistry; D051379:Mice; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D013053:Spectrophotometry / Q000379:methods*; D013552:Swine
D029968:Escherichia coli Proteins; D009243:NAD; D008773:Methylphenazonium Methosulfate; D001224:Aspartic Acid; D026603:D-Aspartic Acid; C023530:1-methoxy-5-methylphenazinium methyl sulfate; D000594:Amino Acid Oxidoreductases; C025745:aspartate dehydrogenase; C031955:L-aspartate oxidase, E coli; D000591:Amino Acid Isomerases; C019749:aspartate racemase; D019342:Acetic Acid