Postcolumn co-immobilized leucine dehydrogenase-NADH oxidase reactor for the determination of branched-chain amino acids by high-performance liquid chromatography with chemiluminescence detection.
A liquid chromatographic system with a co-immobilized leucine dehydrogenase-NADH oxidase reactor is described for the determination of branched-chain amino acids such as I-leucine, I-isoleucine and I-valine. The enzymes were simultaneously immobilized on tresylate-containing poly(vinyl alcohol) beads. The separation was achieved by means of an ODS column with elution with phosphate buffer (pH 7.5). The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The system gave a linear response from 0.3 to 300 mu M for each amino acid and the detection limit was 0.1 mu M.
Kiba
N
N
Department of Applied Chemistry and Biotechnology, Yamanashi University, Kofu, Japan.
Oyama
Y
Y
Kato
A
A
Furusawa
M
M
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000597:Amino Acids, Branched-Chain / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D050803:Leucine Dehydrogenase; D008163:Luminescent Measurements; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D012016:Reference Values
D000597:Amino Acids, Branched-Chain; D004800:Enzymes, Immobilized; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; D009247:NADH, NADPH Oxidoreductases