Flow-Injection Analysis of Amino Acids and Their Metabolites by Immobilized Vitamin B6-Dependent Enzymes.

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Abstract
Sensitive flow-injection analyses of aspartate, glutamate, 2-oxoglutarate, and oxaloacetate were developed. The analytes were enzymatically coupled with NADH which was monitored by light emission from immobilized bacterial bioluminescence enzymes. Aspartate (or oxaloacetate) was assayed on the basis of NADH consumption by introducing the sample through a coimmobilized aspartate aminotransferase-malate dehydrogenase column. The assay responded linearly from 100 pmoles to 5 nmoles per assay. Glutamate (2-oxoglutarate) was determined by formation of NADH in the glutamate dehydrogenase reaction. The measuring range for glutamate was from 10 pmoles to 100 nmoles per assay. The precision of the flow-injection method was generally excellent, and the sensitivities of the described assays were 100-1000-fold higher than with spectrophotometric methods. The immobilized enzyme preparations were stable for several months in storage, and the enzyme columns could be used for 600-800 analyses. Flow-injection analyses of amino acids and related compounds by NADH/bioluminescence-coupled reactions provide a sensitive, fast, and inexpensive assay method for a wide variety of purposes.