Dual-enzyme assay of glutamate in single cells based on capillary electrophoresis.
A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Glutamate dehydrogenase and glutamic pyruvic transaminase were used to catalyze the glutamate reaction. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. Glutamate dehydrogenase catalyzed the formation of NADH, and glutamic pyruvic transaminase drives the glutamate dehydrogenase reaction by removing a reaction product and regenerating glutamate. The detection limit of glutamate is down to the 10(-8) M level, which is 1 order of magnitude lower than previously reported detection limits based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most amino acids. The glutamate content in single human erythrocytes and baby rat brain neurons were determined with this method and the results agreed well with literature values.
Wang
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Department of Chemistry and Ames Laboratory-USDOE, Iowa State University, 50011, USA.
Yeung
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D000328:Adult; D000410:Alanine Transaminase / Q000378:metabolism*; D000818:Animals; D019075:Electrophoresis, Capillary; D004912:Erythrocytes / Q000737:chemistry; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D018698:Glutamic Acid / Q000032:analysis* / Q000097:blood; D006624:Hippocampus / Q000737:chemistry; D006801:Humans; D007834:Lasers; D009474:Neurons / Q000737:chemistry; D051381:Rats; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase; D000410:Alanine Transaminase