Enzyme-based flow injection analysis system for glutamine and glutamate in mammalian cell culture media.
We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the technical setup are presented in this paper.
Osterreichische Akademie der Wissenschaften-APART, Institut für Biochemie und Molekulare Zellbiologie der Universität Wien und Ludwig Boltzmann Forschungsstelle für Biochemie, Dr. Bohrgasse 9, Vienna, A-1030, Austria.
C
C
Mayer
A
A
Frauer
T
T
Schalkhammer
F
F
Pittner
D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry*; D004800:Enzymes, Immobilized; D004885:Erwinia / Q000201:enzymology; D017022:Flow Injection Analysis / Q000379:methods*; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D018698:Glutamic Acid / Q000032:analysis*; D005973:Glutamine / Q000032:analysis*; D008322:Mammals
D003470:Culture Media; D004800:Enzymes, Immobilized; D005973:Glutamine; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase; D001219:Aspartate Aminotransferases