Analysis of complexes of metabolites with europium tetracycline using capillary electrophoresis coupled with laser-induced luminescence detection.

Abstract

Glycolysis and Krebs cycle intermediates were incubated with Eu3+-tetracycline and separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. 3-phopshoglycerate, phosphoenolpyruvate, adenosine diphosphate, phosphate, citrate and oxaloacetate were detected at a concentration of 100μM or lower. When all these detected metabolites were contained within the same sample it was found that they interfered with one another. Of all the metabolites, oxaloacetate showed the highest detectability. The system was found to yield a linear response for oxaloacetate from 50nM to 10μM. The injected volume of sample was 400 pL. This corresponds to 2×10-17mol of injected oxaloacetate from the 50nM sample. As an application, the system was used to assay the enzyme aspartate aminotransferase, for whom oxaloacetate is a product. After a 1h incubation period, 1.2×10-13 M (3.3 μU/mL) enzyme was sufficient to form a detectable product signal. Extension of this incubation to 18h permitted the detection of the activity of 1.2×10-14 M (330 nU/mL) enzyme. This is the equivalent of 4.8 ymoles (2.9 molecules) of enzyme in the 400 pL injection volume. The enzyme's catalytic rate was determined to be 240s-1 under the conditions used. In a second application, homogenates of Drosophila melanogaster were analyzed for metabolites, providing several peaks, including one which had the same retention time as citrate.