Ultramicro enzyme assays in a capillary electrophoretic system.
This paper describes an ultramicro method for achieving enzyme assays. Enzyme saturating concentrations of substrate, coenzyme when appropriate, and running buffer were mixed and used to fill a deactivated fused-silica capillary in a capillary zone electrophoresis apparatus. The enzyme glucose-6-phosphate dehydrogenase was injected by either electrophoresis or siphoning and mixed with the reagents in the capillary by electrophoretic mixing. Enzyme activity was assayed by electrophoresing the product, reduced nicotinamide adenine dinucleotide phosphate, to the detector where it was detected at 340 nm. Under constant potential, the transport velocity of enzyme and the product was generally different. This caused product to be separated from the enzyme after it was formed. Because product formation was much faster than the rate of enzyme-product separation, product accumulated. The amount of accumulated product was inversely related to operating potential. In the extreme case, the operating potential was zero. Zero potential assays were generally carried out by electrophoresing the enzyme partially through the capillary and then switching to zero potential. This capillary was left at zero potential for several minutes to allow additional product to accumulate. After this additional amplification step, potential was again applied and the product transported to the detector. Product formed under constant potential appears as a broad peak with a flat plateau. When the voltage is switched to zero at intermediate migration distance, a peak will be observed on top of this plateau. Either the eight of the plateau or the area of the peak may be used to determine enzyme concentration. The lower limit of detection was 4.6.10(-17) mol of glucose-6-phosphate dehydrogenase.
Bao
J
J
Department of Chemistry, Purdue University, West Lafayette, IN 47907.
Regnier
F E
FE
D004586:Electrophoresis; D004798:Enzymes / Q000032:analysis*; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D007202:Indicators and Reagents; D009249:NADP / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D004798:Enzymes; D007202:Indicators and Reagents; D009249:NADP; D005954:Glucosephosphate Dehydrogenase