PubMed · 491159
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T Sakai. 1979-06-29. [LAP isozyme].. https://pubmed.ncbi.nlm.nih.gov/491159/
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Intracellular contents reflect the specific history of a cell including innate physiological heterogeneity as well as differing levels of exposure to environmental influences. A method capable of analyzing a variety of species from within a single human erythrocyte is demonstrated. Guided by a microscope, individual cells can be drawn into open capillaries of 10-microns i.d. On contact with a low ionic strength buffer solution, the cell lyses and releases its intracellular fluid. The ionic components are then separated by capillary electrophoresis. For glutathione, microderivatization with a fluorescent reagent can be accomplished in vitro with monobromobimane. The effects of extracellular oxidizing and reducing agents on the glutathione levels can thus be followed. For sodium and potassium, or any other ionic species, charge displacement of a fluorescent cation results in indirect fluorescence detection. The two detection modes are suitable for intracellular components present at low-attomole and sub-femtomole levels, respectively.
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.
Capillary isoelectric focusing (cIEF) and free zone capillary electrophoresis were evaluated for separation of native hemoglobins and globin chains. High-resolution separations of adult human hemoglobin A, fetal human hemoglobin F, and hemoglobin variants S and C were obtained using cIEF with cathodic mobilization. Absorbance detection in the UV and visible regions were compared, and on-line fast UV or visible-wavelength scanning detection was used to obtain spectral information on separated components. Globin chain analysis was performed on the same hemoglobin species by free zone capillary electrophoresis following precipitation of the protein with acidic acetone. Free zone separations were carried out at low pH in the presence of 7 M urea.