Simultaneous determination of enzyme activity and enzyme quantity in single human erythrocytes.
An integrated laser-based fluorescence enzyme assay and particle-counting immunoassay system with zeptomole sensitivity has been developed for simultaneous determinations of activity and concentration of enzymes in single erythrocytes. The product NADPH of the reaction between glucose-6-phosphate and NADP+ catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) is monitored. Simultaneously, the agglutination of antibody-coated particles in the presence of G6PDH produces large particles which are counted by light scattering. The correlation between the two parameters indicates that on average only about 35% of the enzyme in individual cells is active. There is more than a 10-fold cell-to-cell variation in the ratios of the two parameters. This indicates that there are multiple mechanisms for the degradation of intracellular enzymes as a function of cell age.
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Ames Laboratory-U.S. Department of Energy, Iowa 50011.
Yeung
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D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods*; D004586:Electrophoresis; D004912:Erythrocytes / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000097:blood*; D006801:Humans; D007124:Immunoenzyme Techniques; D007700:Kinetics; D007834:Lasers; D009249:NADP / Q000032:analysis
D009249:NADP; D005954:Glucosephosphate Dehydrogenase