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.