Simultaneous Determination of Enzyme Activity and Enzyme Quantity in Single Human Erythrocytes

✍ Scribed by W.H. Tan; E.S. Yeung

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 511 KB
• Volume: 226
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1193

✦ Synopsis

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. 1995 Academic Press, Inc.