A theoretical approach to some analytical properties of heterogeneous enzymatic assays.
Heterogeneous enzymatic assays (HEA), where an enzyme in solution acts upon an immobilized substrate, are been increasingly used. Given their high throughput and versatility they hold great potential for developing massive enzyme inhibitor screening. However, current HEA lack, in general, rigorous quantitative use. This is in part due to technical problems as a multiplicity of suboptimal substrate populations achieved with traditional immobilization techniques but, more importantly, is due to a poor understanding of the particular kinetic behavior of these systems. This paper addresses the kinetic features of HEA that arise from the very low amount of solid-phase substrate and the resulting inalterability of the free enzyme concentration during the assay, which classify HEA as enzyme quasi-saturable systems (EQSS). We assessed the optimal enzyme concentration working range and time of reaction. We also considered certain attributes of HEA for evaluating isosteric inhibitors. These studies were done on the basis of a simplified model for the kinetics of EQSS and a formal splitting of the functional factor of the analytical sensitivity of an enzymatic assay into [E(o)]/K(m)-dependent and temporal components.
Gutiérrez
O A
OA
Centro de Estudio de las Proteinas, Facultad de Biología, Universidad de la Habana, 25 # 455 entre J e I. CP 10 400, Vedado, Ciudad de la Habana, Cuba. gutierrez.arenas@biokem.uu.se
Chavez
M
M
Lissi
E
E
D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology; D004798:Enzymes / Q000032:analysis* / Q000737:chemistry* / Q000187:drug effects; D007124:Immunoenzyme Techniques / Q000379:methods*; D007700:Kinetics; D012680:Sensitivity and Specificity
D004791:Enzyme Inhibitors; D004798:Enzymes