Double mixing technique for linked enzyme assays

Abstract

A rapid double mixing technique has been applied to the reaction kinetics of enzymes usually analyzed by linked assay. The test enzyme is allowed to perform in the absence of the linking enzyme but then the convenience of enzymatic analysis of accumulated produce is exploited when a second mixing adds the linking enzyme. The analytical method is presented which enables the determination of the rate of product accumulation during the delay between mixings. Various advantages of the system in both steady-state and transient kinetic analyses are described. As an example, 3-phosphoglycerate kinase is assayed. The results address the proposal that the kinase interacts with the usual linking enzyme, glyceraldehyde-3-phosphate dehydrogenase, and also raise questions about product inhibition.