Premium
Potentiometric determination of coenzyme‐dependent oxidoreductase enzymes with recycling of the coenzyme
Author(s) -
Wallace Thomas C.,
Coughlin Robert W.
Publication year - 1978
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260200907
Subject(s) - cofactor , nad+ kinase , chemistry , ferricyanide , enzyme , oxidoreductase , biochemistry , potentiometric titration , coenzyme a , redox , ferrocyanide , chromatography , reductase , inorganic chemistry , electrode
By enzymatically establishing a rapid (essentially equilibrium) coupling of a redox coenzyme such as NAD with the components of the ferrocyanide–ferricyanide half‐cell (e.g., using excess diaphorase) the half‐cell potential can be used to monitor another enzymatic reaction involving the same coenzyme. This approach provides a general, rapid potentiometric method of assaying coenzyme‐dependent oxidoreductase enzymes. We show that these assay systems can be designed for multiple turnover of coenzyme (in our case NAD) during a single assay thereby amplifying the rate of electromotive force (emf) change with a concomitant increase in sensitivity of enzyme assay. This allows the use of small concentrations of coenzyme and extension of the range of enzyme concentrations that may be assayed.