Application of capillary zone electrophoresis for the study of enzymes

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Project Identification
GA203/03/1125
Project Period
1/2003 - 1/2005
Investor / Pogramme / Project type
Czech Science Foundation
MU Faculty or unit
Faculty of Science
Keywords
Capillary Zone Electrophoresis; Enzymes; Electrophoretically Mediated Microanalysis; EMMA

The aim of the project is to improve and enlarge the applicability of CZE especially its modification EMMA (Electrophoretically Mediated MicroAnalysis) method for the assay and study of enzymes. EMMA utilizes differential electrophoretic mobilities of enzyme, substrate and product. After the application of an electric field, enzymatic reaction takes place and the resultant reaction product and the unreacted substrate are electrophoretically transported towards detector, where they are individually detected. Special attention will be paid on the combination of the EMMA methodology with a partial filling technique. In this set-up the part of the capillary is filled with the buffer best possible for the enzymatic reaction whereas the rest of the capillary with the background electrolyte optimal for separation of substrate and product. The basic limitation of EMMA method – the necessity to have the electrophoretic conditions compatible with both the separation of substrate(s) and product(s) of the enzymatic reaction and the enzymatic reaction – is thus overcome. This methodology will be:

  • apply the EMMA method for assay and study of model enzymes, their basic kinetic parameters and


the kinetic mechanisms, especially in the case of bi-substrate enzymatic reaction

  • apply the EMMA method for study of parameters effecting enzymatic reactions
  • apply the EMMA method for study of inhibition of enzymes and elucidation of the inhibition types
  • used for developing a new method of cyanide determination after its conversion on thiocyanate

Publications

Total number of publications: 17


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