This Userguide will demonstrate measurement of enzyme activity using the Eppendorf BioSpectrometer. In order
to optimize the measurement process, preliminary measurements were performed using the method “single Λ
continuous”. The actual activity measurements were then performed based on these results. Enzyme activities were
determined via linear regression.
For the measurements, a coupled reaction of a hexokinase and a glucose-6-phosphate-dehydrogenase from the
baker’s yeast ‚ was used. Since the Eppendorf BioSpectrometer kinetic is equipped with a temperature controlled
cuvette chamber, temperature dependence of this reaction could also be demonstrated. The highest activity was
detected at 37 °C.
In order to demonstrate the development of a measurement method for a colorimetric assay in the Eppendorf BioSpectrometer, Ponceau-S was scanned across a range of wavelengths from 220 nm to 800 nm to determine its absorbance maxima. Using the novel analysis methods available on the Eppendorf BioSpectrometer, a maximum could be detected at 498 nm using the Eppendorf “SpectraZoom” function.
For the colorimetric assay, a standard curve was generated using Ponceau-S as an example, and measurements were performed. Subsequently, this programmed standard curve was used to calculate an unknown sample concentration.