BIOL 200 Lecture Notes - Lecture 15: Protein Structure, Calorimeter, Fluorescent Tag

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Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Here, the product of one reaction is used as the substrate of another, easily detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-phosphate to nadph production, using glucose-6- phosphate dehydrogenase. Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase.

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