FILM-6010 Lecture Notes - Lecture 16: Enzyme Assay, Sodium Hydroxide, Absorbance
14 Jun 2020
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The aim of this practical was to perform an enzyme assay to analyse the rate of enzyme activity vs. substrate concentration. Enzyme activity is determined by the rate at which an enzyme is able to bind with a substrate to form a product. The rate at which this process occurs can be determined by performing an assay with varying concentrations of a given substrate. In this practical pnpp (p-nitrophenol phosphate) is used as the substrate and alkaline phosphatise for the enzyme. 4 t a e c n a b r o s b. Figure 1: p-nitrophenol standard curve, absorbance at 440nm. Volume of 5 mm p-nitrophenyl phosphate (pnpp) to add (ml) Mol pnp/sec (eg. divide value above by 300) Corrected mol pnp/sec for tubes with enzyme (eg. 2*10^-4 1*10^-4) Attach graphs: rate (nkat) versus substrate concentration [s, lineweaver-burk plot. Rate vs. substrate concentration t a k n y t i v u t c a e m y z n.
