Alkaline Phosphatase Lab

An enzyme biologically defined is a catalyst produced by cells to speed up specific chemical reactions without changing the chemical reaction at the end of the reaction.1 There are several factors that affect the rate an enzyme speeds up reactions; temperature, pH, substrate concentration and enzyme concentration.2 However, when there is too much or not enough of these factors (depending on the enzyme) it can destroy the enzyme entirely. In this experiment we tested how temperature affects enzymes. We observed the enzyme activity for the enzyme Alkaline phosphatase when it was put in an environment of 33°C and 86°C. Because Alkaline phosphatase has the ability to extract phosphate groups from substrates, once the Alkaline phosphatase was in the specifically heated environment for five minutes, we measured it’s activity by inserting para-nitrophenyphosphate. Para-nitrophenyphosphate is a substrate that goes from being colorless to being the color yellow when it’s phosphate group is removed. By using a spectrophotometer, we discovered the absorbance of the solutions which therefore

The concentration of para-nitrophenol (pNP) was then quantified, following the procedure below. The solution at 33 degrees celcius was also tested to act as a negative control sample.

A standard curve for pNP concentration was generated using 3ml of 0.0, 0.1, 0.5 and 1.0 mM, recording absorbance at a wavelength of 410 nm using a spectrophotometer (SpectrovisPlus Vernier). The absorbance of experimental samples was determined in an identical manner and then converted into concentration values via the standard curve.

The relative enzyme activities were calculated as μmoles pNPP/minute and analyzed via a students’ T-test (Microsoft Excel 2010, Microsoft, Redmond, WA,