bio lab 3 - Kinetic Studies of Alkaline Phosphatase Enzyme Measure of the Time Course of a Reaction and the Effect of Substrate PNP Concentration and

bio lab 3 - Kinetic Studies of Alkaline Phosphatase Enzyme...

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Kinetic Studies of Alkaline Phosphatase Enzyme: Measure of the Time Course of a Reaction and the Effect of Substrate PNP Concentration and Temperature on the Rate Absorbance of Standard Solutions 1. Hypothesis: As the PNP concentration increases, the absorbance at 405 nm will increase according to Beer’s Law. 2. Procedure: Place different concentrations of PNP separately into four test tubes. Fill the test tubes about two thirds of the way full. Using a spectrophotometer, measure the absorbance of the PNP standards at wavelength of 405 nm. 3. Results: Tube PNP conc. (µmoles/L) Absorbance at 405 nm 1 0 0.000 2 12.5 0.436 3 25.0 0.935 4 50.0 Above max (>1.999) 4. Discussion: Beer’s Law states that the absorbance is equal to the path length times the concentration times the coefficient. In other words, absorbance depends on the total quantity of the absorbing compound in the light path through the test tube; therefore, my hypothesis was correct. This is why the graph shows a linear
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relationship for the standard curve. The Law is not obeyed at high concentrations, which is why test tube 4 had an absorbance that could not be read on the spectrophotometer. Effect of Time of an Enzymatic Reaction 1. Hypothesis: As reaction time increases, the concentration of PNP will increase. 2. Procedure: Add 2 ml of buffer and 1 ml of substrate solutions to each of five test tubes. Equilibrate the tubes in a water bath of 37° for two minutes and keep them there. Add 0.2 ml of enzyme to tubes 2-5 and incubate them at 37° for the reaction times indicated on the table. Make sure to keep track of the times the enzyme is added to each tube and later the NaOH. Staggering the additions of
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  • Spring '10
  • Coombs
  • Enzyme, ml

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