Staggering the additions of enzyme and NaOH makes it easier to keep accurate

Staggering the additions of enzyme and naoh makes it

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enzyme is added to each tube and later the NaOH. Staggering the additions of enzyme and NaOH makes it easier to keep accurate time measurements. Add 1 ml NaOH at indicated times to stop the reactions. Also add 1 ml to test tube 1 to use to zero the spectrophotometer. Measure the absorbance of each tube at a wavelength of 405 nm. Find PNP conc. values by using the standard curve. 3. Results: Tube Buffer Substrate Reaction Time Enzyme +add time NaOH +add time A 405nm PNP (µmoles/L) 1 2 ml 1 ml ------- 0 ml ---- 1 ml 0.000 0 2 2 ml 1 ml 2 min 0.2 ml 1 ml 0.638 16.5 3 2 ml 1 ml 4 min 0.2 ml 1 ml 0.692 18.0 4 2 ml 1 ml 6 min 0.2 ml 1 ml 0.965 25.0 5 2 ml 1 ml 8 min 0.2 ml 1 ml 1.280 32.5
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4. Discussion: An enzyme reaction over time will lead to a higher substrate concentration as stated in my hypothesis. The substrate has more opportunities to move into the active sites on the enzyme when time is increased. Enzymes at low concentrations especially take more time to break down. Enzymes are very specific and will only convert certain substrates into products. Enzymes reduce the activation energy required for a reaction to occur, which increases the chance for molecules to collide with one another. This increases the concentration of the substrate. This is why when the time increases, the concentration of PNP increases to provide the enzyme with more substrate to act on. Effect of Substrate Concentration on the Rate of an Enzymatic Reaction 1. Hypothesis: As substrate concentration increases, the rate of an enzymatic reaction will increase. 2. Procedure: Each mixture of buffer and substrate in the tubes will have the same buffer concentration. Add the indicated amounts of buffer and substrate solutions on the table to each tube. Add 0.2 ml of enzyme to each tube. Stagger the additions of enzyme and NaOH to note start and stop times. Each enzymatic reaction should run exactly 5 minutes long. 1 ml of NaOH should be added to
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  • Spring '10
  • Coombs
  • Enzyme, ml

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