Enzyme Peroxidase Lab Report

Each were labeled and paired up with one containing the other set of ingredients. To determine if temperature affected the performance of peroxidase each set was isolated in a specific temperature. Two of the test tubes were set in a refrigerator of 4 degrees Celsius, another pair was left to sit in room temperature of 22 degrees Celsius, the third pair was left in an incubator of 32 degrees Celsius, and the last pair was left in a water bath of 60 degrees Celsius. All four sets were left in their designated temperature for 15 minutes. Before the 15 minutes were up the spectrophotometers were set at 470 nm and zeroed out using the blank.

As pH increases or decreases to get closer to the optimal pH --in this case it is 7 for this particular enzyme-- the rate of reaction peaks and is highest at that point, which is described by the molecular shape and structure of the enzyme at its optimal pH. When turnip peroxidase is at pH 7, the active site is able to fit perfectly with the substrate, therefore explaining why the reaction rate is fastest at this point. Accordingly, if the active site is disrupted, the substrate cannot fit perfectly causing the reaction rate to slow down. This can be supported by the data because the reaction rate gradually increased from pH 3 to pH 7 and reached its maximum at pH 7. Once it did reach the optimal pH, the reaction rate continuously decreased

Using two test tubes, label one “s” for substrate and the other “e” for enzyme. The substrate tube should contain 7 mL of distilled water, 0.3 mL of hydrogen peroxide, and 0.2 mL guaiacol and the enzyme tube should contain 6 mL of distilled water and 1.5 mL of peroxidase. Combine the materials of the substrate and enzyme tubes, mix the two using a clean transfer pipette, transfer a portion into a cuvette so that the cuvette is about half-full then cover the top of the cuvette with Parafilm and then place it in the spectrophotometer and record absorbance. Remove the cuvette and repeat recording absorbance at 1, 2, 3, and 4 minutes. Be sure to mix the cuvette and clean its surface with Kimwipes before each reading.

It was hypothesized that the optimal pH for the enzyme was pH 7 while the 1.0 ml peroxidase would have the best reaction rate. At the end of the experiment the results prove the hypothesis to be incorrect. INTRODUCTION Enzymes are proteins that allow a reaction to speed up. These proteins are made up of monomers known as amino acids.

Fill each cuvettes with its respective solution. Turn on the spectrophotometer, so it can warm up then calibrate it to 0% absorbance. Put the corresponding extract blank and set the spectrophotometer to 100% transmittance, then calibrate it to 540 nm. Once catechol is added in the cuvettes, make sure the solution is mixed. Place carrot cuvette in the spectrophotometer and record the resulting transmittance.

1 “substrate” and another “ enzyme.” Instead of using the distilled water, this time you are going to use different pH buffer in the enzyme test tube. In the substrate tube, add 7 mL of distilled water, 0.3 mL of hydrogen peroxide, and 0.2 mL of guaiacol for a total volume of 7.5 mL. For the enzyme tube, instead of distilled water add the pH solution (3) and 1.5 mL of peroxidase which equals a total volume of 7.5 mL. Use the dH2O syringe for our pH solution. To clean the syringe, flush it by drawing 6 mL of distilled water.

LABORATORY REPORT Activity: Enzyme Activity Name: Natalie Banc Instructor: Elizabeth Kraske Date: 09.26.2016 Predictions 1. Sucrase will have the greatest activity at pH 6 2. Sucrase will have the greatest activity at 50 °C (122 °F) 3.

In order to find the answer, an experiment was conducted in which 7 test tubes filled with equal amounts substrate and enzyme were subjected to various temperatures (4˚C, 22˚C, 37˚C, 45˚C, 65˚C, and 95˚C) for ten minutes. It was hypothesized by the group that difference in temperature will have a significant effect upon enzyme activity based on past knowledge on the anatomy and

3X ENZYME (ABSORBANCE AU) TRIALS 0 SECS 20 SECS 40 SECS 60 SECS 80 SECS 100 SECS 120 SECS TRIAL 1 0.194 0.403 0.531 0.612 0.661 0.691 0.713 TRIAL 2 0.148 0.34 0.463 0.544 0.598 0.629 0.651 TRIAL 3 0.164 0.399 0.54 0.626 0.677 0.709 0.733 ENZYME CONCENTRATION: 3X ENZYME (TRANSMITTANCE %T) TRIALS 0 SECS 20 SECS 40 SECS 60 SECS 80 SECS 100 SECS 120 SECS TRIAL 1 64 39.5 29.43 24.45 21.84 20.35 19.35 TRIAL 2 71.15 45.66 34.47 28.56 25.26 23.52 22.34 TRIAL 3 68.54 39.94 28.87 23.65 21.03 19.54

LABORATORY REPORT Activity: Enzyme Activity Name: Natalie Banc Instructor: Elizabeth Kraske Date: 09.22.2016 Predictions 1. Sucrase will have the greatest activity at pH 6 2. Sucrase will have the greatest activity at 50 °C (122 °F) 3. Sucrase activity increases with increasing sucrose concentration Materials and Methods Effect of pH on Enzyme Activity 1. Dependent Variable amount of product (glucose and fructose) produced 2.

By using a spectrophotometer to measure absorbance at 420 nm, the rate of enzyme activity after all reactions have come to a stop can be

5 water bath were set up each to10 °C. (5 were used do the experiment faster) 5 cm3 of starch solution were added into the 5 test tubes that were labeled test tubes. Then 5 cm3 of amylase enzyme was added into the other 5 test tubes that were labeled. Put one of the starch solution test tube (preferably the one labeled 1) and one of the test tube containing amylase into the water bath (10 °C).

H20 + 2 O2 This experiment will use 1% catalase solution and 3% hydrogen peroxide solution, both diluted into water so the reaction slows down. Temperature will be controlled in this experiment to change the reaction speed of the enzyme and the substrate, this is what the experiment is looking at. The effect of the temperature will be determined by how much gas is released in two minutes, which will change the pressure inside the test tube and will be measured by a gas

By observing figure 3, the more enzyme that is available, the faster the reaction rate is. The optimal enzyme concentration was chosen based on the R2 values from figure 2. The highest observable rate also had the best R2 number, which was closest to one. This enzyme concentration was used in part 2.

ABSTRACT: The purpose of the experiments for week 5 and week 6 support each other in the further understanding of enzyme reactions. During week 5, the effects of a substrate and enzyme concentration on enzyme reaction rate was observed. Week 6, the effects of temperature and inhibitor on a reaction rate were monitored. For testing the effects of concentrations, we needed to use the table that was used in week 3, Cells.