Materials: Water Milk Lactaid tablets available from grocery or drug store Glucose test strips available from drug store Table Sugar Solution Preparation: Lactose solution: Milk, about ml. Sucrose Solution: Add 5 grams of sugar to ml of water. Stir until the sugar has dissolved. Enzyme Solution: Add 1 lactase tablet to ml of water.
Stir until the tablet has dissolved. Denatured Enzyme Solution: Place 20 ml of Enzyme Solution into a glass container such as a canning jar. Add ml of water, and then seal the lid. Add the canning jar to a pot of water, and bring the pot of water to a boil. Let the solution cool to room temperature. Procedures: Divide the lactose solution and sucrose solution into two containers each. Be sure to label the containers.
Add the enzyme solution to one, and the denatured enzyme solution to the other. Dip an unused glucose test strip into each container and record whether glucose is detected in the table below. Glucose Detected? What must have happened? What must have happened to the enzyme solution when boiled? Repeat the assay and record the results. Compare with other groups to check that results are similar. To determine the course of an enzymatic reaction, how much substrate is disappearing over time must be measured.
First, set up the cups with the times and the word acid up. Add 10 mL of H2SO4 to each of the cups marked acid. Then put 10 mL of 1. Add 1 mL of catalase extract to this cup.
Swirl gently for 10 seconds. Calculate time using the timer for accuracy. At 10 seconds, add the contents of one of the acid filled cups. Remove 5 mL and place in the second cup marked 10 sec. Assay the 5-mL sample by adding KMnO4 a drop at a time until the solution obtains a pink or brown color.
Repeat the above steps except allow the reactions to proceed for 30, 60, , , and seconds, respectively. Record all results and observations.
Readings Final Reading of Syringe 1. Results Final Reading of Syringe 1. What is the enzyme in this reaction? The enzyme in this reaction is the catalase solution. How could you show that the gas evolved is oxygen? How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference. With the boiled catalase, there was no sign of bubbling because the catalase was denatured by the heat and caused no reaction.
What do you think would happen if the liver were boiled before being added to the hydrogen peroxide? I think that no signs of a reaction occurring would be shown. The catalase that occurs naturally within the liver would have been denatured.
Determine the initial rate of the reaction and the rates between each of the time points. Record the rates in the table below. When is the rate the highest? Explain why. The rate is the highest in the first ten seconds because the rate decreases as the concentration of the catalase decreases over time. Its function is to break down hydrogen peroxide H 2 O 2 , which is one of the toxins produced as a byproduct of using oxygen for respiration.
The fact that it's toxic is what makes hydrogen peroxide useful in first aid kits. We drip dilute hydrogen peroxide on cuts and scrapes to kill any bacteria which have gotten in, and so prevent infection. The peroxidase reaction is as follows:. Water and oxygen are much less toxic than H 2 O 2 , and thus don't damage the interior of the cell.
Experimental Setup We extracted catalase from turnips, and investigated the effects of four factors on the speed of the enzymatic reaction. The factors were: temperature, pH, substrate here, H 2 O 2 concentration, and enzyme concentration. To track the rate of the reactions, we used the spectrophotometers and a reagent called guiacol. In the presence of oxygen, guiacol oxidizes from clear to brown. The more oxygen produced, the darker brown the guiacol becomes.
We set up 10 mL reaction mixtures including guiacol, hydrogen peroxide, turnip extract, and a pH 7 buffer. We then took an absorbance measurement every second for a minute, and graphed absorbance y versus time x. The slope of the resulting line was the rate of the reaction.
We began by running a standard reaction. Cold water may be used. Warm water must be used. Use goggles and gloves for personal protection. Dispose of guaiacol-containing solutions in special container. You only need to pour about 20 mL of each solution. Make sure the beakers are properly labeled.
Obtain two 1-mL pipets and two 5 or 10 mL pipets. Label one 1 ml pipet for Guiacol, and the other 1ml pipet for turnip extract.
Label one 5 ml pipet for Buffer pH5 and the other 5 ml pipet for H2O2. Use of the wrong pipet will cross contaminate your reagents and introduce errors into your subsequent experiments in this exercise.
Do not Cross Contaminate. Using tape or wax pencil, label seven test tubes from 1 to 7. Prepare these tubes according to Mixing Table 1. Amounts given are in mL. These tubes will be used for the following purposes:. Tube 1: Control without hydrogen peroxide; to be used in calibrating the spectrophotometer. At the spectrophotometer, adjust the wavelength to nm. In your lab groups, one person should start the reactions, one person should be the Timer, another the Spectrophotometer Reader, and another the Data Recorder.
Follow the instructions from the previous lab for calibrating and blanking. Note the time to the nearest second and mix the contents of tubes 2 and 3 by pouring the contents of tube 3 into tube 2 two or three times. Mixing should be completed within ten seconds. The contents of this tube will now be referred to as the reaction mixture. Immediately after mixing, add the reaction mixture to a cuvette, wipe the outside of the cuvette with a Kimwipe, and place the cuvette in the spectrophotometer.
Read the absorbance at 20 sec intervals from the start of mixing. Record your measurements in Table 2. After two minutes 6 readings remove the tube from the spectrophotometer and visually note the color change. Discard in waste container. Mix the contents of tubes 4 and 5, pour into a cuvette, and repeat your measurements for two minutes at 20 second intervals.
Record the results in Table 2. Mix the contents of tubes 6 and 7, pour into a cuvette, and record the absorbance measurements in Table 2. Q: Which amount of enzyme gave the most linear absorbance change from 0 to 1 in the full seconds?
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