ENZYME LAB - Part 1 - Temperature Test Procedure –
Enzyme Preparation –
Obtain some liver, and weigh out 10 grams. The liver will need to be cut into small pieces. Measure out 50 mL of water from the container provided – this is a saline solution, prepared to approximately match tissue fluid.
Place the liver, with 10-20 mL of the water, into a large mortar and grind the pieces with the pestle. Tilting the mortar can help the process. Make sure that you get the largest pieces pulverized. The fluid should pick up a lot of color, and pieces should become very small flakes. Once the grinding is done, add the rest of the water to the mortar, rinsing the pestle and the sides. This will be your enzyme solution, but every group will wind up with slightly different concentrations – this preparation will have to last you through all of the tests, since you will need it to be “standard” throughout.
Place filter paper in the bottom of the filter and place the filter in a collection flask. Pour the all of enzyme prep into the filter and swirl. Filtrate will eventually drip down into the flask. Keep a second flask handy – dripping will continue, and when you need your prep, you’ll move the filter to the second flask.
Optain a plastic dropper, and familiarize yourself with the markings – you will be using a lot of single milliliter doses of the filtrate. Practice picking up single milliliters without bubbles in the column.
Temperature Preparation –
There will be four beakers of water at different temperatures – you’ll need to find two and set up two. Set up one beaker on a hot plate, and get the water boiling. Get a “room temperature” beaker and record the temperature on the data sheet. Put several ice cubes in a beaker and surround with cold water. There will be an incubator beaker set up in the incubator.
Each temperature trial will be done three times – get three test tubes designated as “1 mL” and put them in a test tube rack. Organize the rack so that you have a clear spot for the different 3-sets for each temperature.
Temperature Test -
There should be 12
test tubes, designated “1mL,” in sets of 3 in the rack. Into each test
tube, place 1mL of the catalase preparation – be sure to keep
swirling the filtrate to keep it evenly mixed.
Each set of 3 test tubes will be placed in one of the temperature beakers. They should be there for at least 10 minutes, but not too much longer than that. During that time, some of the rate-comparison test can be set up.
After 10 minutes at temperature, transfer the test tubes from the boiling water to the rack (use test tube tongs!); tubes will be left in the room-temperature and ice bath until their tests. Get the incubator tubes – these will be the first ones tested, then the boiled sample, then the ice bath, then the room temperature.
Set up the tubes so that the marked lines are easy to see - you’re going to look for a line of reaction bubbles to reach that line. Get a stopwatch and make sure you know how to start it, stop it, read it, and reset it.
For one tube at a time, pick up 1mL of hydrogen peroxide (no bubbles) and squirt it into the bottom of a test tube with a follow-up swirl – try to do this the same way every time. The timer will start the stopwatch as the fluids make initial contact, then stop the watch when the top surface of small bubbles reached the marked line. The elapsed time will be recorded on the data sheet. If there is no reaction, note this on the sheet.
For each temperature, average the three results. Use “Comments” for any observations or explanations that you think might be important (there might be none). Interpret your results in the space under the table.
ENZYME LAB - Part 2 - Substrate Concentration Test Procedure –
Catalase Prep –
Get five sets of three test tubes each – designated “1mL,” “2mL,” “3mL,” “4mL,” and “5mL.” Place them in racks so that the marked lines can be clearly seen. Into each tube, place 1mL of catalase preparation.
Peroxide Prep -
Set up a second row of unmarked test tubes behind the catalase tubes. Into these tubes, place the appropriate amount (the amount designated on the marked tubes) of hydrogen peroxide, using the dropper.
Make sure everything is prepared for timing. For each trial, pour the hydrogen peroxide from the tube behind into the catalase tube in front of it. Swirl the contents, trying to do it the same way for every trial. The stopwatch will be started as soon as the fluids make contact, then stopped when the top of the small bubbles reach the line marked on the tubes. Record the elapsed time for each trial.
Average the trials for each substrate amount and compare. Use “Comments” for any observations or explanations that you think might be important (there might be none). Interpret your results in the space under the table.
General Biology 2 - Molecules and Cells
Lab Exercise Index
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