Enzymes

Experiment 1: In preparing the effects of environmental conditions on enzyme reactions, the altering of enzyme in solutions vary upon how abundant a substrate is. This experiment will produce how much enzyme solution should be used in the following assay. To begin this procedure a mortar and pestle should be used to crush a lactide pill. This will be seen in the introductory steps as well as two small one hundred milliliter beakers, a timer, paper towels and 0.1 M phosphate buffer. The resulting use of this will be put into four test tubes. To accomplish this, three plunger pipettes, the stock solution and ONPG will be important as well as for the use of eight cuvettes that will be placed in the spectrophotometer.

Experiment 2: To conduct how changes in pH will influence a lactase catalyzed effect, gloves and goggles will need to be worn for the three pH solutions (4,7,10) obtained for this experiment. Also, a lactide pill, mortar and pestle, and phosphate buffer from the initial procedure will be used in a small one hundred milliliter beaker. To conduct this trial three more pipette plungers will be used to create the solution into four cuvettes. With this the spectrophotometer will also be needed to receive the results of change in absorption over time.

In experiment one you will begin by crushing one lactide pill in a mortar in pestle. Once it is in a powder form deposit all of it in the small one hundred milliliter beaker and use a plunger pipette to add ten milliliters of 0.1 M phosphate buffer to the powder. Allow it to dissolve for 1-2 minutes by placing a timer. Once it begins to look cloudy and no powder is at the bottom place a paper towel over the opening and filter the enzyme solution into the other small one hundred milliliter beaker to remove insoluble material. This will now be the stock solution for the experiment. Thereafter, make a serial dilution into four test tubes. To achieve this, you will start-off with adding 4.5 ml of 0.1 phosphate buffer with a plunger pipette into four test tubes. In tube 1, add .5ml of the stock enzyme that was just produced to the buffer. In tube 2, transfer 0.5 ml of the solution within Tube 1 into Tube 2. Continue to transfer 0.5 from Tube 2 into Tube 3 and repeat from Tube 3 to Tube 4. The transfer 0.5 ml from Tube 4 into the waste. Now there is a series of dilutions where each concentration is more dilute then the preceding one. To then measure absorbance, obtain four cuvettes and add 1.5 ml of 2.5 ONPG solution to each. Take 1.5ml of the enzyme solution from Tube 1 with the 1/10 dilution and add it to the ONPG. As soon as it is added to the enzyme solution it should begin to turn yellow. Place the cuvette in the spectrometer with a