Prac 3 Enzymatic Report - 89% HD Result PDF

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Title Catalase reactions in relation to decomposition of hydrogen peroxide.

Introduction In many organisms, metabolism creates the by-product of hydrogen peroxide, which needs to be further broken down, or decomposed. Enzymes are a protein catalyst, comprised of hundreds of amino acids, that can increase the rate of reactions without being consumed (Urry et al p. 155, 2018). The addition of enzymes to this equation can speed up the decomposition rate, however enzymes require a specific environment in order to operate effectively. Enzymes work in partnership with substrates, attaching to the substrate and then through a reaction, converting it to products. The decomposition of Hydrogen Peroxide produces oxygen gas and water (Denby 1995, p. 30) 2H2O2

H2O + O 2

As enzymes can be greatly affected by external environmental factors such as temperature, substrate concentration and pH levels, this experiment is aimed toward finding the optimal conditions for enzymatic reaction through decomposition of hydrogen peroxide by studying the effects of the pH levels, H2O2 concentration and temperature.

Method The initial experiment conducted followed the primary procedure. This experiment was conducted at room temperature.

Celery was finely sliced using a razor blade, then using a balance, was weighed into 5g piles. 20mL of pH7 Universal Buffer fluid was pipetted into a 100mL measuring cylinder. To the cylinder, 3 drops of detergent was added and then gently mixed into the buffer by swirling so as not to create any bubbles. In this experiment, detergent trapped any oxygen gas that is released as a result of the decomposition of hydrogen peroxide, allowing for accurate recording of results. Once the detergent was mixed in, 5g of finely sliced celery was added, pushing down any pieces stuck to the side of the cylinder into the fluid. Once the celery had been added, the starting volume was recorded and as several tests were conducted, the measuring cylinder was labelled. Once labelled, set a timer for 15 minutes, starting the timer as soon as the substrate, 2mL of 10% Hydrogen Peroxide, was added to the measuring cylinder. Once the timer was finished, the total volume of the foam was recorded.

The primary procedure was then repeated several times with minor changes, adjusting the three variables as follows.

Variable: Substrate Concentration: The primary procedure was followed as per the above, however when the substrate was added, the concentration of the Hydrogen Peroxide was altered. Each experiment that tested the effects of substrate concentration was run a total of seven times; one controlled test with no enzyme, and six additional times following the amended primary procedure with the relevant substrate concentrations. The substrate concentrations used were as follows; 0% H2O2, 5% H2O2, 10% H2O2 (as noted in the primary procedure), 20% H2O2 and 30% H2O2.

Variable: pH Levels The primary procedure was followed as per the above, however, for each test under this variable, the pH Buffer was altered. Each experiment that tested the effect of pH levels was run a total of 10 times, three controlled tests with no enzyme, and seven additional times following the amended primary procedure with the relevant pH Buffers. The pH Buffers used were as follows; pH 4, pH 6, pH 7 (as noted in the primary procedure), pH 8 and pH 10.

Variable: Temperature The primary procedure was followed as per the above, however for each trial, the temperature of the solution was altered. The primary procedure tests were conducted at room temperature (19°C), however each experiment that tested the effect of temperature was conducted in a water bath of the following temperatures; ice (...