Investigating the effect of pH of Hydrogen Peroxide upon the activity of Catalase

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Experiment: Investigation as to whether the pH level of Hydrogen Peroxide

                     will influence the activity of the enzyme Catalase.

Variables involved:

Different pH levels will affect the Catalase enzyme by determining whether the enzyme activity will increase, decrease or if it will denature altogether, so the pH level of the Hydrogen Peroxide, which Catalase breaks down into oxygen and water, will be our independent variable.

The temperature of the Hydrogen Peroxide will affect the rate of reaction with the Catalase, thus this will be a controlled variable and will need to be kept constant.

The substrate concentration can also affect enzyme activity in regards to its rate of reaction, so this will also need to be kept constant.

The amount or volume of the chicken liver needed to get the Catalase enzyme can also affect the rate of reaction; therefore this will also be needed to be kept constant.

I will also be using buffer solutions to change the pH of the Hydrogen Peroxide, thus the amount or volume used will also be kept constant.

All these variables can affect the rate at which oxygen is broken down from Hydrogen Peroxide by the enzyme Catalase, so the measurement of how much oxygen is diffused will be our dependent variable.

How to deal with the variables:

The pH level of the Hydrogen Peroxide is the independent variable and so must be adjusted. This can be achieved by the use of buffer solutions that are able to change the pH levels. Thus in this experiment, they will be used to change the pH level to the pH’s 1, 4, 10 and 14. A pH probe will be used to show this occurring.

The temperature of the Hydrogen Peroxide will be kept constant as it will be placed in a water bath that has its temperature level set to 30˚С. This will accurately and effectively ensure that the Hydrogen Peroxide is all at the same temperature.

The substrate concentration will be also be regulated by using a measuring cylinder to measure the amount so that it is consistent in every experiment. We measure this by the readings at the side of the measuring cylinder. For this experiment, 5ml of the substrate concentration will be used.

To prevent the Catalase enzyme from having too much of a significant affect on the rate of reaction, this will also be kept constant. The chicken liver will be measured to 1cm² and weighing 0.5g for every test-tube of an experiment.

The volume of the buffer solutions used shall also be regulated so that it is constant. Again, a measuring cylinder will be used to measure the amount used to 5ml before it is added to the Hydrogen Peroxide.

To measure the amount of oxygen that is diffused after the Catalase enzyme has reacted with the Hydrogen Peroxide, a gas syringe will be utilised to measure this.

Apparatus:

        -Hydrogen Peroxide (10%)                -Chicken Liver (Catalase)

        -Buffer solutions (pH 1, 4, 10, 14)     -water bath

        -Test-tubes/beaker                        -measuring cylinders

        -pH probe                                -Test-tube stand

        -gas syringe                                -scalpel

        -cutting board                                -ruler

        -paper                                        -pencil

        -electronic weighing machine                -computer with scientific software

        -bunsen burner                        -tripod stand/thermometer

Method:

First, the Catalase from the chicken liver will be obtained. It will be cut up with a scalpel and measured by a ruler to 5 pieces of 1cm² and weighed with the weighing machine so that it weighs 0.5g. Then, Hydrogen Peroxide will be measured to 25 ml in a measuring cylinder and 5 ml will be poured into 5 separate test tubes, thus there will be five different measurements. Then, buffer solutions will be added to the Hydrogen Peroxide solutions in the test tubes. Four of the test tubes will be given different buffer solutions with different pH levels, and these include pH 1, 4, 10 and 14. The buffer solutions will first be measured with measuring cylinders so that there is 5 ml to be added to each of the four test tubes. The last of the test tubes will not have the Hydrogen Peroxide change in pH level (through my preliminary work, I have found out that the pH level of the Hydrogen Peroxide, 10 % to be approximately pH 7.5. This was found out through the use of the pH probe). Once the buffer solutions have been added, it will be stirred so that the solution mixes with the Hydrogen Peroxide. Then the pH probe will be used to check whether the pH level of the Hydrogen Peroxide in the 4 different test tubes have changed before proceeding to put the test tubes in the water bath that has its temperature set to 30˚С. They will be left heated for 5 minutes to ensure that they reach the temperature before the experiment continues. After 5 minutes, one at a time, one of the cut up pieces of chicken liver will be added to a test tube and quickly a gas syringe will be connected to that test tube to measure how much oxygen is produced from the reaction between the Hydrogen Peroxide and the Catalase in the chicken liver. The measurement on the gas syringe (how much oxygen produced) and the pH level of the Hydrogen Peroxide in the test tube will be recorded after 5 minutes before the next Catalase is added to another test tube, and this will be repeated until all five test tubes have been covered. Once I have all the recordings of all five test tubes, the equipment will be cleaned and I will do the experiment again. I will do the experiment 3 times, so therefore I shall have 3 sets of 5 measurements.

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Predictions:

Each enzyme has its own optimum pH, and I can deduce from my discovery of Hydrogen Peroxide being pH 7.5 during my preliminary work that the enzyme Catalase would have an optimum pH level of around that figure and this would show that it would work in slightly alkaline solutions. Therefore, I would predict that the Catalase enzyme would denature when it is put into the Hydrogen Peroxide solutions that have been mixed with the more acidic buffer solutions (pH 1 and 4) because if enzymes are put into pH’s that are significantly lower or above from their ...

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