This is due to the ionization of carboxyl, amino and other groups on either the substrate or the enzyme. In neutral or basic solutions, carboxyl groups (– COOH) release H+ ions to become negatively charged carboxylate groups (– COO-). Similarly, amino groups (– NH2 ) accept H+ in neutral or acidic solutions becoming positively charged – NH3+ groups. Thus in a neutral solution, a molecule with an amino group will be attracted electrically to another molecule that has a carboxyl group, because both groups are ionized and they have opposite charges.
If the PH changes, however, the ionization of these groups may change. For example, at a low PH (high H+ concentration) the excess H+ ions may react with the – COO- to form COOH. If this happens, the group is no longer charged and cannot interact with other charged groups in the protein, so the folding of the protein is altered. If this occurs at the active site of an enzyme, the enzyme may no longer have the correct shape to bind to its substrate.
The PH environment of the reaction can be altered and maintained using a ‘buffer solution.’ This can be added with the hydrogen peroxide.
The various PHs of the buffer solutions will be PH1 (strong acid), PH4 (moderate acid), PH10 (moderate alkalis), PH14 (strong alkalis). The buffer solution for the control of the experiment will be substituted with 5cm3 water as water has a neutral PH of 7.
The experiment must be set up as shown below.
Method
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Measure using a measuring cylinder 10cm3 H2O2 and 5cm3 of the appropriate buffer solution to test tube A
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Dice a raw potato using a sharp bladed knife into ruler measured 1cm3 sided cubes so mass, volume, surface area and shape remain constant in every cube. The cubes must be from the same potato and must not include and part of the skin as the enzyme concentration may be different here.
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Ensure the trough is not overfilled, as there should be space for the displaced water. The increase of water in the trough indicates how much O2 has entered the test tube.
- Ensure that test tube B is clamped firmly into position as any movement could distrupt the oxygen flow into the tube.
- Add using a tweezer one of the cubes of potato to test tube A
- Insert a bung into test tube A
- Time using a stopwatch the reaction for ten minutes
- After this time has passed, using the marked scale on the trough, record the increase in water to identify the amount of oxygen obtained in the reaction.
- Release Test tube B out of the trough vertically and place a glowing splint under the test tube (as shown below). The splint should relight with the presence of oxygen.
To make the experiment fair test the following factors must remain constant.
- Temperature: This must not change. An increase gives the molecules more kinetic energy so collisions between enzyme and substrate are more frequent and so the rate of reaction increases.
- Surface area (as well as mass, shape and volume): These must remain constant. An increase in surface area would enable more enzymes to react with the substrate (hydrogen peroxide).
- The experiment will be repeated 10 times for each PH and the mean average will be recorded.
Safety precautions
- Ensure goggles are worn at all times as toxic hydrogen peroxide is harmful .
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Gloves should be worn when handling H2O2
- Bags and stools should be tucked under desks
- Work surface should be clear in case of any spillages of the hydrogen peroxide
Lab coats must be worn
- Long hair should be tied back
Ethical implications
There are no ethics involved in this experiment as animals or rare substances are being used in the experiment.
Table of results
*Control of experiment
