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THE EFFECT OF A CHANGE IN pH ON THE ACTIVITY OF ENZYMES
Background Information:
Enzymes are organic catalysts that speed up chemical reactions within the body. Enzymes are specific for one particular reaction or group of related reactions. Enzymes are large globular protein molecules with one or more indentations on their surface called active sites, an active site is the part of the enzyme that binds to the substrate, the active site has a specific shape that will bind to a specific substrate. For an enzyme to catalyse a reaction the small substrate molecules must temporarily bind to the active sites of the enzymes were the bonds in the substrate are broken and the products released.
Rennin is an enzyme secreted mainly by the kidney that breaks down protein and produces a rise in blood pressure. Each enzyme has an optimum range to which it functions most efficiently, the optimum for rennin is around 3. When the enzyme–substrate solution is subjected to a high level of alkalinity or acidity this causes the enzyme in the solution to denature, hence increasing the rate of reaction. Most known enzymes function at or near neutral, although enzymes specific to the digestive tract function in an acidic or alkaline medium. Rennin is found in the gastric juice of the stomach – it requires an acidic medium, rennin starts to denature as the food passes from the stomach and into the intestines as the intestines have very high alkaline pH mediums, hence subjecting rennin to a high pH will cause it to denature and increasing the rate of reaction.
Aim:
To investigate and determine the effect of a change in pH on the activity of enzymes.
Hypothesis:
Once the rennin (enzyme) is added to the milk (substrate), the milk will curdle (coagulate). A pH of 3 will cause the rennin to react with the milk the fastest as it is the optimum pH for the reaction. If the pH varies too much from the optimum, the enzyme (rennin) will start to denature and the reaction will not occur.
Equipment:
- Styrofoam cups
- Test tubes
- Test tube rack
- Thermometer
- Syringe
- Milk (Full cream)
- Kettle
- Water
- Ice - Crushed
- Junket powder (contains rennin)
- Universal indicator and charts
- Acid (Hydrochloric acid)
- Base (Sodium Hydroxide)
- Stopwatch
- Safety Goggles
- Closed in shoes
Risk Assessment:
Category
Hazards / Risks
Control measures
Category D –
Hazardous chemicals
e.g. reactions involving acids, bases, metals and a variety of salts
Substances used in experiment getting into eyes.
Falling glassware or broken glass ware.
Inhalation of hazardous fumes
Safety goggles
Closed in shoes
Insure area where the experiment is taking place has adequate ventilation
Hair tied back – prevention of hair interfering with experiment.
Variables:
Controlled
Dependent
Independent
The amount of milk used – 20 ml
The amount of rennin solution - 1 ml
The concentration of rennin solution
Test tube size
Same stop watch or same make and model
The experiment is carried out in the same environmental conditions.
Type of milk (Full cream)
Same person timing
Time for milk to curdle (minutes/second)
The concentration of pH solution used:
pH of 3
pH of 5
pH of 6
pH of 8
pH of 11
Method:
1. Insure all safety measures are met; application of safety goggles, hair tied back, closed in shoes.
2. Make rennin (enzyme) solution by dissolving ¼ teaspoon of junket powder in 20 ml of cold water and insure rennin does not denature by keeping on ice with a temperature between 0-4oC.
pH of Three
3. Create water bath by adding boiling water and cold water to reach a temperature of 37oC (optimum) in a Styrofoam cup.
4. Fill four test tubes with 20 ml of full cream milk (substrate), labeling with tt1, insure one test tube is also labeled control.
5. Place test tubes into water bath (Styrofoam cup) and insure substrate reaches a temperature of 37o C by placing a thermometer into each test tube to monitor the temperature changes.
6. When temperature reaches 37oC add universal indicator to monitor pH changes, also add sufficient amounts of acid (Hydrochloric acid) to each test tube to reach a pH of three.
7. Once the pH of test tube reaches desired reading, using a syringe, add 2 ml of rennin (enzyme) to the test tubes and start timing, do not add enzyme to the test tube allocated as control, observe control test tube to notice if any coagulation occurs.
8. Stop timing once substrate coagulates, you will notice coagulation when test tube contents shows signs of visible lumpiness.
9. Record results in a table format.
pH of Five
10. Create water bath by adding boiling water and cold water to reach a temperature of 37oC (optimum) in a Styrofoam cup.
11. Fill four test tubes with 20 ml of full cream milk (substrate), labeling with tt2, insure one test tube is also labeled control.
12. Place test tubes into water bath (Styrofoam cup) and insure substrate reaches a temperature of 37o C by placing a thermometer into each test tube to monitor the temperature changes.
13. When temperature reaches 37oC add universal indicator to monitor pH changes, also add sufficient amounts of acid (Hydrochloric acid) or base (Sodium hydroxide) to each test tube to reach a pH of five.
14. Once the pH of test tube reaches desired reading, using a syringe, add 2 ml of rennin (enzyme) to the test tubes and start timing, do not add enzyme to the test tube allocated as control, observe control test tube to notice if any coagulation occurs.
15. Stop timing once substrate coagulates, you will notice coagulation when test tube contents shows signs of visible lumpiness.
16. Record results in a table format.
pH of Six
17. Create water bath by adding boiling water and cold water to reach a temperature of 37oC (optimum) in a Styrofoam cup.
18. Fill four test tubes with 20 ml of full cream milk (substrate), labeling with tt3, insure one test tube is also labeled control.
19. Place test tubes into water bath (Styrofoam cup) and insure substrate reaches a temperature of 37o C by placing a thermometer into each test tube to monitor the temperature changes.
20. When temperature reaches 37oC add universal indicator to monitor pH changes, also add sufficient amounts of acid (Hydrochloric acid) or base (Sodium hydroxide) to each test tube to reach a pH of six.
21. Once the pH of test tube reaches desired reading, using a syringe, add 2 ml of rennin (enzyme) to the test tubes and start timing, do not add enzyme to the test tube allocated as control, observe control test tube to notice if any coagulation occurs.
22. Stop timing once substrate coagulates, you will notice coagulation when test tube contents shows signs of visible lumpiness.
23. Record results in a table format.
pH of eight
24. Create water bath by adding boiling water and cold water to reach a temperature of 37oC (optimum) in a Styrofoam cup.
25. Fill four test tubes with 20 ml of full cream milk (substrate), labeling with tt4, insure one test tube is also labeled control.
26. Place test tubes into water bath (Styrofoam cup) and insure substrate reaches a temperature of 37o C by placing a thermometer into each test tube to monitor the temperature changes.
27. When temperature reaches 37oC add universal indicator to monitor pH changes, also add sufficient amounts of acid (Hydrochloric acid) or base (Sodium hydroxide) to each test tube to reach a pH of eight.
28. Once the pH of test tube reaches desired reading, using a syringe, add 2 ml of rennin (enzyme) to the test tubes and start timing, do not add enzyme to the test tube allocated as control, observe control test tube to notice if any coagulation occurs.
29. Stop timing once substrate coagulates, you will notice coagulation when test tube contents shows signs of visible lumpiness.
30. Record results in a table format.
pH of eleven
31. Create water bath by adding boiling water and cold water to reach a temperature of 37oC (optimum) in a Styrofoam cup.
32. Fill four test tubes with 20 ml of full cream milk (substrate), labeling with tt5, insure one test tube is also labeled control.
33. Place test tubes into water bath (Styrofoam cup) and insure substrate reaches a temperature of 37o C by placing a thermometer into each test tube to monitor the temperature changes.
34. When temperature reaches 37oC add universal indicator to monitor pH changes, also add sufficient amounts of acid (Hydrochloric acid) or base (Sodium hydroxide) to each test tube to reach a pH of eleven.
35. Once the pH of test tube reaches desired reading, using a syringe, add 2 ml of rennin (enzyme) to the test tubes and start timing, do not add enzyme to the test tube allocated as control, observe control test tube to notice if any coagulation occurs.
36. Stop timing once substrate coagulates, you will notice coagulation when test tube contents shows signs of visible lumpiness.
37. Record results in a table format.
Results:
pH of Substrate
Test tube
Time taken for substrate to coagulate
( min/sec )
3
6 sec
Tt 1 – Replicate 1
27 sec
Tt 1 – Replicate 2
25 sec
Tt 1 – Replicate 3
28 sec
5
15 sec
Tt 2 – Replicate 1
51 sec
Tt 2 – Replicate 2
53 sec
Tt 2 – Replicate 3
49 sec
6
No coagulation
Tt 3 – Replicate 1
1.02 min
Tt 3 – Replicate 2
1.03 min
Tt 3 – Replicate 3
1.02 min
8
No Coagulation
Tt 4 – Replicate 1
1.14 min
Tt 4 – Replicate 2
1.12 min
Tt 4 – Replicate 3
1.15 min
11
No Coagulation
Tt 5 – Replicate 1
>10 min
Tt 5 – Replicate 2
>10 min
Tt 5 – Replicate 3
>10 min
pH of Substrate
Average time taken for substrate to coagulate ( min/sec )
3
27 sec
5
51 sec
6
1.02 min
8
1.14 min
11
> 10 min
Discussion:
Aspects such as using the same methodology and equipment lead to an increased reliability of the experiment as using the same methodology will insure that all tests are done under the same conditions such as temperature. Adhering to the same methodology will also result in increased accuracy of results as external factors that affect results will apply for each test performed meaning that unexplained variations within the experiments results can be determined more easily as the methodology used will factor in the control of known external influences.
Using the same equipment when performing the experiment will increase reliability. This involves test tubes of the same size, stop watches measuring time in the same increments, syringes and other measuring equipment such as beakers or thermometers, to insure that results are being recorded in the same measuring increments which will improve reliability as there is fairness in results.
When the experiment was being performed it was observed that the addition of acid (Hydrochloric acid) to the substrate (Full cream milk) caused the substrate to coagulate even without the enzyme (rennin) in the solution. This vastly affected results as it was difficult to determine the coagulation of the substrate and acid compared to the coagulation when the enzyme is added, the only way to determine the effect of the acidic pH on the enzyme functioning is to see if there is a visible increase in the amount of coagulation occurring in the test tube, hence affecting the reliability of results as accuracy of results comes down to the observer which is easily effected by human error in judgment.
When revising the literature on the effect of pH on the activity of enzymes, as the results are graphed there is a distinct curve, although when the results observed in the school laboratory were graphed there was a different curve produced due to the fact that equipment supplied in testing the pH of the substrate did not further then a pH of 3 – 11. Hence the results may prove unreliable as proper conditions and requirements needed to observe correct results could not be met.
This experiment is valid as the method fairly tests the hypothesis. This experiment is also valid as the variables are kept constant, for example test tube size etc apart from those being investigated (dependent). Most systematic errors were eliminated as external factors that may influence results were taken care of, for example the influence of temperature was eliminated with the use of a water bath set at 37o C (optimum temperature), and random errors usually caused by human error were reduced as there was a mean of the replicates calculated.
Conclusion:
We can conclude that changing the pH solution in the substrate effects the activity of rennin, rennin’s optimum pH is 3 – proven with the results indicating that at a pH of 3 it only took 27 seconds in comparison to longer times recorded at other pH readings. As the pH varies further and further from that of its optimum the enzyme starts to denature – denaturing is when due to the change in pH hydrogen bonds in the molecule break apart which alters the shape and structure of the molecule, denaturing it – This is seen when the time recorded for the milk to coagulate becomes more and more delayed.
Evaluation:
Although the experiment’s method is relatively sound there are still aspects that could be improved, although due to past experiences with other experiments of the same nature alterations to method and equipment have proved beneficial as more reliable results have been provided.
It was suggested that instead of using a measuring cylinder to measure the solutions in the experiment that a syringe would be more advisable as it produces more accurate quantities and reduces the time spent on collecting the desired amount of solution. Due to the benefits of using a syringe I would include it in further experiments of the same nature.
To improve the quality and reliability of results in future experiments I would have more then one stop watch timing the amount of time until the substrate coagulates when the enzyme is added as this would increase reliability of results as a mean could be calculated for the time taken for the coagulation.
