3C_Investigating+the+effect+of+pH+on+enzyme+action

__**INVESTIGATING THE EFFECT OF pH ON ENZYME ACTION**__

__**Aim:**__ To find out the effect that various pH levels of solutions have on enzyme action, hence determining the most ideal pH level for enzyme action to occur.

__**Hypothesis:**__ The solutions in test tubes W, Y and Z would turn the iodine solution blue-black during the first and second iodine test, indicating the presence of starch. The solution in test tube X would turn the iodine solution brown during the first and second iodine test, suggesting that starch is not present in the solution. This is because the solution in test tube X has contents that have a neutral pH level, similar to that of the body, therefore being the most ideal pH level for enzyme action to occur.

__**Method:**__
 * 1) Label 4 test tubes W, X, Y, Z using the white labels provided
 * 2) Fill a beaker (water bath) with warm water, and with the aid of a thermometer, add tap water to the beaker until the temperature read on the thermometer is 37 ºC.
 * 3) Add 2cm 3 of starch solution to tubes using the dropper provided in the starch solution (taking care not to use droppers from other solutions so as to avoid contamination of the solutions) to drop the amount into a measuring cylinder before pouring it into the test tubes. This will ensure more accurate readings.
 * 4) Place the test tubes in the water bath at 37 ºC.
 * 5) Add 2cm 3 of Hydrochloric Acid (HCL) to test tube W, measuring the exact amount of solution in the measuring cylinders provided before adding it to the test tube. Add 2cm 3 of 1% amylase solution to test tube W, using the dropper provided in the solution, (taking care not to use droppers from other solutions so as to avoid contamination of the solutions) to drop the amount into a measuring cylinder before pouring it into the test tube.
 * 6) Add 2cm 3 of distilled water to test tube X, measuring the exact amount in the measuring cylinders provided before adding it to the test tube. Add 2cm 3 of 1% amylase solution to test tube X, using the dropper provided in the solution, (taking care not to use droppers from other solutions so as to avoid contamination of the solutions) to drop the amount into a measuring cylinder before pouring it into the test tube.
 * 7) Add 2cm 3 of ammonium hydroxide (NH 4 OH) to test tube Y, measuring the exact amount of solution in the measuring cylinders provided before adding it to the test tube. Add 2cm 3 of 1% amylase solution to test tube Y, using the dropper provided in the solution, (taking care not to use the droppers from other solutions so as to avoid contamination of the solutions) to drop the amount into a measuring cylinder before pouring it into the test tube.
 * 8) Add 4cm 3 of distilled water to test tube Z, measuring the exact amount in the measuring cylinders provided before adding it to the test tube.
 * 9) Test the solutions in all 4 test tubes by dropping 4 drops of iodine solution onto a white tile and using a dropper to drop one drop of each solution onto each drop of iodine. (Be sure to use either a new dropper for each solution, or to wash the dropper thoroughly before placing it into another solution). Record any colour change in the drops of iodine solution, and the time that they were tested (keep time using a stopwatch).
 * 10) Leave the solutions in the water bath for 10 minutes, using a stopwatch to keep time.
 * 11) After 10 minutes, test the solutions again by dropping 4 drops of iodine solution onto a white tile and using a dropper to drop one drop of each solution onto each drop of iodine. (Be sure to use either a new dropper for each solution, or to wash the dropper thoroughly before placing it into another solution). Record any colour change in the drops of iodine solution.

__**Apparatus and materials:**__
 * 1) Warm water
 * 2) 1- 37ºC water bath
 * 3) Tap water
 * 4) Beaker
 * 5) Thermometer
 * 6) Measuring cylinder
 * 7) White tile
 * 8) 4 droppers
 * 9) Distilled water
 * 10) Stop watch
 * 11) 2% starch solution
 * 12) 1% amylase solution
 * 13) 1 M Hydrochloric acid solution
 * 14) 1 M Ammonium Hydroxide solution
 * 15) Iodine solution

__**Results:**__

__**Analysis:**__ The concentration of hydrogen ions in solution affects the enzyme activity. Each enzyme has maximal efficiency under an optimum pH. Since pH is one of the factors for the denaturation of proteins, if an enzyme is submitted to a pH level under which it is denatured there will be no enzymatic activity. Most enzymes act in pH between 6 and 8, a range that corresponds to the general acidic level of cells and blood. There are enzymes, however, that act only under very acid or very basic pH. So enzyme activity depends on pH interval. From the results of the experiment, test tube X left the iodine solution brown (original colour) in both the first and second iodine tests, suggesting that starch was no longer present in the solution. This shows that starch digestion had occurred, the amylase digesting the starch, as there was starch in the solution originally. The contents of test tube X had a neutral pH level (pH 7.0), one similar to that of the human body, therefore is the most suitable pH level for enzyme activity to occur. In test tubes W, Y and Z, the iodine turned blue-black when tested with the solutions, indicating that starch was present. In tube W, the solution contained hydrochloric acid (HCL), making the solution have a pH less than a neutral pH of 7.0. The starch in this solution was not digested by the amylase, therefore indicating that the pH level of the solution was not suitable for enzyme activity to occur. In test tube Y, the solution contained ammonium hydroxide (NH4OH), making the solution have a pH more than a neutral pH of 7.0. The starch in the solution was not digested by the amylase, therefore turning the iodine solution blue-black, indicating that solutions with pH levels higher than a neutral pH of 7.0 are not suitable for enzyme activity to occur. Tube Z was used as a control in the experiment, as no enzyme (amylase) was added to the solution. This would definitely turn the iodine solution blue-black, as the starch would not have been digested, hence acting as a reference to determine if other solutions contained starch when they are tested with iodine solution. In conclusion, the pH level suitable and most ideal for enzyme activity to occur is that of a neutral pH, about 7.0. pH levels lower or higher than the neutral pH level (7.0), are not suitable for enzyme activity to occur as they are either too acidic or too alkaline.
 * **Tube** || **Contents** || **Temp. (ºC)** || **Time of first iodine test (s)** || **Time of second iodine test (min)** |||| **Iodine test** || **Conclusion** ||
 * ^  ||^   ||^   ||^   ||^   || **First** || **Second** ||^   ||
 * W || 2cm3 of starch solution + 2cm3 of HCL + 2cm3 of amylase || 37 || 15 || 10:17 || Iodine turned blue-black || Iodine turned blue-black || Solution contained starch during the first and second iodine test ||
 * X || 2cm3 of starch solution + 2cm3 of distilled water + 2cm3 of amylase || 37 || 30 || 10:34 || Iodine turned brown || Iodine turned brown || Solution did not contain starch during the first and second iodine test ||
 * Y || 2cm3 of starch solution + 2cm3 of NH4OH+ 2cm3 of amylase || 37 || 45 || 10:48 || Iodine turned brown || Iodine turned brown || Solution contained starch during the first and second iodine test ||
 * Z || 2cm3 of starch solution + 4cm3 of distilled water || 37 || 60 || 11:05 || Iodine turned blue-black || Iodine turned blue-black || Solution contained starch during the first and second iodine test ||

__**Questions:**__ Tube X. It left the iodine solution remaining brown, therefore suggesting that starch was no longer present in the solution. Starch digestion occurred as there was starch in the tube originally, but starch was no longer present at the end of the experiment, proving that the starch digestion had occurred. Tubes W, Y and Z. In tubes W and Y, the solutions were too acidic and alkaline, respectively for enzyme activity to occur, as the pH levels were lower and higher than neutral pH 7.0. In tube Z, there was no enzyme present in the mixture, therefore the starch could not have been digested. Neutral pH 7.0 is the most suitable and ideal pH level for enzyme activity to occur, and when the pH level is too high or too low, the enzymes can no longer work on the solutions. Enzyme activity is the most effective at temperatures and pH levels that are similar to the levels in the human body. If the temperatures are too low or high, enzyme activity no longer occurs, and if the pH levels are too high or low, enzyme activities also cannot occur.
 * 1) Which tube shows that starch digestion has occurred? Give your reasons.
 * 1) Which of the test tubes contained mixtures that gave a blue-black colour?
 * 1) Why did the mixtures in tubes stated in (2) remain blue black?
 * 1) What conclusion(s), based on the observations in this experiment, can be made about the effect of pH on enzyme action.
 * 1) How does temperature and pH affect enzyme action?