Lab+Report+5B+-+Enzyme+Experiment

Biomolecules
To investigate the effect of pH on enzyme action
 * Aim**

Most enzyme reaction will occur at pH 7.0 [distilled water], with little or no reaction at high or low pH levels.
 * Hypothesis**

- 1 water bath - Thermometer - 4 test tubes and rack - Measuring cylinder - 1 250ml beakers - 1 white tile - 2 droppers - Distilled water - Stop watch - 2% starch solution - 1% amylase solution - 1 M hydrochloric acid solution - 1 M ammonium hydroxide solution - Iodine solution
 * Appparatus/Materials**

2. To each of the test-tubes, add 2 cm3 of starch solution. Place the tubes a water bath at 37 °C 3. Make up the contents of the 4 test tubes as shown in the table below ** 4. Test the solution with iodine solution at the beginning of the experiment and after 10 minutes 5. Record your observations and conclusions in a table below
 * Methodology**
 * 1. Label 4 test tubes W, X, Y and Z
 * || Contents ||
 * W || 2 cm3 of starch solution + 2 cm3 of HCL + 2 cm3 of amylase solution ||
 * X || 2cm3 of starch solution + 2cm3 of distilled water + 2cm3 of amylase ||
 * Y || 2cm3 of starch solution + 2cm3 of NH4OH + 2cm3 of amylase ||
 * Z || 2cm3 of starch solution + 4cm3 of distilled water ||


 * Results**
 * Test tube || Contents || Iodine test at 0 minutes || Iodine test at 10 minutes || Conclusion ||
 * W || 2 cm3 of starch solution + 2 cm3 of HCL + 2 cm3 of amylase solution || Turned blue-black || Turned blue-black || Starch is present ||
 * X || 2cm3 of starch solution + 2cm3 of distilled water + 2cm3 of amylase || Remained brown || Remained brown || Starch is absent ||
 * Y || 2<span style="-webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; border-collapse: separate; font-family: sans-serif;">cm3 of starch solution + 2<span style="-webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; border-collapse: separate; font-family: sans-serif;">cm3 of <span style="-webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; border-collapse: separate; font-family: Verdana,sans-serif; font-size: 12px; line-height: 16px;">NH4OH + 2<span style="-webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; border-collapse: separate; font-family: sans-serif;">cm3 of amylase || Turned-blue-black || Turned blue-black || Starch is present ||
 * Z || <span style="font-family: sans-serif,helvetica,sans-serif;">2<span style="-webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; border-collapse: separate; font-family: sans-serif;">cm3 of starch solution + 4<span style="-webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; border-collapse: separate; font-family: sans-serif;">cm3 of distilled water || Turned blue-black || Turned blue-black || Starch is present ||

1. Which tube shows that starch digestion has occurred? Give reasons. Test-tube X. Test-tubes W, Y and Z turned blue-black when tested with iodine solution both before and after 10 minutes, showing that starch was still present in the test-tubes, and that the starch had not been digested by the amylase. On the other hand, test-tube X remained brown when tested with iodine solution, showing that starch is absent and therefore had been digested.
 * Interpreting the results

2. Which of the test-tubes contained mixtures that gave a blue-black colour? Test-tubes W, Y and Z.

3. Why did the mixtures in tubes stated in Question 2 remain blue-black? Even after 10 minutes, little or no starch has been digested, therefore starch was still present in these test-tubes, which turned the iodine solution blue-black.

4. What conclusion(s), based on the observations in this experiment, can be made about the effect of pH on enzyme action? Similar to the effect of temperature on action, an increase in pH will cause a increase in enzyme action until it reaches the optimum pH level, after which the rate of enzyme action will be inversely proportional to the level of pH. Most enzymes are sensitive to pH as the hydrogen ions in highly acidic solutions or the hydroxide ions in alkali solutions will denature the enzymes, thus stopping enzyme activity.

Every enzyme has a optimum pH, as shown in this table: ** Table II: pH for Optimum Activity mostly ranging from pH 6-8, with a few exceptions [e.g. Pepsin, Trypsin].
 * Enzyme || Optimum pH ||
 * [|Lipase (pancreas)] || 8.0 ||
 * Lipase (stomach) || 4.0 - 5.0 ||
 * Lipase (castor oil) || 4.7 ||
 * [|Pepsin] || 1.5 - 1.6 ||
 * [|Trypsin] || 7.8 - 8.7 ||
 * [|Urease] || 7.0 ||
 * Invertase || 4.5 ||
 * [|Maltase] || 6.1 - 6.8 ||
 * [|Amylase (pancreas)] || 6.7 - 7.0 ||
 * [|Amylase (malt)] || 4.6 - 5.2 ||
 * [|Catalase] || 7.0 ||

5. How does temperature and pH affect enzyme action? Extreme conditions will result in low rate of enzyme action as the enzymes are either denatured [as in high and low pH, and high temperatures] or do not possess enough kinetic energy to react with the substrates.