Investigation of the effect of the pH value on the enzyme controlled reaction

Background

•A buffer is a mixture of a weak acid and a soluble salt of that acid. It is used to maintain the pH value of a solution.
A weak acid can dissociate in solution to form H ions and the acid group ions. The concentration of the H ions decides the pH value of the solution.

The H ions and the weak acid molecules are at an equilibrium state. If either of them increases the concentration, the reaction will go towards the side at which the molecules or ions with low concentration.

To insure the solution with an almost constant pH value, the little amount H ions or OH ions should be removed if they are added to this solution. OH ions are easy to remove because they will react with H ions to form water. (Remember the amount of OH ions mustn’t too great or the weak acid solution will not available to remove it.) This disturbs the equilibrium and as predicted by Le Chatelier’s principle more acid molecule will dissociate to restore the situation, so the pH tends to remain the same. If more H ions are added, it will combine with the acid group ions to form the acid molecule in order to maintain the equilibrium. Because it is a weak acid, the supply of group ions soon runs. At this time buffer solution can be used to provide acid group ion to remove the excess H ions. And so the pH is maintained well. H ions are released during the breaking down of the starch.

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•The H ions will interact with R group of amino acids and affect the way in which they bond with each other and therefore affect their 3D arrangement. Neither too many nor too little is good for the shape of the enzyme (depend on the type of the enzyme). An enzyme with the distorted shape will not fit the substrate well so lose the ability to catalyse the reaction. As pH value gets higher and higher, the H ions get more and more and fewer and fewer enzymes can work properly. In consequence the rate of reaction is lower ...

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Safety

a). Goggles essential as the solution with pH value 5 is irritant

b). Almost all the enzymes all potentially irritant so avoid contact with skin.

Apparatus

2 x Goggles / 10 x test tube / 6 x 2ml syringes / marker pen / 2 x iodine solution in dropper bottle (full) / 2 x stop clocks / 4 x spotting tiles / 1x thermometer / 4 x glass rods /10 x tissues

Access to
Starch suspension(1.0%)–about 25ml per group ( for wastage) / Amylase (0.5%) buffered at five different pH that in the range 5 – 8. about 5ml per group.

N.B

Make up the buffered amylase with buffer instead of distilled water at the required pH levels.

Plan

1.Using a thermometer take and record the temperature of the laboratory.

2.Using 2ml syringe place 2ml of buffered amylase with different concentrations into each of the
five test tubes.

3.Using 2ml syringe place 2ml of starch suspension into each of the five test tubes.
4.Pour the amylase into the starch, shake and immediately start the stop clock. Simultaneously

remove one drop of the mixture with a clean glass rod and test with orange iodine solution on a
spotting tile.

5.Repeat step 4 at intervals e.g. every 10 seconds. Once the mixture fails to turn the orange iodine
solution blue / black you will be able to work out how long it took for the amylase to break down
the substrate starch.
6.Repeat step 5 for each pair of starch and amylase, record the time for amylase to break down the
starch.

Results

Note: To time the Average reciprocal is simply to make the value easier to read.

Graph plotted on the graph paper

Interpretations

The graph shows as the pH value increases from 0 to around 7.7, the rate of reaction increases. As pH value increases from 7.7 to further, the rate of reaction decreases. This shows that amylase has an optimum pH around 7.7 (neutral). The interfering of the enzyme by H ions is described in the background.