Test for effect of enzyme concentration on catalysis

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The aim of this laboratory exercise was to test the effect of enzyme concentration on catalysis by means of having varying concentrations of amylase (saliva) to act or catalyze a mixture of starch and buffer solution of pH 6.8 in a solution of water.

Enzymes are known as biological catalysts. These catalysts speed up the rate of chemical reactions that occur in the body by lowering the activation energy of these reactions. Enzymes play a very important role in the body, since the very vital reactions that these enzymes speed up, may take too long, or may not even occur at all if the particular enzyme was not present.

There are various conditions or ranges in the body, within which enzymes work well. Too far beyond this range and the enzyme will change its three dimensional shape therefore, denaturing the enzyme and rendering it unable to perform its task. Some of these conditions are pH, temperature, concentration of enzyme, concentration of substrate. For this labaratory session, we will be focusing on the concentration of enzyme and its effect on catalysis. Therothecially it is seen that the more enzymes that are available at any particular time at a fixed area with a fixed number of substrates would increase catalysis, since more enzymes are available for substrates to act on. Although there would b a point where the level of catalysis would reach a constant and cease to increase, simply because all of the active sites of the enzymes would be occupied at that given time, therefore preventing further reactions until a site becomes free. As a result of that, the hypothesis presented states that the increase in concentration of the enzyme would increase the rate of catalysis.

APPARATUS/MATERIALS

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1% starch solution

1% amylase solution (Saliva)

Water

Buffer solution (pH 6.8)

12 test tubes

A timer

Iodine solution

Pipette

Small measuring cylinder

METHOD

4 test tubes were labeled A-D, a serial dilution of amylase solution was generated for each test tube; the amylase solution was generated as follows.

2 ml of water was placed into test tubes A through to D

2 ml of amylase (saliva) solution was placed in to test tube A, bringing its total contents to 4 ml.

2 ml of the contents of test tube A was added to the 2 ml of water in test tube B

2 ml of the contents of test tube B was added to test tube C

2 ml of the contents of test tube C was then added to test tube D

2 ml of the resulting mixture from tube D was removed and discarded; tubes A-D now had 2 ml of solution each.

40 drops of buffer solution (pH 6.8) was added to tube A.

8 test tubes were obtained, which were labeled 1-8; 2 drops of iodine solution was then placed into each tube 1-8.

0.5 ml of 1% starch solution was added to tube A.

A dropper was used to immediately transfer one drop of solution from tube A to Iodine tube #1.

The dropper was rinsed

1 minute later, the dropper was used to transfer one drop of solution from tube A to iodine tube #2.

This was repeated at 1-minute intervals until the iodine solution was light yellow brown, or until iodine tube # 8 was reached.

The contents of iodine tubes were discarded; the tubes were then rinsed properly and dried for use again.

Steps 3-10 were repeated for tubes B-D.

RESULTS

Test Tube

Time

(mins)

Time that change occurred

Observations

A

1

*

Light Yellow/Brown

2

-

3

-

4

-

5

-

6

-

7

-

8

-

B

1

Dark Brown

2

Dark Brown

3

Dark Brown

4

Dark Brown

5

*

Light Yellow/Brown

6

-

7

-

8

-

C

1

Dark Brown

2

Dark Brown

3

Dark Brown

4

Dark Brown

5

Dark Brown

6

Dark Brown

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7

*

Light Yellow/Brown

8

-

D

1

Dark Brown

2

Dark Brown

3

Dark Brown

4

Dark Brown

5

Dark Brown

6

Dark Brown

7

Dark Brown

8

*

Light Yellow/Brown

DISCUSSION

For this lab, there was an incremental dilution of the amylase in the respective test tubes A to D, this allowed for a means of measuring the effect of concentration, since there was a decrease in concentration of the enzyme going from tube A to tube D. The buffer solution was added to the starch in order to maintain a constant pH of about 6.8, since it is known that most enzymes have an optimum level of performance at a narrow pH range, out of this range, enzyme activity would be altered; in other words, the buffer solution acts as a control mechanism that allows for an increased level of accuracy of the results. The enzyme amylase catalyzes the reaction where starch is broken down. When this happens, starch is converted back into the sugar, maltose. This breakdown is actually the basis of the reaction, and this is where the iodine comes in. Iodine is known to test for the presence of starch, in which case it changes to a dark brown or purple colour. Therefore, in case where the amylase actually broke the starch down into maltose, there would be less starch present in relation to the units of maltose; the less starch that is present would give rise to a lighter shade of brown or a yellowish shade rather than the dark brown or purple. After the experiment was done, it was seen that the solution of starch and the highest concentration of amylase (test tube A), had changed to yellow upon addition to the first test tube of iodine, indicating that the starch had began to break down after the first minute. For test tube B, where the concentration was lower than that of test tube A, the colour change took place at the fifth test tube of iodine, indicating that the reaction occurred after five minutes. For the third and forth test tube, the colour change occurred at the seventh and eighth test tubes of iodine respectively. These results show that where the concentration of the amylase was high, the reaction took less time to occur, and where the concentration was lower, the reactions took more time to occur. This indicated that there is a direct link between concentrations of the enzyme and the rate of the reaction. Some sources of error that could have caused inaccurate results would be the fact that the iodine was left exposed for a while before the starch/amylase solution was added to it

CONCLUSION

From the experiment that was conducted, it was found that enzyme concentration is directly proportional to the reaction rate.