Test for the effect of enzyme concentration on catalysis

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Enzymes are homogenous biological catalysts which are made up of globular protein or nucleic acid. A homogenous catalyst is one that is in the same phase as the reaction mixture. Enzymes contain an active site which binds the substrates, the reactants, and processing them into products. Numerous enzymes are present in the body to speed up reactions that would otherwise take very long. They are highly specific and work by lowering the activation energy for the reaction thus increasing its rate. They are highly specific due to the structure of the active site being specific to a certain reaction. In doing so, they are neither consumed nor do they affect the equilibrium of the reaction.

The activities of enzymes are affected by temperature, enzyme concentration, substrate concentration and pH. This laboratory report looks at enzyme concentration where varying dilutions of an enzyme amylase would be used to hydrolyze starch and the time taken for complete hydrolysis recorded.

Amylase is an enzyme which is responsible for breaking down starch into glucose. It is present in saliva of humans and the pancreas.

APPARATUS/MATERIALS

Water

Buffer solution (pH 6.8)

1% starch solution

Saliva

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.

Tube

Water

Amylase

Dilution factor

A

2 ml

2 ml amylase solution (or saliva)

0.5

B

2 ml

2 ml from tube A

0.25

C

2 ml

2 ml from tube B

0.125

D

2 ml

2 ml from tube C

0.063

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.

Discussion

In the experiment, various dilutions of amylase solution were made and the time taken for the reaction to complete with the starch solution was timed. Iodine was placed so as to indicate that the starch has been converted to glucose. Iodine changes colour to blue-black when starch is present. However, when the amylase converts the starch to glucose it would change to a yellow brown colour.

Making a plot of the time taken for complete hydrolysis of the starch against the concentration (dilution factor) is a good indication of how concentration of the enzyme affects the reaction.  From the graph obtained, it is feasible to state that a linear relationship exists between the concentration of the enzyme, amylase, and the time taken for hydrolysis of the starch. Only few points deviated by a small margin from the best fit line which had a negative gradient. This same relationship was observed by the University of North Dakota when hydrolyzing starch with different concentrations of amylase. From the negative gradient of the line, it can be inferred that as the concentration of the enzyme increases, the time taken for complete hydrolysis decreases. This is true when compared with theoretical data obtained from Biochemistry 5th Edition by Stryer. More molecules of amylase would be present as the concentration increases, to catalyze the same amount of starch, as such having more molecules of amylase present, it would have more active sites to bind substrates and hence a faster reaction. Hence experimental results can be said to be true at this point.

Although various expected results were obtained, the experiment was not without errors. 

Firstly, pure amylase solution was not used. Saliva was used as a substitute directly from the student's mouths. This could well contain inhibitors that could slow down the rate of the reaction. Hence it may not be accurate to say that the saliva is 100% amylase. In relation, a lot of bubbles in the saliva were present when measuring it; as such it would not be accurate to say it is 2ml. However, for this experiment, as a unit of measurement, a dilution factor was used, not concentration, so it would not affect results.

Moreover, the original dilution "dilution A" was not used to derive all other solutions. It was diluted & re-diluted. Hence any error made in dilution B would carry over to the other dilution. More significantly, it would not be accurate to say that the dilution (concentration) of the enzyme was halved during each dilution. This is due to the fact that any errors made in making one solution would carry over to the next solution and it would not be plausible to say that the components of the solution are equally distributed. As such when removing the 2ml it would be inaccurate for one to say that it is 50% saliva and 50% water.

This leads to another error. It was noted that some of the colour of the iodine did not change. It is important to note here that amylase may not have been in that sample tested; as such it would not be a valid case for an argument of experimental success. This again goes back to the solution not being evenly distributed.

Disregarding whether the solution is a true 2ml amylase to 2ml water, it is possible to extrapolate from the graph the time taken for the hydrolysis of starch at different concentrations.

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