How does the concentration of a substrate affect the activity of an enzyme?

Variables:

Independent variable: substrate concentration (1.5%, 1.0%, 0.5%)

Dependent variable: the amount of indicator (KMNO4) needed to neutralize the hydrogen

                                   peroxide/ catalase solution

        Controlled variables:  amount of water used, amount of catalase in each solution, amount                                         of sulfuric acid used to stop reaction, amount of solution, type of                                         substrate, type of enzyme, type of indicator, dilution of substrate

Planning (b)

Materials: a burette of potassium permanganate attached to a ring stand by butterfly clamps* (see figure 1), an Erlenmeyer flask, four stirring rods, six small test tubes for pre-measuring the sulfuric acid (to ensure that stopping happens at the exact correct time), test tube rack, an ice cup (to keep catalase cold and reduce denaturing), several pipettes, graduated cylinders for measuring substrate, a stop watch, food processor/blender, potato, distilled water, one cup

Figure 1*

   

Hazards: Caustic chemicals will be used (sulfuric acid, to stop the reaction between catalase and hydrogen peroxide, for example); safety clothing (goggles, apron, close-toed shoes) should be worn to ensure protection of skin. Beakers should be handled with care to avoid potential glass breakage.

Procedure:  

        

1. First, a catalase sample was prepared by combining a potato portion with 3 mL of water in a food processor or blender. This was placed in a cup, then placed in a separate cup full of ice in order to keep the sample cool and reduce denaturing of the enzyme tested.
2. Next, the different substrate samples were prepared. To prepare 1.5% hydrogen peroxide, 10 mL of distilled water was added to 10 mL of 3.0%  peroxide in a graduated cylinder. Three individual 5 mL samples were measured from this and set aside, and the excess was discarded in the waste container. To prepare 1.0% hydrogen peroxide, similar steps were taken, only 6.7 mL of 3.0% peroxide was combined with 13.3 mL of water. To prepare 0.5% peroxide, the same steps were taken, only 3.2 mL of 3.0% peroxide was combined with 16.8 mL of water. (For calculations, see Data Processing/Presentation.)
3. Eighteen 2 mL samples of sulfuric acid were measured with a graduated cylinder and set aside in small test tubes, to be used to stop the reactions.
4. Then, 5 mL of the 1.5% hydrogen peroxide solution was combined with 0.5 mL of the prepared catalase sample in an Erlenmeyer flask.
5. This reaction was timed using a stopwatch and proceeded for 3 minutes, after which it was stopped using the prepared 2 mL sulfuric acid.
6. The solution was then titrated using potassium permanganate (2%) and a burette. The amount of indicator needed to neutralize (it turned a slight pink color when this occurred) was recorded in a data table.
7. Steps 4-6 were repeated twice.
8. Steps 4-6 were repeated three times, only the 1.0% hydrogen peroxide was used instead of the 1.5%.
9. Steps 4-6 were repeated three times, only the 0.5% hydrogen peroxide solution was used instead.
10. All results were then recorded in data table #1.
11.  Lastly, all materials were cleaned and/or disposed of in the proper manner.

Data Collection

Raw Data Table #1: Measuring the amount of indicator (mL of KMNO4) needed to neutralize solutions involving substrates of varying levels of concentration

        

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Data Processing/ Presentation:

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Conclusion/ Evaluation

        Conclusion:

        Because the solution using the most concentrated hydrogen peroxide solution required

the highest amount of indicator to titrate, it has been proven that the hypothesis was correct; increased substrate concentration does in fact yield higher enzyme activity. The above data supports this inference because while catalase mixed with 0.5% hydrogen peroxide only required an average of 1.1 mL of potassium permanganate to neutralize; the 1.5% required an average of 5.3 mL, much higher.

        Evaluation:

        While the procedures used were successful in that they proved the hypothesis correct and         

were for the most part clear and consistent, they were far from perfect. For example, the enzyme used was diluted with distilled water, which could have limited its capability to catalyze. The reactions were not all stopped after exactly three minutes; there could have been a few seconds of inconsistency, which would skew the data. Not enough pipettes were used, therefore contamination could be another issue that would skew the data.

Nevertheless, the results produced by experimentation were relatively consistent. Perhaps if it was repeated, more attention could be paid to the issue of contamination, more consistent timing could be observed, and the enzyme tested could remain pure.