An Investigation of the Effects of Substrate Concentration on Enzyme Activity

Purpose / Personal Engagement

Substrate concentration is an important factor which affects the activity of enzymes. I became interested in the effect of substrate concentration on enzymes during our classroom examinations of heat and pH on enzyme activity. Substrate concentration was one of factors that I had explored and researched the least, so I chose this particular experiment partially to enhance my understanding of this enzyme-affecting factor.

I chose catalase’s catalysis of the decomposition of hydrogen peroxide for this experiment firstly, because I previously experimented with bovine catalase (from beef liver) and wanted to study catalase again, but this time using catalase from a potato, and having a broader knowledge of enzymes with which to interpret and understand my data. Secondly, I find catalase to be an interesting enzyme due to its natural use as a protectant against oxidative damage to cells, and wanted to observe and find out more about it through research and experimentation. Lastly, catalase and hydrogen peroxide were easy to obtain and were flexible enough to allow for a straightforward implementation of my investigation, a factor which I considered in order to get the clearest and most presentable results possible.

Background Information

Enzymes are important biological molecules which speed up and slow down chemical reactions within cells while remaining unchanged. Substrates bind to enzymes at their active sites, where the enzyme weakens bonds to decrease the activation energy of the reaction of the substrate, and thus can control reaction rate.

Catalase is a common enzyme which facilitates the decomposition of hydrogen peroxide into oxygen and water (2H_2 O〖_(2 )〗 -

Catalase→ 2H_2 O + O_2 ). Catalase can be found in the peroxisomes of most aerobic cells, and protects the cells against oxidative damage from hydrogen peroxide, a toxic by-product of the breakdown of food molecules into energy(Mcdowall). There are actually a number of different catalases, each one essentially specific to a particular species and varying by an NADP molecule or a heme group (Boon, Downs, Marcey). Catalase has one of the highest turnover numbers of any enzyme, and can break down millions of molecules of H2O2 per second(“Catalase”), making it a good candidate for observable results in this experiment.

In general, reaction rate for enzyme catalyzed reactions increases with substrate concentration, but as active sites of the enzyme become increasingly occupied, the change in reaction rate from substrate concentration increase asymptotically approaches zero, as can be seen from Figure 1(“Energy, Enzymes, and Catalysis”). The point at which enzyme concentration, rather than substrate concentration becomes the limiting factor in a reaction is called the point of saturation(“Biomolecules: Enzyme Kinetics”).

Hypothesis

If substrate(H2O2) concentration is increased, the reaction rate will increase until the catalase active sites becomes saturated with H2O2 molecules, at which point the rate of change of reaction rate with respect to substrate concentration will begin to decrease, since this had been shown to be the general effect of substrate concentration on enzyme catalysis of reactions(“Biomolecules: Enzyme Kinetics”). The graph of substrate concentration vs reaction rate for this experiment should resemble that of Figure 1, since enzymic reactions all follow the same general model(“Energy, Enzymes, and Catalysis”).

Variables

Independent Variable: Concentration of hydrogen peroxide solution (measured in moles/L).

The independent variable was manipulated by diluting a solution of 3% H2O2 to produce five different solutions.

Dependent Variable: Reaction rate of hydrogen peroxide decomposition(in molesO2/second).

The dependent variable was measured by measuring the volume of O2, in mL produced over a constant time interval(12▁0 seconds), and converting it to moles/second.

Controlled Variables:

Approximate number of moles of H2O2

Changing the number of moles of the substrate between trials would change the amount of substrate molecules available to collide with enzyme active sites without changing the concentration of the solution. The ratio of moles to volume must be the only variable changed, otherwise the experiment measures the effects of both substrate concentration and substrate amount conjunctly. Moles of H2O2 were kept as constant as possible by measuring a constant 20 mL of 3% H2O2 solution before the dilutions when manipulating the independent variable.

Concentration of puréed potato solution

Not controlling for the concentration of the catalase source would introduce enzyme concentration as a confounding variable in the experiment, and would make it difficult to measure the effect of substrate concentration on the reaction rate. This variable was controlled for by taking all of the puréed potato solution from the same beaker of blended potato solution, which was stirred periodically to maintain homogeneity.

Milliliters of puréed potato solution

The amount of catalase source would skew the data because the number of molecules of catalase would affect reaction rate since more active sites would be available to act on H2O2 molecules. To control for enzyme amount, a constant volume of 5mL of puréed potato solution was added to each dilution of the hydrogen peroxide solution.

Initial and final volumes of air and water in the O2 transfer tube (see Figure 2)

The initial air in the tube is forced into the graduated cylinder when the reaction begins, and the tube’s volume of produced O2 is left in the tube at the end of the reaction. Any amount of water in the transfer tube at the beginning of the experiment decreases the volume of air in the tube that is pushed into the graduated cylinder, and since this amount isn’t measured, would add an extra degree of variance in the data which may render it uninterpretable. The volume of O2 at the end of the reaction could affect the data if the tube is removed before measurement of the dependent variable, since various amounts of O2 may escape the tube and not be included in the measurement. Both these variables were controlled for by keeping all water out of the tube before the reaction, and measuring the dependent variable before removing the tube from the beaker(see Figure 2). This way, the initial volume measurements all have the same subtracted value(the volume of the tube), and all the final measurements have the same added value(the volume of the tube plus the volume of the beaker).

Temperature of reaction

Temperature affects enzyme activity since different speeds of particles result in different numbers of particle collisions per interval of time, and therefore affect reaction rate. This would result in a change in the dependent variable that isn’t due to the independent variable. Temperature also affects the volume that a number of moles of gas occupies, so measurements of the dependent variable where trials occurred at different temperatures would vary as a result of the ideal gas law rather than substrate concentration. This was controlled for by collecting all data in one temperature-controlled location(a classroom) over a short period of time.

Time for reaction to occur(measured in seconds)

If a different amount of time is given for each reaction to occur, reaction rate cannot be measured. Additionally, if too long a time interval is given for each trial, all of the substrate will have decomposed, and reaction rate will also not be measured, since all data points of the dependent variable would theoretically have the same value. Time was eliminated as a confounding variable by allowing 12▁0seconds for each reaction to occur.

Control: The control was a 3.0%(0.0013M) solution of hydrogen peroxide dissolved in water.

Procedure

Materials:

Two 400 mL glass beakers

Two 100 mL graduated cylinders

5000 mL plastic tub

10 mL plastic syringe with large nozzle

Plastic wrap(25 9cm x 9cm sheets)

Rubber band(with a diameter less than that of the beaker)

0.4m plastic tube, with .01m diameter

Solution of 3.0% hydrogen peroxide dissolved in water(500 mL or more)

Potatoes(with a volume of at least 125cm3)

Electric Blender

Digital stopwatch(which measures in seconds)

Methodology:

A potato was cut into small pieces and puréed in a blender until near liquid consistency was obtained. The puréed potato solution was stirred periodically to maintain homogeneity during the experiment.

5 Solutions of H2O2(aq) were prepared using the following chart:

20mL of 3.0% H2O2(aq) were measured with a graduated cylinder and added to each solution initially, and then the dilution specified by Column 3 of the table was performed by measuring the specified volume of water and adding it to the existing solution.

20mL of 3.0% H2O2(aq) = 0.6mL H2O2(aq)

0.6mL H2O2(aq) x (1.45g〖H_2 O_2〗

)/(1 mL H_2 O_2 ) x (1moleH_2 O_2)/(34.02gH_2 O_2 ) ≈ 0.026molesH2O2

Solution Calculation for dilution Dilution required Calculation for molarity of solution

3.0% H2O2 20mLH_2 O_2 (0.03) = 0.6mLH2O2(aq) Initial concentration, no dilution required (0.026molesH_2 O_2)/(20mlH_2 O_(2(aq)) ) = 0.0013M

2.5% H2O2 (20mLH_2 O_2 (0.03))/0.025 = 24mLH2O2(aq) Add 4mL H2O (0.026molesH_2 O_2)/(20mlH_2 O_(2(aq)) ) = 0.0011M

2.0% H2O2 (20mLH_2 O_2 (0.03))/0.020 = 30mLH2O2(aq) Add 10mL H2O (0.026molesH_2 O_2)/(20mlH_2 O_(2(aq)) ) = 0.00086M

1.5% H2O2 (20mLH_2 O_2 (0.03))/0.015 = 40mLH2O2(aq) Add 20 mL H2O (0.026molesH_2 O_2)/(20mlH_2 O_(2(aq)) ) = 0.00065M

1.0% H2O2 (20mLH_2 O_2 (0.03))/0.010 = 60mLH2O2(aq) Add 40mL H2O (0.026molesH_2 O_2)/(20mlH_2 O_(2(aq)) ) = 0.00043M

Approximately 3000 mL of water were added to the plastic tub. The 100mL graduated cylinder was also filled with water, inverted, and inserted into the tub. The initial volume of water in the cylinder was measured and recorded.

The appropriate solution of hydrogen peroxide was poured into the 400 mL beaker, which was then covered by plastic wrap and secured by a rubber band to ensure airtightness.

The plastic tube was inserted into the top of the beaker, through the plastic wrap, and into the inverted graduated cylinder, while maintaining an airtight fit. The tube was emptied of water for each trial.

Using the 10mL syringe, 5mL of the puréed potto solution were measured and the syringe was inserted into the beaker through the plastic wrap.

The plunger of the syringe was pushed down, and the stopwatch was started simultaneously. The reaction of the catalase and H2O2 produced oxygen bubbles, which were transferred to the graduated cylinder via the plastic tube. The solutions were allowed to react for 12▁0seconds, during which time, qualitative observations could be made, such as the nature of the O2 bubbles produced by the reaction, or apparatus insufficiencies which could affect data.

After the time limit was reached, the tube was immediately removed, and the volume of water in the graduated cylinder was measured and recorded a second time.

The beaker was cleaned out and the graduated cylinder was refilled with water(initial volume was recorded again).

Steps 4-9 were repeated five times each for each of the five independent variable conditions.

The data points for the dependent variable were taken by dividing the absolute value of the difference between the initial and final water volumes by the time interval of 120 seconds(3 minutes), and converting mLO2 to molesO2 :

Reaction Rate = |(V_(initial ) - V_final)/(120 seconds)| *(32gO_2)/(22400mLO_2 ) *(1 moleO_2)/(32gO_2 )

The data for each trial were averaged using ▁X=(∑_(k=1)

N X_k)/N where N = 5, and the standard deviation for the discrete random variable, X(reaction rate) was taken by using σ=√((∑_(k=1)

N(X-μ)

2)/N) where μ=▁X.

Diagram of Lab Setup:

Safety, Ethical and Environmental Concerns:

Precautions should always be taken when using any kind of chemicals. Even at low concentrations, hydrogen peroxide can pose a health risk if ingested, or if applied to the eyes. If possible, safety goggles are recommended when using hydrogen peroxide. If hydrogen peroxide comes in contact with the eyes, rinse immediately, if ingested, contact a poison control center. Caution must also be exercised when using equipment such as blenders. Care should be taken to unplug the blender before opening it to maintain safety.

No ethical concerns are relevant to this particular study, and none of the materials or products pose an environmental risk.

Data Tables

Time was 120 seconds ± 0.5 seconds for every trial

Relative uncertainties for Column 5 of each table was calculated using the sum of the relative uncertainties of the tables respective ∆Volume ε_C5=ε_H2OV0+ε_H20VF.

Relative uncertainties for Column 6 of each table were calculated using the sum of the table’s uncertainties for MolesO2 and the relative uncertainty for ∆Time(.42%).

Raw Data Tables: Molarity of H2O2 solution vs. Reaction rate

Data Table A:

0.0013M H2O2(aq)

3.0% Solution H2O Initial Volume(mL ± 0.5mL) H2O Final Volume(mL ± 0.5mL) ∆Volume(mL ± 1mL)

(|H2O V0 - H2O Vf|) MolesO2 Produced

(∆Volume x (1mol/22400mL) Reaction Rate(sec)

MolesO2/120

Trial 1 98 72 26 0.0012 ± 3.8% 9.7 x 10-6 ± 4.2%

Trial 2 65 41 24 0.0012 ± 4.2% 8.9 x 10-6 ± 4.6%

Trial 3 68 39 29 0.0013 ± 3.4% 1.1 x 10-5 ± 3.9%

Trial 4 76 55 21 0.00094 ± 4.8% 7.8 x 10-6 ± 5.2%

Trial 5 59 30 29 0.0013 ± 3.4% 1.1 x 10-5 ± 3.9%

Data Table B:

0.0011M H2O2(aq)

2.5% Solution H2O Initial Volume(mL ± 0.5mL) H2O Final Volume(mL ± 0.5mL) ∆Volume(mL ± 1mL)

(|H2O V0 - H2O Vf|) MolesO2 Produced

(∆Volume x (1mol/22400mL) Reaction Rate(sec)

MolesO2/120

Trial 1 75 60 15 0.00067 ± 6.7% 5.6 x 10-6 ± 7.1%

Trial 2 84 68 16 0.00071 ± 6.3% 6.0 x 10-6 ± 6.7%

Trial 3 75 61 14 0.00063 ± 7.1% 5.2 x 10-6 ± 7.6%

Trial 4 67 45 22 0.00098 ± 4.5% 8.2 x 10-6 ± 5.0%

Trial 5 90 77 13 0.00058 ± 7.7% 4.8 x 10-6 ± 8.1%

Data Table C:

0.00086M H2O2(aq)

2.0% Solution H2O Initial Volume(mL ± 0.5mL) H2O Final Volume(mL ± 0.5mL) ∆Volume(mL ± 1mL)

(|H2O V0 - H2O Vf|) MolesO2 Produced

(∆Volume x (1mol/22400mL) Reaction Rate(sec)

MolesO2/120

Trial 1 78 68 10 0.00045 ± 1▁0% 3.7 x 10-6 ± 1▁0%

Trial 2 90 77 13 0.00058 ± 7.7% 4.8 x 10-6 ± 8.1%

Trial 3 65 51 14 0.00063 ± 7.7% 5.2 x 10-6 ± 7.6%

Trial 4 66 57 9.0 0.00040 ± 11% 3.3 x 10-6 ± 12%

Trial 5 93 80 13 0.00058 ± 7.7% 4.8 x 10-6 ± 8.1%

Data Table D:

0.00065M H2O2(aq)

1.5% Solution H2O Initial Volume(mL ± 0.5mL) H2O Final Volume(mL ± 0.5mL) ∆Volume(mL ± 1mL)

(|H2O V0 - H2O Vf|) MolesO2 Produced

(∆Volume x (1mol/22400mL) Reaction Rate(sec)

MolesO2/120

Trial 1 96 93 3.0 0.00013 ± 33% 1.1 x 10-6 ± 34%

Trial 2 57 52 5.0 0.00022 ± 2▁0% 1.9 x 10-6 ± 21%

Trial 3 80 77 3.0 0.00013 ± 33% 1.1 x 10-6 ± 34%

Trial 4 75 71 4.0 0.00018 ± 25% 1.5 x 10-6 ± 26%

Trial 5 77 74 3.0 0.00013 ± 33% 1.1 x 10-6 ± 34%

Data Table E:

0.00043M H2O2(aq)

1.0% Solution H2O Initial Volume(mL ± 0.5mL) H2O Final Volume(mL ± 0.5mL) ∆Volume(mL ± 1mL)

(|H2O V0 - H2O Vf|) MolesO2 Produced

(∆Volume x (1mol/22400mL) Reaction Rate(sec)

MolesO2/120

Trial 1 70 64 6.0 0.00027 ± 17% 2.2 x 10-6 ± 17%

Trial 2 63 60 3.0 0.00013 ± 33% 1.1 x 10-6 ± 34%

Trial 3 89 88 1.0 0.00045 ± 1▁0 0% 3.7 x 10-7 ± 1▁0 0%

Trial 4 92 92 0 0 0

Trial 5 71 68 3.0 0.00013 ± 33% 1.1 x 10-6 ± 34%

Processed Data Table: Average value and standard deviation of dependent variable

Data Table F: Statistics for raw data Average Reaction Rate(molesO2/second)

1/5 ∑_(k=1)

5(Reaction Rat〖e)〗_k Standard Deviation of Reaction Rate(molesO2/second)

√(1/5 ∑_(k=1)

5(Rxn Rate - μ)

2 )

Data Table A: 0.0013MH2O2 Solution 26 3.1

Data Table B: 0.0011M H2O2 Solution 16 3.2

Data Table C: 0.00086M H2O2 Solution 12 1.9

Data Table D: 0.00065M H2O2 Solution 3.6 0.80

Data Table E: 0.00043M H2O2 Solution 2.6 2.1

Standard Deviation of Dependent Variable Set: Sx = √(1/25 ∑_(k=1)

25(X - ▁X )

2 ) = 3.3molesO2/sec

Average ∆Reaction Rate for .5% dilution of H2O2 Solution: 1/4 ∑_(k=1)

4 〖∆X〗_k= 5.9 molesO2 / sec

Qualitative Data:

-Oxygen bubbles begin to form immediately when catalase is added to the H2O2 solution, then oxygen travels through the tube. Observable water level changes began occurring after about a minute for all reactions.

-Some oxygen escapes between the plastic wrap and the syringe, especially if pressure is applied to the plastic wrap.

-Rate of bubble production was noticeably decreased between the 0.0013M solution and the 0.0011M solution, as well as between the 0.00086 and 0.00065M solutions.

-Any agitation of the beaker seemed to increase the rate of bubble production, and likely affected reaction rate.

Graphs

This is the graphical representation of the data. The mean dependent variable points(reaction rates) were plotted against the independent variable([H2O2]). Error bars were added showing plus or minus one standard deviation of the dependent variable data set. An approximation of the line of best fit was drawn using the area method

Conclusion

The data suggests that reaction rate increases as substrate concentration decreases, which is consistent with the hypothesis, however, there is no evidence of the asymptotic decrease in the rate of change of reaction rate as predicted by the hypothesis. This lack of evidence of substrate saturation is probably due to the constraints of the materials. The highest concentration used was 3.0% (0.0013M) H2O2(aq), so only a portion of the predicted graph(Figure 1) could be achieved.

The average change in reaction rate for a .5% dilution of the substrate solution was 5.9 molesO2/sec(see Data Table F). The statistical difference between the data points in this experiment were calculated by the difference in the numerical areas enclosed by the data points and their uncertainties(represented by error bars on the graph) of one standard deviation in either direction. As can be seen by the graph(Figure 3), although there was no statistically significant difference in the reaction rate produced by 0.00043M H2O2 compared to 0.00065M H2O2, or 0.0011M H2O2 compared to 0.00086MH2O2, the rest of the data points show a significant increase in the reaction rate which exceeds that which could have been due to uncertainty, as indicated by Figure 3 and Data Table F. The conclusion that substrate concentration and reaction rate have a positive correlation is statistically valid based on the data. The qualitative observations of the data(that bubble volume increased with substrate concentration) also supports this conclusion. No statistical or qualitative evidence was obtained from the experiment which supports an eventual decrease in the effect of substrate concentration, although this portion of the hypothesis can be tentatively maintained due to the constraints of the range of data collected.

Limitations and Improvements

Although this lab obtained discernable results, it still had many limitations. One limitation was measurement, both due to the apparatus used, and human error. The degree to which this could have affected the data, however, was calculated and listed in the Data section.

Besides the statistical uncertainties of the data already mentioned, other factors may have somewhat influenced the data. Some oxygen escaped from the beaker without entering the tube, for example, as was observed during the experiment. Some agitation of the beakers also occurred during some of the trials, which perhaps affected the collision of substrate molecules with enzyme active sites, and could have increased reaction rate. Non-constant applied pressure to the top of the beaker also may have forced oxygen into the tube, affecting measurement of the dependent variable. Since none of these possible factors were controlled for or factored into the uncertainty calculations, these may have affected the observed relationship between the independent and dependent variables, and thus, add a degree of uncertainty to the data and the conclusion. In order to eliminate these sources of error, steps could be taken in future experiments to increase the quality of the experiment. To account for both oxygen escape and acute pressure applied to the plastic top of the beaker, a bung could be acquired for the beaker. Holes for the tube and the syringe could be drilled with more permanence and accuracy, which would make dependent variable measurements more accurate. Agitation of the beaker could be accounted for by using an apparatus to affix the beaker to a surface for the duration of the reaction, so that molecular motion is the only things causing collisions of particles within the solution, and reaction rate isn’t affected by beaker movement.

Another significant limitation of this experiment was the failure to measure concentrations greater than 3% hydrogen peroxide to see if enzyme active site saturation would occur at higher concentrations. Since this limitation was due to availability of materials, This could be achieved by obtaining a higher concentration of hydrogen peroxide, and manipulating the independent variable over a wider range of values.