Gena GorinBlock II Biology 14 October ‘11

Lab #1: Catalase Reaction

Research

The goal of this experiment is to determine 1. the effect of pH changes on the activity of the hydrogen peroxide breakdown reaction as conducted by yeast catalase and 2. its relation to the human optimum reaction pH.

The procedure will involve conducting serial dilutions of strong acids and combining them with the hydrogen peroxide to be broken down by catalase produced by yeast. Given that enzymes normally have a highly specific range of optimum pH activity, a gradient of acidity values must prompt drastic changes in rate at some specific point.

The relevant reaction is: 2H2O2 2H2O + O2 ; catalase accelerates its process in most organisms.

Given that this thesis specifically compares the yeast and human catalase pH values, their range will be limited to all .5-pH increments between 5.5 and 9, as the optimum human range is between 6.8 and 7.2.

Variables

The independent variable is the pH of the dilute acid/base added to the hydrogen peroxide.

The dependent variable is the rate of the reaction as measured by O2 gas production.

The controlled variables include the calibration of the instruments, the amounts of active reactants and yeast, and environmental factors.

Materials

5 mL 1M HCl 5 mL .1 M NaOH Multitude ( ≈ 20) test tubes Disposable transfer pipettes Micropipettes with disposable tips

Distilled water Non-distilled water Excess of H2O2 and yeast Single-hole test tube stopper with pipette tip Containers for water, distilled and not Biohazard waste disposal container Graduated cylinder

Safety

The experiment uses strong (though made dilute in the process) bases and acids in uncovered containers; gloves and goggles are necessary. To avoid skewing results or releasing caustic materials, micropipette tips should be disposed of as biohazards and replaced if not functioning.

Procedure

Fill large container (box) with tap water; fill beaker with distilled water Prepare H2O2 and yeast for use Acquire 5 mL HCl and NaOH Prepare dilutions of acids and bases

o Use serial dilution techniques to neutralize the solutions

o Dilute 5 mL 1M ( = 0 pH) HCl to 5 pH by repeatedly combining one part acid

with nine parts distilled water (i.e. .5 mL HCl + 4.5 mL dH2O). In total, the process should be done five times cumulatively, yielding 5 mL of 5 pH solution

o Dilute 5 mL 1M ( = 13 pH) to 9 pH in the same fashion. In total, the process

should be done four times, yielding 5 mL of 9 pH solutiono Continue diluting each solution. To bring either .5 pH closer to 7, a 31.6%

solution must be made; that is, add 1.58 mL of solution to 3.42 mL dH2O. In total, the process should be done four times (cumulatively) to both the 9 pH and the 5 pH solutions

o Add 5 mL dH2O to the last test tube to test 7 pH

o Mark all test tubes and test tube racks with their acidity values

In a separate test tube, add 3 mL hydrogen peroxide Submerge graduated cylinder under water, remove all gas from the tube Add 3 mL of dilute acid/base to H2O2; add 1 mL yeast to begin reaction; close the tube

with a single-hole stopper Submerge tube under mouth of graduated cylinder and record amount of gas at 10-second

intervals; repeat with all solutions between 5.5 and 9 pH (i.e. 8 trials)

Data

Note well that any uncertainty in time is negligible: in most cases, the gradients are (overall) small enough to negate any difference between, for example, +.2 s and -.2 s. In all trials, the experiment was run for at least seventy seconds, providing a reasonable margin of accuracy.

Also, all gas displacement readings are accurate to ± .1 mL, making all ∆V values accurate to approximately ± .2 mL.

Gas displacement at 5.5 pH

Time (s) Volume (mL)

ΔV (mL)

0 3.810 3.9 0.120 4 0.130 4.3 0.340 4.6 0.350 4.9 0.360 5.1 0.270 5.2 0.180 5.3 0.190 5.3 0

Gas displacement at 6 pHTime (s) Volume

(mL)ΔV (mL)

0 0.510 0.5 020 0.7 0.230 0.8 0.140 0.9 0.150 1.2 0.360 1.4 0.270 1.6 0.280 1.8 0.290 1.95 0.15

0 10 20 30 40 50 60 70 80 90 1000

1

2

3

4

5

6

f(x) = 0.0193939393939394 x + 3.76727272727273

5.5 pH Hydrogen Peroxide Decomposition Gas Output v. Time

Time / s

Gas V

olum

e /

mL

0 10 20 30 40 50 60 70 80 90 1000

0.5

1

1.5

2

2.5

f(x) = 0.0174242424242424 x + 0.350909090909091

6 pH Hydrogen Peroxide Decomposi-tion Gas Output v. Time

Time / s

Gas V

olum

e /

mL

Gas displacement at 6.5 pHTime (s) Volume

(mL)ΔV (mL)

0 010 0 020 0.2 0.230 0.3 0.140 0.3 050 0.4 0.160 0.5 0.170 0.5 080 0.6 0.190 0.7 0.1

Gas displacement at 7 pHTime (s) Volume

(mL)ΔV (mL)

0 1.510 1.6 0.120 1.7 0.130 1.8 0.140 1.9 0.150 2.2 0.360 2.4 0.270 2.6 0.280 2.8 0.290 2.9 0.1

Gas displacement at 7.5 pHTime (s) Volume

(mL)ΔV (mL)

0 010 0.1 0.120 0.15 0.0530 0.3 0.1540 0.5 0.250 0.7 0.260 0.9 0.270 0.95 0.0580 1 0.05

0 10 20 30 40 50 60 70 80 90 1000

0.51

1.52

2.53

3.5

f(x) = 0.0167272727272727 x + 1.38727272727273

7 pH Hydrogen Peroxide Decomposition Gas Output v. Time

Time / s

Gas V

olum

e /

mL

0 10 20 30 40 50 60 70 80 90 1000

0.2

0.4

0.6

0.8

1

f(x) = 0.0076969696969697 x + 0.0036363636363636

6.5 pH Hydrogen Peroxide Decomposition Gas Output v. Time

Time / s

Gas V

olum

e /

mL

0 10 20 30 40 50 60 70 80 90 1000

0.20.40.60.8

11.21.4

f(x) = 0.014 x − 0.05

7.5 pH Hydrogen Peroxide Decomposi-tion Gas Output v. Time

Time / s

Gas V

olum

e /

mL

90 1.2 0.2Gas displacement at 8 pH

Time (s) Volume (mL)

ΔV (mL)

0 010 0.15 0.1520 0.2 0.0530 0.4 0.240 0.6 0.250 0.9 0.360 1.1 0.270 1.3 0.280 1.5 0.290 1.8 0.3

Gas displacement at 8.5 pHTime (s) Volume

(mL)ΔV (mL)

0 010 0.8 0.820 1.5 0.730 2.3 0.840 2.9 0.650 3.5 0.660 4.2 0.770 4.9 0.780 5.5 0.690 6 0.5

Gas Displacement at 9 pHTime (s) Volume

(mL)ΔV (mL)

0 010 0 020 2.4 2.530 3.7 1.340 4.7 150 5.4 0.760 6.4 170 7.4 180 7.9 0.590 8.5 0.6

0 10 20 30 40 50 60 70 80 90 1000

0.5

1

1.5

2

f(x) = 0.0203333333333333 x − 0.12

8 pH Hydrogen Peroxide Decomposi-tion Gas Output v. Time

Time / sGa

s Vol

ume

/ m

L

0 10 20 30 40 50 60 70 80 90 10001234567

f(x) = 0.0667878787878788 x + 0.154545454545455

8.5 pH Hydrogen Peroxide Decomposi-tion Gas Output v. Time

Time / s

Gas V

olum

e /

mL

0 10 20 30 40 50 60 70 80 90 1000123456789

f(x) = 0.100363636363636 x + 0.123636363636363

9 pH Hydrogen Peroxide Decomposi-tion Gas Output v. Time

Time / s

Gas V

olum

e /

mL

Total Gas Displacement (mL) v. Time and pHTime (s) 5.5 pH 6 pH 6.5 pH 7 pH 7.5 pH 8 pH 8.5 pH 9 pH

0 3.8 0.5 0 1.5 0 0 0 010 3.9 0.5 0 1.6 0.1 0.15 0.8 020 4 0.7 0.2 1.7 0.15 0.2 1.5 2.430 4.3 0.8 0.3 1.8 0.3 0.4 2.3 3.740 4.6 0.9 0.3 1.9 0.5 0.6 2.9 4.750 4.9 1.2 0.4 2.2 0.7 0.9 3.5 5.460 5.1 1.4 0.5 2.4 0.9 1.1 4.2 6.470 5.2 1.6 0.5 2.6 0.95 1.3 4.9 7.480 5.3 1.8 0.6 2.8 1 1.5 5.5 7.990 5.3 1.95 0.7 2.9 1.2 1.8 6 8.5

Note well that for rate calculations the volume uncertainties are dismissed as negligible due to convergence of results resulting from a large sample of time values. The next data set makes use of ∆V values; however, the final one uses an automatic linear regression based directly on gas output values.

Standard deviation is applicable to none of the data, as they are not expected to stay around the same value.

0 10 20 30 40 50 60 70 80 90 1000123456789

Hydrogen Peroxide Decomposition Gas Output v. Time

5.5 pH6 pH6.5 pH7 pH7.5 pH8 pH8.5 pH9 pH

Time / s

Gas V

olum

e /

mL

Gas Displacement Rates (mls-1 x 10-1) v. Time and pHTime (s) 5.5 pH 6 pH 6.5 pH 7 pH 7.5 pH 8 pH 8.5 pH 9 pH

10 0.1 0 0 0.1 0.1 0.15 0.8 020 0.1 0.2 0.2 0.1 0.05 0.05 0.7 2.530 0.3 0.1 0.1 0.1 0.15 0.2 0.8 1.340 0.3 0.1 0 0.1 0.2 0.2 0.6 150 0.3 0.3 0.1 0.3 0.2 0.3 0.6 0.760 0.2 0.2 0.1 0.2 0.2 0.2 0.7 170 0.1 0.2 0 0.2 0.05 0.2 0.7 180 0.1 0.2 0.1 0.2 0.05 0.2 0.6 0.590 0 0.15 0.1 0.1 0.2 0.3 0.5 0.6

Reaction rates v. pHpH Regression rate

5.5 0.01946 0.0174

6.5 0.00777 0.0167

7.5 0.0148 0.0203

8.5 0.06689 0.1004

Qualitative data includes release of gas bubbles of various sizes, becoming smaller with time.

0 10 20 30 40 50 60 70 80 90 1000

0.5

1

1.5

2

2.5

3

Hydrogen Peroxide Decomposition Gas Output Rate v. Time

5.5 pH6 pH6.5 pH7 pH7.5 pH8 pH8.5 pH9 pH

Time / sGas

Vol

ume

Per S

econ

d /

mLs

-1 x

.1

5 5.5 6 6.5 7 7.5 8 8.5 9 9.50

0.02

0.04

0.06

0.08

0.1

0.12

Hydrogen Peroxide Decomposition Gas Output Rate v. pH

pH of solution

Gas V

olum

e pe

r Sec

ond

/ m

s-1

Conclusion

Several trends may be seen in evaluating the final data set.

Firstly, there is a marked trend of very low reaction rates until the pH reaches the 8-9 interval, wherein it increases fivefold. This implies that any solution more acidic than 8 pH inhibits (though does now stop!) the catalase activity to approximately the same extent (constant rate), and starting at 8 pH it nears optimal environmental acidity.

Secondly, the ∆V values generally decrease with time. This is expected, as hydrogen peroxide is a limiting reactant and a decrease in quantity naturally leads to a decrease in its conversion into oxygen gas and water.

Thirdly, after the pH does reach 8, the trend seems to shift rapidly from a constant rate of inhibition to decline in such, accelerating the reaction considerably in a linear fashion (?). However, the absolute optimal temperature appears to be outside the range of this experiment in particular.

Fourthly, the constant rate of inhibited catalase is approximately 0.015917, with a standard deviation of 0.00459, judging from data in the range [5.5, 8].

Fifthly, regarding the thesis and comparison between the optimal environments of yeast and human catalase enzymes, it must be said that the yeast’s preferred reaction base concentration is at least two orders of magnitude (or 2 pH) higher than that seen in humans (to reiterate, approximately neutral).

Improvements

The most significant improvement that can be used in this lab is covering a greater range of pH values, e.g. all values from 0 to 14 at (at most) .5 increments. This would aid in finding an absolute optimal concentration, not necessarily relatively to the human one.

Also, greater precision is always sought, so conducting the experiments with less-error prone tools would considerably help determine the degree of non-negligibility of uncertainties.