Gena Gorin Block 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 solution o 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) 0 10 20 30 40 50 60 70 80 90 Volume (mL) 3.8 3.9 4 4.3 4.6 4.9 5.1 5.2 5.3 5.3 V (mL)
6

5.5 pH Hydrogen Peroxide Decomposition Gas Output v. Time

Gas Volume / mL 5 4 3 2 1 0 0 20 40 Time / s 60 80 100

0.1 0.1 0.3 0.3 0.3 0.2 0.1 0.1 0

y = 0.0194x + 3.7673

Gas displacement at 6 pH Time (s) Volume V (mL) (mL) 0 0.5 10 0.5 0 20 0.7 0.2 30 0.8 0.1 40 0.9 0.1 50 1.2 0.3 60 1.4 0.2 70 1.6 0.2 80 1.8 0.2 90 1.95 0.15

6 pH Hydrogen Peroxide Decomposition Gas Output v. Time

2.5 Gas Volume / mL 2 1.5 1 0.5 0 0 20 40 Time / s y = 0.0174x + 0.3509 60 80 100

Gas displacement at 6.5 pH Time (s) Volume V (mL) (mL) 0 0 10 0 0 20 0.2 0.2 30 0.3 0.1 40 0.3 0 50 0.4 0.1 60 0.5 0.1 70 0.5 0 80 0.6 0.1 90 0.7 0.1

6.5 pH Hydrogen Peroxide Decomposition Gas Output v. Time

1 Gas Volume / mL 0.8 0.6 0.4 0.2 0 0 20 40 Time / s y = 0.0077x + 0.0036 60 80 100

Gas displacement at 7 pH Time (s) Volume V (mL) (mL) 0 1.5 10 1.6 0.1 20 1.7 0.1 30 1.8 0.1 40 1.9 0.1 50 2.2 0.3 60 2.4 0.2 70 2.6 0.2 80 2.8 0.2 90 2.9 0.1

7 pH Hydrogen Peroxide Decomposition Gas Output v. Time

3.5 3 2.5 2 1.5 1 0.5 0 0 20 40 Time / s 60 80 100 y = 0.0167x + 1.3873

Gas displacement at 7.5 pH Time (s) Volume V (mL) (mL) 0 0 10 0.1 0.1 20 0.15 0.05 30 0.3 0.15 40 0.5 0.2 50 0.7 0.2 60 0.9 0.2 70 0.95 0.05 80 1 0.05 90 1.2 0.2

Gas Volume / mL Gas Volume / mL

7.5 pH Hydrogen Peroxide Decomposition Gas Output v. Time

1.5 1 0.5 0 0 20 40 Time / s y = 0.014x - 0.05 60 80 100

Gas displacement at 8 pH Time (s) Volume V (mL) (mL) 0 0 10 0.15 0.15 20 0.2 0.05 30 0.4 0.2 40 0.6 0.2 50 0.9 0.3 60 1.1 0.2 70 1.3 0.2 80 1.5 0.2 90 1.8 0.3

8 pH Hydrogen Peroxide Decomposition Gas Output v. Time

2 Gas Volume / mL 1.5 1 0.5 0 0 20 40 Time / s 60 80 100

y = 0.0203x - 0.12

Gas displacement at 8.5 pH Time (s) Volume V (mL) (mL) 0 0 10 0.8 0.8 20 1.5 0.7 30 2.3 0.8 40 2.9 0.6 50 3.5 0.6 60 4.2 0.7 70 4.9 0.7 80 5.5 0.6 90 6 0.5

8.5 pH Hydrogen Peroxide Decomposition Gas Output v. Time

7 6 5 4 3 2 1 0 0 20 40 Time / s 60 80 100 Gas Volume / mL

y = 0.0668x + 0.1545

Gas Displacement at 9 pH Time (s) Volume V (mL) (mL) 0 0 10 0 0 20 2.4 2.5 30 3.7 1.3 40 4.7 1 50 5.4 0.7 60 6.4 1 70 7.4 1 80 7.9 0.5 90 8.5 0.6

9 pH Hydrogen Peroxide Decomposition Gas Output v. Time

10 Gas Volume / mL 8 6 4 2 0 0 20 40 Time / s 60 80 100

y = 0.1004x + 0.1236

Time (s) 0 10 20 30 40 50 60 70 80 90

5.5 pH 3.8 3.9 4 4.3 4.6 4.9 5.1 5.2 5.3 5.3

Total Gas Displacement (mL) v. Time and pH 6 pH 6.5 pH 7 pH 7.5 pH 8 pH 8.5 pH 9 pH 0.5 0 1.5 0 0 0 0 0.5 0 1.6 0.1 0.15 0.8 0 0.7 0.2 1.7 0.15 0.2 1.5 2.4 0.8 0.3 1.8 0.3 0.4 2.3 3.7 0.9 0.3 1.9 0.5 0.6 2.9 4.7 1.2 0.4 2.2 0.7 0.9 3.5 5.4 1.4 0.5 2.4 0.9 1.1 4.2 6.4 1.6 0.5 2.6 0.95 1.3 4.9 7.4 1.8 0.6 2.8 1 1.5 5.5 7.9 1.95 0.7 2.9 1.2 1.8 6 8.5

Hydrogen Peroxide Decomposition Gas Output v. Time

10 Gas Volume / mL 8 6 4 2 0 0 20 40 Time / s 60 80 100 5.5 pH 6 pH 6.5 pH 7 pH 7.5 pH 8 pH 8.5 pH 9 pH

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.

Time (s) 10 20 30 40 50 60 70 80 90

5.5 pH 0.1 0.1 0.3 0.3 0.3 0.2 0.1 0.1 0

Gas Displacement Rates (mls-1 x 10-1) v. Time and pH 6 pH 6.5 pH 7 pH 7.5 pH 8 pH 8.5 pH 9 pH 0 0 0.1 0.1 0.15 0.8 0 0.2 0.2 0.1 0.05 0.05 0.7 2.5 0.1 0.1 0.1 0.15 0.2 0.8 1.3 0.1 0 0.1 0.2 0.2 0.6 1 0.3 0.1 0.3 0.2 0.3 0.6 0.7 0.2 0.1 0.2 0.2 0.2 0.7 1 0.2 0 0.2 0.05 0.2 0.7 1 0.2 0.1 0.2 0.05 0.2 0.6 0.5 0.15 0.1 0.1 0.2 0.3 0.5 0.6

Hydrogen Peroxide Decomposition Gas Output Rate v. Time

Gas Volume Per Second / mLs-1 x .1

3 2.5 2 1.5 1 0.5 0 0 20 40 60 80 100 Time / s 5.5 pH 6 pH 6.5 pH 7 pH 7.5 pH 8 pH 8.5 pH

Gas Volume per Second / ms-1

Reaction rates v. pH pH Regression rate 5.5 0.0194 6 0.0174 6.5 0.0077 7 0.0167 7.5 0.014 8 0.0203 8.5 0.0668 9 0.1004

Hydrogen Peroxide Decomposition Gas Output Rate v. pH

0.12 0.1 0.08 0.06 0.04 0.02 0 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 pH of solution

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

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 yeasts 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.