bio formalz auto saved)

8/8/2019 Bio Formalz Auto Saved)

1/16

Effect of pH on Enzyme Activity

Logan Hansen, Biology A1

Submitted 11-22-10


2/16

Hansen

Table of Contents

EFFECT OF PH ON ENZYME ACTIVITY .............. ............... .............. .............. .............. .............. ...... ....1

LOGAN HANSEN, BIOLOGY A1 ............. .............. .............. .............. .............. ............... .............. ..... ...... ..1

TABLE OF CONTENTS ............. .............. ............... .............. .............. .............. .............. ............. ...... ...... ....2

INTRODUCTION ............. .............. .............. .............. ............... .............. .............. .............. ......... ..... ..... ..... ..3

METHODS ............. .............. .............. .............. .............. ............... .............. .............. .............. .............. ...... ....7

RESULTS .............. .............. .............. ............... .............. .............. .............. .............. .............. ........... ...... ...... ..9

2


3/16

Hansen

Introduction

The basic function of an enzyme is to increase the rate of a reaction. Most cellular

reactions occur about a million times faster than they would in the absence of an enzyme.

(Ophardt, 2003) Enzymes speed up this rate of reaction so much because they lower the

activation energy that is required for a reaction to occur, therefore allowing it to happen

much quicker than it would on its own. An excellent example of the usefulness of one

enzyme, catalase, is if we look at the decomposition of hydrogen peroxide with and

without catalase present. When hydrogen peroxide is simply left alone, depending on the

temperature and other factors, it decomposes very slowly. Due to specific properties of

hydrogen peroxide, if it entered the body and was not able to be decomposed quickly, it

would kill cells and cause damage to body systems. However with catalase inside human

body cells, the activation energy needed for the reaction is decreased, and the rate of

decomposition is increased greatly and thus the cells are able to decompose the H2O2 in a

relatively quick manner, surviving the exposure to the chemical.

Also the nature of catalase in its ability to break down hydrogen peroxide into

oxygen gas and water gives it several useful medical purposes. If a wound is deep and

has dirt and potentially harmful bacteria in it, we can pour a diluted solution of hydrogen

peroxide in it. Not only is it toxic to the bacteria that are unable to produce catalase, the

catalase in the cells of the human body start to decompose it creating bubbles of oxygen

gas which then push dirt and other contaminants to the surface so as to cleanse and

sterilize the wound.

3
http://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Oxygen


4/16

Hansen

One thing that has an effect on the activity of an enzyme is the salt concentration

of the solution that it is in. However this is a little more complex than just having a

change of shape in the enzyme. If the salt concentration is close to zero, the charged

amino acid side chains of the enzyme molecules will attract each other. ..The enzyme will

denature and form an inactive precipitate. If, on the other hand, the salt concentration is

very high, normal interaction of charged groups will be blocked. New interactions will

occur, and again the enzyme will precipitate. ..An intermediate salt concentration such as

that of human blood (0.9%) or cytoplasm is the optimum for many enzymes. (science-

projects.com, n.d.)

Another thing that would have an effect on the activity of an enzyme is substrate

and enzyme concentration. It makes sense that if the concentration of a substrate is

increased, the speed of the reaction is going to increase up to a point, because by

increasing the concentration of the substrate you are increasing the chances of the

substrates coming in to contact with the active site of the enzyme and then being able to

be put into a reaction. However, once that point is reached, the reaction wont go any

faster because there are only so many enzymes that can catalyze reactions.

However if the concentration of enzymes is increased, then the speed of the

reaction will increase drastically. This is because then there are more enzymes to catalyze

the reaction between specific substrates. However, all of the substrates will be used up

eventually and the reaction will not be able to continue.

We know that enzymes are adapted to operate at a specific pH or pH range.

(Farabee, 2010) And a change in pH that is too great will denature the enzyme by

changing the shape of the enzyme. (Farabee, 2010) Too much of a change in the shape of

4


5/16

Hansen

the enzyme will possibly leave the enzyme functionless, due much to the fact that

enzymes are dependent on their shape for the job that they perform. This scenario is

much the same with temperature, if it is changed too much from the original level, the

enzymes shape will change and it will no longer be able to react with the same

substrates.

This then leads to the reason that we are testing the change in activity of catalase

after a change in pH; to determine at what level of change is necessary to give such a

change in shape so as to render the catalase unable to decompose hydrogen peroxide.

Enzymes are very specific in the reactions that they catalyze. According to our

text book, the active site and the substrates have complementary shapes. The fit is so

precise that the active site and substrates are often compared to a lock and key. (Levine &

Miller, 2003) This theory is often called the Lock and Key Theory. However there is

another theory that is out there that also attempts to explain the reaction between enzyme

and substrate. The induced fit model suggested by Daniel Koshland in 1958 is the more

accepted model for enzyme-substrate complex than the lock-and-key model. (Biology-

online.org, 2008) Unlike the lock-and-key model, the induced fit model shows that

enzymes are rather flexible structures in which the active site continually reshapes by its

interactions with the substrate until the time the substrate is completely bound to it

(which is also the point at which the final form and shape of the enzyme is determined).

(Biology-online.org, 2008)

Generally when the substrates necessary for a reaction come into contact with the

active site of the enzyme the reaction is started, and when this reaction is finished, the

products are released from the enzyme and the whole process can be started again with

5
http://www.biology-online.org/dictionary/Enzymeshttp://www.biology-online.org/dictionary/Substratehttp://www.biology-online.org/dictionary/Enzymehttp://www.biology-online.org/dictionary/Enzymeshttp://www.biology-online.org/dictionary/Substratehttp://www.biology-online.org/dictionary/Enzyme


6/16

Hansen

new reactants. This active site is only compatible with specific substrates, this is

explained by the theories above, generally the substrates have to be a perfect or near

perfect fit with the active site, or the enzyme is unable to bond with these materials and

by default, unable to catalyze a reaction.

Enzymes are simply proteins, however they are very important to ensure that a

cell functions. When there is a specific reaction that needs to occur in a cell or in an

environment, the nucleus of the cell uses its DNA to code to tell specific parts of the cell

to start producing these proteins that function as enzymes. Like all other proteins,

enzymes are made up of amino acids. They start with a primary structure which is merely

a one-dimensional string of amino acids with a hydrocarbon on one end and a carboxyl

group on the other end. (Primary Structure, 2006) This then builds to the secondary

structure which is a three dimensional figure made up of multiple amino acid strings, and

which generally assumes the shape of a helix. (Secondary Structure, 2010) And then

into the tertiary structure, upon which the function of a protein (except as food)

depends and if this is disrupted, the protein is said to be denatured, and it loses its

activity. (Tertiary Structure, 2009)

When formulating my hypothesis, I looked at several different facts that would

suggest certain things about the functions and workings of enzymes. First of all, the

enzyme that we are testing is catalase which is found in many different cells throughout

the entire human body. In order to function properly in the human body, the catalase

enzymes would have to be able to function under the conditions that are present in the

body. (pH, temperature, etc.)

6


7/16

Hansen

The Purpose of this experiment was to determine the effect that an elevation of

pH would have on the activity of catalase in decomposing hydrogen peroxide. (H2O2)

And to determine the pH level at which the catalase works with maximum efficiency.

I believe that the catalase will perform at max efficiency at its original pH level in

the liver. That is I believe that this enzyme will work best without the addition of a base

or acid. In fact, to be even more specific, I believe that the enzyme will work best at a pH

of around 7 due to the fact that that is the pH level that is most commonly found in the

human body which is where the catalase is found, thus reasonably allowing us to

conclude that the catalase would work best at the pH level in which it is found.

Methods

The following procedure was adapted from The catalyzed rate of decomposition

of Hydrogen Peroxide (Carolina Biological Supply, 2006)

The first step is to establish the baseline for the hydrogen peroxide. To do this, we

label a syringe H2O2 and draw out 10 mL of hydrogen peroxide from the bottle and put it

into a plastic 60 mL cup labeled baseline. Next using another syringe, we took 1 mL of

distilled water and added it to the hydrogen peroxide solution in the cup. (The distilled

water is to replace the 1 mL of catalase solution that will be used in future trials) Next we

labeled another syringe H2SO4 and drew out 10 mL of sulfuric acid and added that to the

hydrogen peroxide mixture. We then gently swirled the cup so as to completely mix the

contents. Once the solution in the cup was completely mixed, we drew out 5 mL and put

it into another plastic cup labeled titration. We then took that plastic cup and put it under

a burette filled with potassium permanganate suspended on a ring stand. We then opened

7
http://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Oxygen


8/16

Hansen

the burette so that the KMnO4 would drip into the contents of our titration cup one drop at

a time. We continued to let the potassium permanganate drip into the cup, swishing the

contents the entire time, until the liquid turned and stayed pink. At which point we closed

the end of the burette so as to stop the flow and then measured the change in the level of

the KMnO4 in the burette.

Now that the baseline is established, our next step was to determine how active

the catalase sample is at its normal pH. To do this first we needed to have the catalase

solution diluted, so we added water until it was at a concentration of 20% catalase in

solution. We then took the pH probe and plugged it into the Labquest device and put the

probe into the catalase. We recorded the pH of the liver puree solution as the starting pH.

After taking this measurement we rinsed off the pH probe with distilled water and then

put it in a beaker of tap water to keep it wet. Next we used the H2O2 syringe to put 10 mL

of hydrogen peroxide into another cup. Now with a timer ready, we added 1 mL of the

catalase solution to the hydrogen peroxide and at the same time started the timer. We let

our catalase decompose the hydrogen peroxide for 60 seconds in this experiment. As we

approached the time limit, we drew out 10 mL of H2SO4 and when the time was up, added

the sulfuric acid to the H2O2 and catalase to stop the reaction. After mixing the contents

of the cup, we then proceeded to titrate the mixture of sulfuric acid, hydrogen peroxide

and catalase using the same procedure as above; so as to determine how effective the

catalase was at its normal pH level.

Now after both the baseline and neutral pH trials were completed, we proceeded

to put 10 mL of the catalase solution into several different beakers. It is in these beakers

that the sodium hydroxide was added to determine the effect of an elevation of pH on the

8


9/16

Hansen

effectiveness of catalase. To the first beaker we added 5 drops of sodium hydroxide,

using an eye dropper; and 10 to the next and so on until a pH of 12.5 was reached. After

we added and mixed in the sodium hydroxide into each of the different beakers of

catalase we repeated the steps taken in the trials before substituting in the appropriate

catalase sample for each.

In this experiment there is both an independent and a dependent variable. The

independent variable in this case was the pH of the solution. We chose the number of

drops that were added to the catalase so therefore we controlled this variable thus making

it the independent variable. The dependent variable was the amount of hydrogen peroxide

left after the addition of catalase which translates to the amount of potassium

permanganate used to titrate the solution until it was a pink color. The KMnO4 was used

to measure the hydrogen peroxide content of the solution because when it was added to

the mixture containing the catalase and H2O2, it reacted with the hydrogen peroxide, so

after all of the H2O2 is used up, there is nothing to react with the KMnO4 thus turning the

solution purple. This is dependent because it depends on how active the catalase was

which in turn depends on what the pH of the catalase solution was.

In this experiment, all of the things that we used were reagents. The catalase,

sodium hydroxide, sulfuric acid, hydrogen peroxide and potassium permanganate were

all reagents that we used to get our data.

Results

These results model what is happening when the catalase is added to the hydrogen

peroxide and begins to catalyze its decomposition. If we look at table three, the averages,

we see that as the pH increases higher than the start value, the amount of KMnO4 needed

9


10/16

Hansen

to titrate the solution increases. This suggests that there is more of the hydrogen peroxide

left over from the reaction and therefore that the catalase was less active in decomposing

the H2O2.

10

Catalase Decomposing Hydrogen Peroxide, Trial Two, Table Two

Level of Potassium Permanganate

MaterialBefore

Titration (mL)After Titration

(mL)Change in Level of Potassium

Permanganate

Baseline Trial 12.6 16.4 3.8

Catalase (pH 6.7) 19.3 20.1 0.8

Catalase (pH 8.1) 20.1 21.2 1.1

Catalase (pH 9.1) 21.2 22 0.8

Catalase (pH 11.0) 22.8 25.6 2.8

Catalase (pH 12.5) 27.6 31.3 3.7

Catalase Decomposing Hydrogen Peroxide, Trial One, Table One

Level of Potassium Permanganate

MaterialBefore

Titration (mL)After Titration

(mL)Change in Level of Potassium

Permanganate

Catalase (pH 6.7) 22 22.8 0.8

Catalase (pH 8.1) 25.6 26.6 1

Catalase (pH 9.1) 26.6 27.6 1

Catalase (pH 11.0) 31.3 34.2 2.9

Catalase (pH 12.5) 34.2 38 3.8

Table Three Average Change in level of PotassiumPermanganate (Final Level Initial Level)

Catalase (pH 6.7) 0.8





Potassium Permanganate Used vs. pH Level

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10 12 14

pH Level of Catalase

MnO4UsedSubtract

edfromBaseline


11/16

Hansen

Looking at the graph, there is obviously a negative trend going on after the pH of

the catalase passes a value of around 9.1. However in the range of pH from around 6.7 to

9.1 we see little or no change in the activity of the catalase. This suggests that the catalase

was able to adapt to the change and did not have its shape changed so much as to render

it unable to catalyze the decomposition of the hydrogen peroxide. And when the pH value

reaches around 12.5 we see that the activity of the catalase is just above zero. We know

this because the baseline would be when the y-value of a point is zero, and at a pH of

12.5 the y-value is 0.05 which suggests that barely any of the hydrogen peroxide was

decomposed.

Discussion/Conclusion

When we first started this lab, we wanted to find out what would happen to the

level of activity of catalase in decomposing hydrogen peroxide if we were to change the

pH of the liquid that it was in. Through the experiment that we devised we explored that

topic and came out with some data that shows trends that can explain what happened to

the enzyme very well. My original hypothesis was that the catalase would perform at

maximum efficiency when it was at or near its original pH level. If we are to look at

figure 1, we see that the catalase is able to keep its regular amount of activity even when

the pH is increased up to around 9.1, we see justification of this when we look at table

three; we see that for these pH values between 6.7 and 9.1 all required around 1 mL of

potassium permanganate for titration. When the pH is elevated to any level higher than

9.1 we see that the activity of the enzyme in decomposing the hydrogen peroxide drops

off very steeply and the activity is just above zero at a pH of 12.5; and when the pH

11


12/16

Hansen

increases the amount of potassium permanganate used to turn the solution pink is

drastically increased. And when these amounts of KMnO4 are subtracted from the

baseline we see that they get closer and closer to the established baseline value, which

can be interpreted as the amount of enzyme activity. This drop off can be explained as

being the point at which the shape of the enzyme was changed to such and extreme

amount that it was no longer able to catalyze the decomposition of hydrogen peroxide.

However all of the molecules of catalases shapes were not changed when the acid was

added, thus even when the pH was far above its normal level some of the hydrogen

peroxide was still able to be decomposed.

There were many variables in this experiment and almost all of those were. We

controlled the pH of the catalase by adding Sodium Hydroxide to the catalase samples

before they were tested, thus resulting in different pH samples which gave us different

results. Another variable which we controlled was the concentration of the catalase. We

controlled this by diluting the catalase samples that we were given with distilled water to

the concentration that we desired. If the concentration of the catalase was greater than

there would be more enzymes floating about in the solution thus making the chances of

contact between the substrates and the enzymes greater and thereby resulting in a greater

amount of the hydrogen peroxide being decomposed. Yet another variable which was

controlled in this experiment was the amount of time that the catalase was allowed to

react with the hydrogen peroxide. We controlled this by using a stopwatch and sulfuric

acid to stop the reaction. At the same time, the reaction and the stopwatch would be

started and then when the chosen amount of time had passed the sulfuric acid was added

to the sample to stop the reaction from happening. We chose 60 seconds but if the

12


13/16

Hansen

allotted time had been longer than more of the hydrogen peroxide wouldve been

decomposed and if the time interval as shortened then of course less of the hydrogen

peroxide wouldve been decomposed.

Before the experiment we thought that the catalase would work best at its original

pH because that was the pH which it had to adapt to working under in the body. Through

the experiment we found that our hypothesis was partially correct. We did indeed find

that the level of activity was near its highest at the original pH; however we also found

that within a range of about 2 on the pH scale the catalase would stay within the range of

its original level of activity. This was not a result which we expected because we thought

that the enzyme would be much more specific in the conditions that it could work under

than we found. So we were not expecting to see such a wide range at which the catalase

stayed at it top level of activity. So as a final concluding note I would say that the data

which we collected in the experiment supports our original hypothesis, but also provides

new evidence to suggest a wider range than we originally thought.

Despite the fact that we had data that showed a trend that we expected, there were

without doubt errors in our experiment and methods. These errors could throw off the

data and perhaps suggest a trend when graphed that wasnt truly what happened, or what

should happen. Within the class we saw some examples of this, one of the common

mistakes was that the baseline trial was done incorrectly, and then when the data was

graphed, the line descended into the negatives producing a response that could not be true

for the experiment. Another possible error is with the titration; too much or too little of

the potassium permanganate was used to titrate the solution resulting in inaccurate values

for the change in level of the KMnO4. One of the biggest things that could be done to

13


14/16

Hansen

improve the accuracy of this experiment would be simply to do more trials. Also perhaps

adding the potassium permanganate to the solution to titrate it slower would result in

greater accuracy as well because there wouldnt be to much or too little added to the

solution. Also, to get better data, more catalase could have been tested, because each

sample of catalase that was taken was unique, so if more and more samples were tested,

perhaps the averages for the data would come out to be more accurate.

We especially saw the possibility for error in our experiment on the first day that

we worked on it. We followed much the same procedure on day one with testing the

effect of pH on our catalase activity, however the data that we got was very inaccurate

compared to the data that we collected on day two. If this day one data had been averaged

with the data that was collected on day two then it wouldve acted as an outlier and

skewed all of the data that we were analyzing. For the sake of our data and analysis of

our data, we decided to omit the gathered data from day one in our graphs and tables. I

suspect that many of these flaws in our day one data originated with over titration as

mentioned previously and unfamiliarity with the equipment and the proper way to run the

experiment.

Seeing the data and how it came out left me with some other questions. We saw

from our results that the catalase had a fairly wide range of pH levels that it could

perform at its best at and another question I had was if the same trend would hold for pH

levels below that of normal catalase. Would the activity be high until about a pH of 4

before it dropped to relatively little activity? Also there are different variables that would

be interesting to try such as salt concentration and temperature. Would these provide data

similar to the lab we just did? With the highest activity being in a range around which the

14


15/16

Hansen

catalase is normally at, these would all provide interesting areas of study relating to

enzymes. They would also not require very large changes to the existing procedure for

this lab. Simply replace the addition of acid with the addition of salt or the change in

temperature.

15


16/16

Hansen

Literature Cited

2010. Induced fit model. Retrieved fromhttp://www.biology-online.org/dictionary/Induced_fit_model

2008. Effect of salts and inorganic ions on enzyme activity. Retrieved fromhttp://academic.brooklyn.cuny.edu/biology/bio4fv/page/salt_enz.htm

2006. The catalyzed rate of decomposition of hydrogen peroxide. Burlington, NorthCarolina. Carolina Biological Company

Primary structure. (2006, March 26). Retrieved from

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/PrimaryStructure.html

Secondary structure. (2010, May 23). Retrieved from

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SecondaryStructure.html

Tertiary structure. (2009, December 18). Retrieved from

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TertiaryStructure.html

Catalase kinetics. (n.d.). Retrieved from http://www.science-

projects.com/catalasekinetics.htm

Farabee, M.J. 2010. REACTIONS AND ENZYMES. Retrieved from

http://www.emc.maricopa.edu/faculty/farabee/biobk/biobookenzym.html

Miller, Kenneth R. & Levine, Joeseph S. 2003.Biology. Saddle River, New Jersey.

Prentice Hall Publishing. (p. 53)

Ophardt, Charles E. 2003. Role of Enzymes in Biochemical Reactions. Retrieved from

http://www.elmhurst.edu/~chm/vchembook/570enzymes.html

16
http://www.biology-online.org/dictionary/Induced_fit_modelhttp://academic.brooklyn.cuny.edu/biology/bio4fv/page/salt_enz.htmhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SecondaryStructure.htmlhttp://www.emc.maricopa.edu/faculty/farabee/biobk/biobookenzym.htmlhttp://www.biology-online.org/dictionary/Induced_fit_modelhttp://academic.brooklyn.cuny.edu/biology/bio4fv/page/salt_enz.htmhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SecondaryStructure.htmlhttp://www.emc.maricopa.edu/faculty/farabee/biobk/biobookenzym.html

bio formalz auto saved)

Documents