1.4 effect of ph on enzyme activity

INTERNATIONAL EDUCATION CENTRE INTECKAMPUS SEKSYEN 17, 40200 SHAH ALAM, SELANGOR DARUL EHSAN.

TEL: 603-55227000

A REPORT ON THE EFFECT OF TEMPERATURE ON MEMBRANES

PREPARED FOR BIOLOGY PRACTICAL 2.1

BY GAAJEEN A/L PARMAL @ PERUMAL

(NRIC: 920621-10-5801)

(SID NO: 2010646046)

IN GROUP 11M7

UNDER MADAM ILYANIE HJ. YAACOB

SUBMITTED ON 04 OCTOBER 2010

1 The Effect of Temperature on Membranes

TEAM MEMBERS

FROM LEFT:

MUHAMMAD HAQIMI BIN ZULKEFLI (920614 – 04 – 5273)

GAAJEEN A/L PARMAL @ PERUMAL (920512 – 04 – 5192)

THERESA NG LEK WEI (920512 – 04 – 5192)


TitleThe effect of pH value on enzyme activity

ObjectiveTo investigate how different pH value can affect rate of enzyme activity

Problem StatementWhat is the effect of different PH value on rate of enzyme activity?

HypothesisThe rate of enzyme reaction is highest when the pH value is at optimum level. The rate of enzyme activity is fastest at pH 6.5, the optimum pH value for potato catalase.

Introduction

Diagram 1: Picture of raw beetroot1 Table 1: Scientific Classification of Beetroot2

Beetroot are biennial plants grown as annuals and harvested for their swollen root tuber and leaves. It tastes like earthy caramel and its freshness is similar to earthy mint. The usually deep-red roots of beetroot (refer to Diagram 1) are eaten boiled either as a cooked vegetable, or cold as a salad after cooking and adding oil and vinegar, or raw and shredded, either alone or combined with any salad vegetable. A large proportion of the commercial production is processed into boiled and sterilised beets or into pickles.

1 Image source: www.greatgrubclub.com/?location_id=702 Information source: http://en.wikipedia.org/wiki/Beet


BeetrootKingdom: PlantaePhylum: MagnoliophytaClass: MagnoliopsidaOrder: CaryophyllalesFamily: AmaranthaceaeGenus: BetaSpecies: B. vulgaris

http://en.wikipedia.org/wiki/Beet

http://www.greatgrubclub.com/?location_id=70

http://en.wikipedia.org/wiki/Beta_(plant)

http://en.wikipedia.org/wiki/Amaranthaceae

http://en.wikipedia.org/wiki/Caryophyllales

http://en.wikipedia.org/wiki/Magnoliopsida

http://en.wikipedia.org/wiki/Flowering_plant

Diagram 2: Chemical structure of beetroot3

The bright red colour in beet roots is caused by betalains, or specifically betacyanin, a water soluble plant pigment replacing anthocyanins in plants of the order Caryophyllales. Betalains contain nitrogen while anthocyanins do not (refer to Diagram 2). Betalains are found in the vacuole. The red-violet vesicles obtained from protoplasts are intact beet vacuoles, so betalain pigments have to cross two membranes. In the living tissues of beet slices, there can be a leakage by a heat shock, by acid treatment or by incubating them in acidified 80% methanol, due to damage of the membrane structure and denatured plasma proteins. Besides, putting beet slices in the deep-freeze will kill them, and the pigments will leak out. On the other hand, a pH of about 3-4 stabilises the pigments and protects them against oxidation. Pigment extracts must also be protected from direct sun light and should be kept in a cool and dark place.

Diagram 3: Betalains present in flowers4

The function of betalains is not provably known, but it is assumed that when present in the flowers (refer to Diagram 3), they serve to attract pollinating insects and when present in seeds they may attract birds to disperse the seeds. Man favours betalains because they are attractive to look at, and the colour may have co-segregated with another useful trait which improves the flavour. This explains why betanin, a red glycoside food dye obtained from beets, is commercially used. There is no indication that betalains protect plants against herbivores and pathogens such as fungal, bacterial or viral, and they do not absorb UV light.

3 Image source: www.absoluteastronomy.com/topics/Betalain4 Image source: www.webexhibits.org/causesofcolor/7H.html


http://www.webexhibits.org/causesofcolor/7H.html

http://www.absoluteastronomy.com/topics/Betalain

As for the goodness for human, betalains are often claimed to have antioxidant properties and are believed to be good for health. Beetroots are good sources of folic acid, potassium and dietary fibre. They also contain sugars that contribute to its calorie content. Beetroot also has stimulating effects on the liver's detoxification processes. The betacyanin content gives beetroot its rich purple-crimson colour and is a potent cancer-fighting agent. Beetroot's fibre promotes both healthy cholesterol levels and bowel function.5

Diagram 4: Structure of plasma membrane6

When beetroot is heated or cooked, there will be lots of red dye in the cooking water. This is because leakage of betalains has occurred as a result of the disruption of the cell membranes. A biological membrane is made of a phospholipid bilayer. A phospholipid bilayer is formed because the phospholipids that line up side by side have a polar "water-loving" (hydrophilic) head and a non-polar “water-hating” (hydrophobic) hydrocarbon tail. The tails pack together and face inwards, exposing only the polar heads to the water. It resembles two blankets one atop the other, with the fatty acid tails towards each other.

In a cell, the phospholipids form sacks. The biggest sack goes all around the whole cell to form the plasma membrane; others may form the tonoplast of vacuoles. In these lipid seas, there will be a number of proteins in various degrees of submersion (refer to Diagram 4). Some span all the bilayer, thus being exposed on both sides, is known as integral, trans-membrane or intrinsic proteins. Others just drift on either of its surfaces, which are called peripheral or extrinsic proteins. Typically, 70% of a cell membrane is protein, mostly for transport. The water in the cytoplasm around and within the compartments formed by the phospholipid bilayers is also crammed with protein.

VariablesManipulated variable (Independent variable): Different pH values

5 Information source: http://blogs.abc.net.au/nsw/2009/09/kitchen-gardener-episode-7-beetroot.html?program=702_drive6 Image source: www.tutorvista.com/.../plasma-membrane.php


http://www.tutorvista.com/content/biology/biology-iii/cell-organization/plasma-membrane.php

http://blogs.abc.net.au/nsw/2009/09/kitchen-gardener-episode-7-beetroot.html?program=702_drive

Responding variable (Dependent variable): Rate of enzyme reaction

Fixed variables: the time for the action of enzyme potato catalase, the volume of buffer solution usedthe volume of hydrogen peroxide used.

ApparatusStopwatch, calibrated tube, test tube, boiling tube, boiling tube rack, 250cm3

beakers ,delivery tube, cork, syringe, spatula, measuring cylinders.

Diagram 5: cuvettes7

MaterialsCitric acid and sodium phosphate buffer solutions, hydrogen peroxide solution 6% w/v, Blended potato, water baths.

7 Image source: www.labdepotinc.com/c-489-spectrophotometer.php


http://www.labdepotinc.com/c-489-spectrophotometer.php

ProceduresPreparation of the materials and apparatus

1. 1 spatula of blended potato is added into a boiling tube.2. 5cm3 of buffer solution of pH 4.4 is transferred into the boiling tube.3. It is swirled to mix.4. Graduated tube is filled with water and is inverted carefully into the beaker.5. 2.5 cm3 of hydrogen peroxide is measured into the syringe and added to the boiling

tube.6. The flask is connected.7. The graduated tube is immediately placed into the end of the delivery tube.8. Stopwatch is started. The volume of oxygen collected is measured every 30 second

for 5 minutes.9. Steps 1 to 8 are repeated using different buffer solution of pH 5.2, 6.5, and 7.5.10. The experiment is repeated twice to obtain the average value.11. All the results obtained are tabulated.12. A graph of rate of catalase activity against pH value is plotted.

Results

Time(second) 30 60

pH value

Trial

Volume of oxygen released (cm3)


1 2 3 Average 1 2 3 Average

4.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

5.2 1.0 1.0 0.0 0.67 1.0 1.0 1.0 1.0

6.5 16.0 5.0 7.8 9.6 23.6 7.6 12.6 14.6

7.5 10.0 10.6 12.0 10.9 16.0 17.8 20.0 18.0

Table 1: Volume of oxygen collected in respective to different pH values


Time(second) 90 120

pH value

Trial




4.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

5.2 2.0 1.8 2.0 1.9 2.6 2.0 2.0 2.2

6.5 27.0 11.4 16.0 18.1 28.0 13.0 19.6 20.2

7.5 21.0 23.0 25.0 23.0 25.0 26.0 29.0 26.7


Time(second) 150 180

pH value

Trial




4.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

5.2 2.7 2.2 2.0 2.3 2.8 2.2 2.4 2.5

6.5 28.4 15.4 21.4 21.7 28.4 16.0 23.0 22.5

7.5 27.0 28.8 31.6 29.1 29.8 30.8 33.4 31.3




pH value

Trial




4.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

5.2 2.8 2.2 2.6 2.5 2.8 2.4 2.8 2.7

6.5 28.4 16.0 23.8 22.7 28.6 16.0 25.0 23.2

7.5 31.4 32.6 34.6 32.9 32.4 32.9 35.6 33.6



pH value

Trial




4.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

5.2 2.8 2.6 2.8 2.7 2.8 2.8 2.8 2.8

6.5 28.6 16.0 26.0 23.5 28.6 16.0 26.0 23.5

7.5 33.4 33.6 36.0 34.3 34.0 35.4 36.4 35.3



4 4.5 5 5.5 6 6.5 7 7.5 80

5

10

15

20

25

30

35

40

pH value of buffer solution

Volu

me

of o

xyge

n re

leas

ed (c

m3)

Graph 1: Graph of volume of oxygen released against pH value of buffer solution


4 4.5 5 5.5 6 6.5 7 7.5 80

0.02

0.04

0.06

0.08

0.1

0.12

0.14

pH value of buffer solution

Rate

of c

atal

ase

reac

tion

(cm

3 S-

1)

Graph 2: Graph of rate of catalase reaction against pH value of buffer solution


DiscussionAnalysis and Interpretation of Results

In this experiment to examine the effect of temperature on cell membranes, the surrounding temperature of the cell membrane of beetroot is modified to see the effect it has on the membrane structure. Graph 1 above which data is plotted using the average value of colorimeter reading in Table 2 shows the general trend of data with increasing temperature. From the graph, we are able to see that the colorimeter reading decreases from 0°C to 27°C and increases from 27°C to 65°C. To understand this, we ought to know the functioning of the colorimeter in this experiment. The colorimeter is used to measure the percentage absorbance of light by the solutions where the beetroot cylinders are immersed into after the heat treatment. The higher the colorimeter reading brings about a higher percentage absorbance and so the higher is the amount of red dye present in the solution because there is more red dye in the solution to absorb the light transmitted from the colorimeter. The increase in amount of red dye in the solution is related to the increase of the permeability of beetroot membrane. Hence, an increase in the reading of colorimeter indicates that the permeability of the beetroot membrane increases.

The colorimeter shows a reading of 0.207 arbitrary units when the beetroot cylinder is immersed into water bath at 0°C. It is accurately drawn in the graph that when the beetroot cylinder is immersed into distilled water at room temperature, that is 27°C, the colorimeter shows a reading of 0.144 arbitrary units. This colorimeter reading is lower compared to reading taken at 0°C. Presumably we understand that the leakage of betalains located within the cell vacuole is due to the damaged membrane structure and denatured plasma proteins at high temperatures. But why is there any pigment released from what should be intact cell membranes at room temperature? This may be due to when washing the beetroot, all we are washing off is the pigment which has leaked out of the damaged cells at that time. The pigment does not necessarily all leak out at once, however, and the leakage is probably continuing when we start the experiment.

While for higher temperatures, from the distilled water which the beetroot cylinder stands in after it is immersed in water bath at 35°C, the colorimeter reading is 0.197 arbitrary units. Similarly when the temperature of water bath is at 45°C, the colorimeter reads a value of 0.442 arbitrary units. The colorimeter shows a reading of 0.969 arbitrary units for the condition where the beetroot cylinder which is being immersed in water bath at 55°C. Lastly from the distilled water which the beetroot cylinder stands in after it has been heat treated at 65°C, we obtain a colorimeter reading of 1.142 arbitrary units.


Diagram 6: Effect of temperature on the packing of the hydrocarbons8

To function correctly a cell needs to be able to control transport across the partially permeable cell surface membrane, so normally the betalains in the cell vacuole cannot pass through it. In addition, in order for the betalain to leave the cell it needs to pass through two different membranes, which are the membrane bounding the vacuole and the membrane enclosing the cell. At normal room temperature, the phospholipid bilayer is in a gel state and tightly packed (refer to Diagram 6). At higher temperatures, the bilayer actually ‘melts’ and the interior is fluid allowing the lipid molecules to move around, rotate, exchange places. As more phospholipids move around, more betalains are able to leak out. The cellulose cell wall on the outside of the cell is fully permeable and so no barrier to the egress of the pigment. Consequently at higher temperature, the red dye in the distilled water where the beetroot cylinder is placed is more concentrated.

Not only that, when we increase the temperature of the water baths, the transport proteins embedded on the plasma membrane is denatured. Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions of the polypeptide chain. This occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted. This damages the vacuole and makes holes in the cell membrane, inducing leakage of cytoplasm and other substances contained within the membrane, which is proven by the presence of red dye in the distilled water.

From the temperature of 27°C to 35°C, the colorimeter reading increases gradually and steadily, meaning that the percentage absorbance rises slowly. This shows there is only a little increase in the amount of betalains present in the distilled water which the beetroot cylinder stands in after it is immersed in water bath. The rise may not seem to be significant because 35°C is not high enough to denature the plasma proteins. The increase in heat

8 Image source: Wolfe S.L., Molecular and Cellular Biology, (1993). Wadsworth Publishing Company.


energy only increases the kinetic energy of the phospholipid molecules to move around, rotate and exchange places which at the same time increases the rate of random movement of betalains out through the cell membrane. Still, more leakage of betalains has occurred at 35°C as compared to that at 27°C. This indicates that the permeability of cell membrane has increased even by an increase in room temperature.

From the temperature of 35°C to 45°C, there is a rapid rise in the colorimeter reading, showing that the percentage absorbance increases in a sudden manner due to a big increase in the amount of betalains present in the distilled water which the beetroot cylinder stands in after it is immersed in water bath. As most proteins denature by which its tertiary structure unravels as a result of the destroy of the strong covalent bond between the R groups of amino acids in the polypeptide chain at a temperature over 40°C, there is less control of transport of substances across the partially permeable cell surface membrane. Hence much more betalains is able to pass out of the membrane, causing the permeability of cell membrane increases so much as shown by the gradient of the line from the temperature of 35°C to 45°C on the graph.

From the temperature of 45°C to 55°C, the gradient of the line increases again. This shows the permeability of the cell membrane is even higher due to more and more plasma proteins have been denatured, in addition to the ‘melting’ of the bilayer because of the violent movement of the phospholipid molecules.

As the temperature goes on to 65°C, the curve starts to flatten out, meaning that the rate of leakage of betalains has decreased. This is probably due to most of the plasma proteins have been denatured by the heat, and the leakage is already at its maximum rate. In other words, the amount of the plasma proteins has become the limiting factor for this experiment. However, there is also another suggestion that says that this happens because although the denaturing of the plasma proteins causes a rapid rise in the amount of betalains released, when the temperature begin to get higher still, the tertiary structure of the protein blocks parts of the holes in the cell membrane9. Therefore, this slows down the leakage of betalains. On the other hand, it could also be the effect of a very high temperature which causes the red dye from the beetroot to change colour, and so influences the colorimeter reading. The red pigment is heat labile, which means it is denatured by heat and would turn into a yellow chemical. Since the colorimeter is set to measure absorbance at one wavelength, a change in colour will result in a lower absorbance at that wavelength and so apparently the amount of betalains decreases.

9 Information source: http://www.123helpme.com/view.asp?id=123054


http://www.123helpme.com/view.asp?id=123054

EvaluationLimitations and Improvements

Laboratory coats were worn during the investigation to prevent chemicals from spoiling clothes. Care was also taken whilst handling the chemicals as hydrogen peroxide is corrosive and the manometer fluid is permanently staining.Assumingly that we have done the experiment under full attention, there are still some techniques themselves which are faulty. One of the limitations of the experiment is that the red dye of betalains may not be uniformly dispersed throughout the distilled water even though the solution has been shaken. In other words, the solution being measured using the colorimeter may not be a homogenous solution. Thus, the reading of the colorimeter obtained may not accurately show the actual percentage absorbance of the red dye in the solution. This might explain why the colorimeter reading at 27°C is lower than that of 0°C. To eliminate this limitation we can possibly stir the solution using a stirrer or glass rod.

Besides that, a little amount of the red dye in the solution may be lost when we transfer the solution into the cuvette. To test for the percentage absorbance we placed 2 cm3

of the dye solution into the colorimeter cuvette by using a dropper. There is a high chance which a small amount of the solution is still left in the dropper, resulting in the volume of solution in the cuvette is less than 2 cm3 and is not constant for the testing of all the dye solution after the beetroot is being heat treated under different temperatures. Consequently, the accuracy of results is affected. So to get rid of this limitation, we could use a pipette for measuring 2 cm3 instead of a dropper.

In addition to that, the reading of colorimeter may not necessarily be perfectly accurate. This is due to another limitation in this experiment using colorimeter that lies in the imperfections in the glass or plastic cuvette. We need to understand that colorimeter functions by simply directing a light through a cell cuvette containing the dye solution to be analyzed. A portion of the light beam is absorbed by the sample and the remaining portion passes through the cuvette to the photodetector means for measurement. Very minor variations in a cuvette, for example any foreign particles or fingerprints on the cuvette will substantially affect measurements made by the photodetector means. So to reduce the effect of this limitation, we shall clean the surface of the cuvette properly by wiping using a clean cloth. We should also make sure the cuvette surface is smooth on all sides.

As explained in the discussion for the flattening of the curve from the temperature of 55°C to 70°C, a very high temperature can cause the red dye from the beetroot to change colour to yellow. Since the filter dial of the colorimeter is set to the blue/green filter, the colorimeter can only measure absorbance at one wavelength; a change in colour will result in a lower absorbance at that wavelength and so apparently the amount of betalains decreases. This limitation is hard to eliminate.

Other than that, when we measure and cut 1 cm length slices from sections of beetroot, we assumed that each of the length slices has roughly the same surface area to


volume ratio, but in actual they are not exactly the same. This would lead to slight inaccuracy of the results because we know the rate of diffusion would increase if the total surface area to volume ratio increases. There might be more betalains diffused into the distilled water for thinner and smaller beetroot cylinders. The best way to eliminate this limitation is to repeat the experiment and get the average of the results.

Besides, as mentioned before why is there any pigment released from what should be intact cell membranes at room temperature, this is may be due to one of the limitations in this experiment too. When we cut the beetroot cylinders and place them in a beaker of distilled water, the limited time may not be able to wash away all the red pigments on the outside of the beetroot cylinder which comes from the damaged cells. What we can possibly do is to leave them in the distilled water for a longer time, perhaps 15 minutes if not left overnight.

Systematic and Random Errors

When we are measuring the length of beetroot cylinders, parallax error may occur when our eye level is not perpendicular to the scale of the ruler. This error would contribute to the limitation of inconstant length of beetroot cylinders as mentioned above.

Parallax error may also occur when we are measuring the volume of distilled water needed to be placed into each boiling tube if our eye level is not perpendicular to the bottom meniscus of the water. When the volume of distilled water is inconstant, the concentration of the betalains that leak out of the beetroot will vary too. To avoid these two parallax errors, we might as well pay total attention and concentration by making sure our eyes is perpendicular to the scale of the measuring tools.

Systematic error may occur when zero error is present on the colorimeter. It is therefore very important when we calibrate the colorimeter. The colorimeter is adjusted to read zero absorbance for clear water. We also made sure no one alter the setting again during the experiment especially when we share the colorimeter with other groups.

In this experiment, we used electric water baths and not prepare our individual water baths. Hence thermometer used in this experiment is just to confirm the temperature of the water bath, as there is minimum fluctuation of temperature of water baths.

Validity and Reliability of Results

There are some apparent anomalies in our results. This can be seen in the colorimeter reading of beetroot placed in water bath of 0°C and 27°C. There are some limitations and sources of error in the experiment that can deem our results unreliable. In order to make sure that the results are reliable and valid, besides adhering to the other constant variables and follow all the necessary precautionary steps as mentioned in the beginning of discussion, replication of experiment is very important. Replication of studies helps to support findings and eliminate random errors. If repeated experiments confirm unexpected results in this experiment, then the hypothesis should probably be revised if there is any hope of it becoming a theory. In this case, since the


colorimeter readings we obtain are about the same for each temperature, and the average values we calculated does not exactly follow the expected trend, we could conclude that our hypothesis should not be accepted.

Safety PrecautionsWhen handling a cork borer to cut sections from a single beetroot, it is necessary to

hold the beetroot securely and ensure that our hand is not in the path of the borer. It is also very important to screw the borer away from our body.

These sections had to be then cut into 1 cm length slices using a sharp knife to cause less damage to the membranes. However it may be dangerous if a person do not apply his full concentration when using the knife. Users must be careful concerning how the knife is held and used.

When we place or remove the boiling tubes into and out of the electric water bath, it is essential to use a pair of boiling tube holder, if not, a wet cloth to protect our hand from the heat. We must not attempt to hold the boiling tubes with bare hands even after they are left over some time to prevent any accidents.

Besides, we should also handle the colorimeter with care as colorimeter is an expensive instrument.

We ought to wear a laboratory coat and a pair of gloves as spillage of beetroot juice will stain our skin and clothing very badly, even though it is harmless to us.

ExtensionThis experiment can be modified to investigate effect of alcohol concentration on

membrane permeability. Solutions with different concentration of alcohol are prepared by mixing the same amount of alcohol with variable amount of distilled water. The beetroot cylinders are placed in solutions with different concentration of alcohol instead of different temperature of water baths. The amount of red dye which leaked out is measured using a colorimeter.

ConclusionAn increase in temperature does not exactly increase the permeability of the cell membrane. The hypothesis is not accepted.


References1. Image on website (The Great Grub Club)

The Great Grub Club – Being Healthy is Fun. Retrieved September 29th, 2010 fromwww.greatgrubclub.com/?location_id=70

2. Image on website (Absolute Astronomy)Absolute Astronomy – Betalains. Retrieved September 29th, 2010 fromwww.absoluteastronomy.com/topics/Betalain

3. Image on website (Causes of Color)Causes of Color – Plants and Flowers. Retrieved September 29th, 2010 fromwww.webexhibits.org/causesofcolor/7H.html

4. Image on website (TutorVista)TutorVista – Plasma Membrane. Retrieved September 29th, 2010 fromwww.tutorvista.com/.../plasma-membrane.php

5. Image on website (The Lab Depot)The Lab Depot – Spectrophotometers. Retrieved September 29th, 2010 fromwww.labdepotinc.com/c-489-spectrophotometer.php

6. Article on website (Wikipedia)Wikipedia – Beet. Retrieved September 29th, 2010 fromhttp://en.wikipedia.org/wiki/Beet

7. Article on website (Kitchen Gardener)Kitchen Gardener – Episode 7 – Beetroot. Retrieved September 29th, 2010 fromhttp://blogs.abc.net.au/nsw/2009/09/kitchen-gardener-episode-7-beetroot.html?program=702_drive

8. Article on website (123HelpMe)123HelpMe - The Effect of Temperature. Retrieved September 29th, 2010 fromhttp://www.123helpme.com/view.asp?id=123054

9. Wolfe S.L., Molecular and Cellular Biology, (1993). Wadsworth Publishing Company.

10. C.J.Clegg, Edexcel Biology for AS, (2008). Hodder Education.

11. Campbell, Reece, Urry, Cain, Wasserman, Minorsky, Jackson, Biology Eighth Edition, (2008). Pearson.

(NUMBER OF WORDS: 4242)


1.4 effect of ph on enzyme activity

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