BIOLOGY LAB REPORT

TITLE PREPARED BY I/C NUMBER STUDENT ID GROUP

: THE EFFECT OF TEMPERATURE ON MEMBRANE : : : :

LECTURERS NAME :
PRACTICAL DATE SUBMISSION DATE : :

OBJECTIVE To investigate the effect of temperature on membrane structure. To practice experimental and investigative skill.

INTRODUCTION

BEETROOT

The beet (Beta vulgaris) is a plant in the Chenopodiaceae family. It is best known in its numerous cultivated varieties, the most well known of which is the purple root vegetable known as the beetroot or garden beet. Beta vulgaris is a herbaceous biennial or, rarely, perennial plant with leafy stems growing to 12 m tall. The leaves are heart-shaped, 520 cm long on wild plants (often much larger in cultivated plants). The flowers are produced in dense spikes; each flower is very small, 3 5 mm diameter, green or tinged reddish, with five petals; they are wind pollinated. The fruit is a cluster of hard nutlets. The usually deep-red roots of beetroot are eaten boiled either as a cooked vegetable, or cold as a salad after Diagram 1 : Beetroot fruits 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. In Eastern Europe beet soup, is a popular dish. Yellow-coloured beetroots are grown on a very small scale for home consumption. The green leafy portion of the beet is also edible. It is most commonly served boiled or steamed, in which case it has a taste and texture similar to spinach. Functions of beetroot Beetroot are a rich source of potent antioxidants and nutrients, including magnesium, sodium, potassium and vitamin C, and betaine, which is important for cardiovascular health. It functions by acting with other nutrients to reduce the concentration of homocysteine, a homologue of the naturally occurring amino

acid cysteine, which can be harmful to blood vessels and thus contribute to the development of heart disease, stroke, and peripheral vascular disease. Betaine functions in conjunction with S-adenosylmethionine, folic acid, and vitamins B6 and B12 to carry out this function. Additionally, several studies on both rats and humans have shown betaine may protect against liver disease, particularly the build up of fatty deposits in the liver caused by alcohol abuse, protein deficiency, or diabetes, among other causes. The nutrient also helps individuals with hypochlorhydria, a condition causing abnormally low levels of stomach acid, by increasing stomach acidity.Beetroot juice has been shown to lower blood pressure and thus help prevent cardiovascular problems. Research published in the American Heart Association journal Hypertension showed drinking 500 ml of beetroot juice led to a reduction in blood pressure within one hour. The reduction was more pronounced after three to four hours, and was measurable up to 24 hours after drinking the juice. The effect is attributed to the high nitrate content of the beetroot. The study correlated high nitrate concentrations in the blood following ingestion of the beetroot juice and the drop in blood pressure. Betanin Betanin, obtained from the roots, is used industrially as red food colourants, e.g. to improve the color and flavor of tomato paste, sauces, desserts, jams and jellies, ice cream, sweets and breakfast cereals. Within older bulbs of beetroot, the colour is a deep crimson and the flesh is much softer. Beetroot dye may also be used in ink. Betanin is not broken down in the body, and in higher concentration can temporarily cause urine (termed beeturia) and stool to assume a reddish color. This effect can cause distress and concern due to the visual similarity to bloody stools or urine, but is completely harmless and will subside once the food is out of the system. It is a rich source of the element boron. Field Marshal Montgomery is reputed to have exhorted his troops to 'take favours in the beetroot fields', a euphemism for visiting prostitutes. From the Middle Ages, beetroot was used as a treatment for a different kind of conditions, especially illnesses relating to digestion and the blood.

Plasma membrane

The cell membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It consists of the phospholipid bilayer with embedded proteins on its surface All living cells, prokaryotic and eukaryotic, have a plasma membrane that encloses their contents and serves as a semi-porous barrier to the outside environment. The membrane acts as a boundary, holding the cell constituents together and keeping other substances from entering. The plasma membrane is permeable to specific molecules, however, and allows nutrients and other essential elements to enter the cell and waste materials to leave the cell. Small molecules, such as oxygen, carbon dioxide, and water, are able to pass freely across the membrane, but the passage of larger molecules, such as amino acids and sugars, is carefully regulated.

Proposal of fluid mosaic model According to fluid mosaic model proposed by S.J.Singer and G.L.Nicholson, the plasma membrane is composed of a double layer (bilayer) of lipids, oily substances found in all cells .Most of the lipids in the bilayer can be more precisely described as phospholipids, which means, lipids that feature a phosphate group at one end of each molecule. Phospholipids are characteristically hydrophilic ("water-loving") at their phosphate ends and hydrophobic ("water-fearing") along their lipid tail regions. The hydrophobic lipid tails are oriented inwards and the hydrophilic phosphate groups are aligned in each layer of a plasma membrane, so they face outwards, either toward the aqueous cytosol of the cell or the outside environment. Phospholipids tend to spontaneously aggregate by this mechanism whenever they are exposed to water. Within the phospholipid bilayer of the plasma membrane, many diverse proteins are embedded, while other proteins simply adhere to the surfaces of the bilayer. Some of these proteins, primarily those that are at least partially exposed on the external side of the membrane, have carbohydrates attached to their outer surfaces and are, therefore, referred to as glycoproteins. The presence of cholesterol and glycolipids, which are found in most cell membranes, can also affect molecular dynamics and inhibit phase transitions. Function of plasma membrane Plasma membrane proteins function in several different ways. Many of the proteins play a role in the selective transport of certain substances across the phospholipid bilayer, either acting as channels or active transport molecules. Other than that. It also carries function as receptors, which bind information-providing molecules, such as hormones, and transmit corresponding signals based on the obtained information to the interior of the cell. Membrane proteins may also exhibit enzymatic activity, catalyzing various reactions related to the plasma membrane.

Diagram 2 : structure of plasma membrane

Structure of plasma membrane

Plasma membrane is called as partially permeable since some small and neutral molecules can pass through the plasma membranes easily whereas big and charged (ionised) particles cannot. The molecules move through the plasma membrane through diffusion. To function correctly, a cell needs to be able to control transport across the partially permeable cell surface. Effect of temperature on plasma membrane

Increased Temperature
As temperatures increase, both the cell membrane and the proteins can be affected. The fatty acid tails of the phospholipid bilayer can "melt" at high temperatures meaning that they become more fluid and allow more movement. This affects the permeability of the cell which may allow molecules into the cells that should not get in, thereby damaging the cell. The peripheral proteins can also be damaged by high temperatures. High temperatures cause proteins to denature, or break down. Increased temperatures also increase the reactions that happen within the cells, which may be acceptable to a point, until the temperature becomes too high which will destroy the protein, the reactions and the cells.

Decreased Temperatures
A decrease in temperature also has an effect on cell membranes and cells. The fatty acid tails of the phospholipids become more rigid at cold temperatures. This affects the fluidity, the permeability and the cells ability to live. When the cells are less fluid, they cannot move or grow. The decrease in permeability means that vital molecules cannot get into the cell. In addition, cold temperatures can cause cellular reactions to slow down or even stop.

Application on betalain pigment

The molecule such as the red betalain pigment inside the beetroot cells cannot pass through the membrane because they are not discrete enough to be diffused out of the its plasma membrane. If the membrane disrupted by high temperatures, then the betalain pigment can move across the membrane and leak out to the solution around it. Therefore, the amount of pigment that leaks out can be assessed, as betalain pigment will colour any water that surrounds the cell. The more the amount of red colour in the water, the higher the permeability of the membrane since the betalain pigment can move across the membranes easily. the spectrophotometer
A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement. A spectrophotometer is arranged so that liquid in cuvette can be placed

between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes

Diagram 3: An idea on how a spectrophotometer works

PROBLEM STATEMENT What is the effect of temperature on membrane structure? HYPOTESIS As the temperature increases, more dye will be released from beetroot. High temperatures may distort the active site of the carrier affecting the shape of the fluid mosaic model membrane. The increase of kinetic energy will speed up the diffusion rate of the red pigment to a point then structural damage of the membrane and the denatured proteins will increase the amount of red pigment escaping out of the cells.

VARIABLES

Types of Variables Manipulated Variable: Temperature of solution ( C)

Ways to control the variables

Use water bath at different temperatures, at 25C, 35C, 45C, 55C and 65C and measure using thermometer

Responding Variables: Spectrophotometer reading (A) Repeat the reading thrice to get an average value by adding all three reading and divide with three

Fixed Variables: Diameter of corer (cm) Use the same diameter of corer which is size 4

Size of beetroot (cm)

Use same size of beetroot which is 1cm cut using a knife

APPARATUS Size 4 cork borer, white tile, knife, ruler, plastic beaker about 250 cm , water bath at 25C, 35C, 45C, 55C and 65C, 2 boiling tube rack, 5 boiling tubes, thermometer (one per water bath), calorimeter, 12 cuvettes, stopclock, pipette (2 cm), small measuring cylinder, sticker, marker pen, automatic water bath MATERIALS Raw beetroot, distilled water PROCEDURE A white tile and size 4 cork borer was prepared. A cylinder of beetroot was collected by pushing the corer into the beetroot and it was withdraw. Since the cylinder was still remains inside the corer, sharp end of a steel rod was used to push it out. Eight 1cm length slices were cut from these beetroot cylinder sections. Those cut sections were placed in a beaker of distilled water to wash away excess dye. Five test tubes were labeled using stickers and marker pen. Then, all test tubes were filled with 7 cm distilled water and placed into water bath at 25C, 35C, 45C, 55C and 65C. Those test tubes were left for 5 minutes (calculated using stopwatch) until the water reaches the required temperature. Then, one of the beetroot sections were placed into each boiling tube using forceps. The test tubes then were left for 30 minutes in the automatic water bath. After 30 minutes, the beetroot sections were removed and the test tubes were shook in order to disperse the dye in solution. The spectrophotometer was switched on and was set up to read % absorbance. Cuvettes smooth surface were cleaned using tissue papers while the cuvette was held on the rougher surface. Accurately 2 cm distilled water was measured using a pipette and placed into cuvette. The cuvette was placed into the spectrophotometer after making sure that the light was shining through the smooth side. The spectrophotometer was adjusted to read zero absorbance of clear water. The setting was not altered again during experiment. 2 cm of the dye solution at 25C was placed into a spectrophotometer cuvette and reading was taken for absorbency. The readings were repeated for 35C, 45C, 55C and 65C thrice for each. All the data is recorded in a table and the graph of reading of absorbance against temperature is plotted.

RESULTS ( DATA COLLECTION) QUALITATIVE DATA As the temperature increases, the solution become darker in colour . Average spectrophotometer reading also increases as the temperature increase. QUANTITATIVE DATA Temperature Observation of solution ( C) Spectrophotometer reading (A) A B C Av era ge (A+ B+ C)/ 3 25 Very pale pink Very Pale pink Pale pink Pale pink Pink 0.397 0.165 0.1 68 0.169 0.1 67

35

0.230

0.2 33

0.243

0.2 35

45

0.311

0.3 20 0.3 26 0.4 02

0.322

0.3 18 0.3 24 0.3 96

55

0.324

0.323

65

0.390

Table 1: The spectrometer reading against the temperature

Average spectrophotometer reading (A)

Temperature of solution ( C)

Graph 1 : Average spectrophotometer reading (A) against temperature of

of solution ( C)

DISCUSSION Analysis of data An experiment was held to investigate the effect of temperature on membrane structure. A beetroot was used as specimen to determine the changes that happen due to increase in temperature. A cylinder of beetroot was cut into 1 cm for 15 pieces and was

soaked in distilled water to remove its excess dye. Five test tubes filled with 7cm and placed in different temperature water bath ( 25C, 35C, 45C, 55C and 65C) and after 5 minutes, those beetroot pieces were placed in the test tubes using forceps. After 30 minutes, the test tubes was removed from water bath and the solution was measured to 2cm using pipette, placed into cuvette and by using spectrophotometer the readings for absorbency was taken thrice per each solution.

The manipulated variable is the temperature of the water bath. Different temperatures of water bath was used so that we can see the effect of temperature on the beetroot cells clearly. The temperatures used are 25C, 35C, 45C, 55C and 65C. The responding variable in this experiment is the reading of absorbance by the spectrophotometer of the dye solution. The results are precise and valid since the measurement of the absorbance reading was taken three times using the spectrophotometer. A higher reading of absorbance shows that there is more dye solution inside the water. This shows that, the more dye solution inside the water, the more the plasma membrane has been disrupted. There are some variables that are kept constant throughout the experiment. Such variable is the length of the beetroot slice is kept constant because we need the same amount of beetroot cells that are to be affected by the temperature. Besides that, the size of cork borer used must also be the same so that we get a better and accurate result which is 1 cm. usage of different size of cork borer may cause results to be invalid because maybe it will affect the reading of absorbance by the spectrophotometer. From the data and the Graph 1, it clearly shows that the higher the temperature, the higher the absorbance reading. Hence, this shows that the disruption of plasma membrane increases with temperature and therefore increases the permeability of the membrane. This shows that the plasma membrane is affected by the temperature.

Generally , my graph plotted shows that as the temperature increases , the amount of pigment released from beetroot increases and thus spectrophotometer reading increases too.

My results increased sharply between the temperature ranges of 25C and 45C, that is from 0.167 arbitrary unit to 0.318 arbitrary units .This is because the amount of random movement of
betalain molecules out through the cell membrane depends on the amount of heat energy the betalain molecules are given to convert into kinetic energy- hence the higher the temperature the more betalain lost from the vacuole. Normally, the betalain pigment of beet root cells sequestered in the vacuole and by means of the cell membrane which maintains the integrity of the cell and the tonoplasts, it does not leak into the cytosol of the beet root. However when we increase the temperature the relatively weak forces holding the different parts of the polypeptide chains together (like hydrogen bonds, sulphur bridges and ionic bonds) can be disrupted very easily causing damages happen to the vacuole and makes holes in the cell membrane, inducing leakage. The cell membrane is also damaged and so diffusion of betalain occurs through the partially permeable membrane by osmosis- the betalain molecules move by diffusion from an area where they are more highly concentrated to an area where they are at a lower concentration, along a concentration gradient.

At ranges of temperature 45C and 55C, the change in arbitrary units is almost insignificant that is from 0.318 to 0.0.324. this is may caused by some mistakes during experiments such as when beetroot sections wasnt handle with care or when the beetroot removed from boiling tube, beetroot dye wasnt fully mixed with the water. At 55C until 65C, reading increase gradually from 0.324 to 0.396 arbitrary units showing that at this point most of the proteins in the beetroot cells are being denatured allowing gaps and the sudden rush of pink pigment to escape.
Basically, it is the input of kinetic energy due to the build up of temperature that increases the rate of diffusion. This in turn will damage and denature the plasma membrane causing substances contained within the membrane to leak out. It is the breakdown of phospholipids in the membrane, which cause gaps to appear allowing red pigment to pass through. As the red pigment particles move faster, they diffuse out of the membrane at a faster rate, increasing more as the temperature increases.

FURTHER INVESTIGATION Effect of alcohol on plasma membranes permeability Further investigation can be made by using different concentration of alcohol to investigate their effects towards the permeability of cell membrane. The beetroot sections are incubated in five test tubes each containing 15 cm3 of different concentration of alcohol ranging from 25% to 100%. The experiment showed that different concentrations of alcohol had a different effect on the cell membranes.

Absorbance (%)

Concentration of alcohol (%)

The graph shows that when the concentration in alcohol increased,the permeability or absorbance of plasma membrane also increases. Discussion
The cell membrane is composed of a phospholipid bilayer and proteins embedded in the bilayer. Alcohols are less polar than water and have similar intermolecular forces as fats, so ethanol diffuses through the outermost phospholipid layer. Ethanols methyl end is also relatively nonpolar, which explains its ability to interact with the amphipathic phospholipids and disrupt the organization of the phospholipids. The cell membrane then loses its structural integrity and could break in water. Greater ethanol concentration means more alcohol-to-phospholipid interactions In addition to the phospholipids, alcohols at high concentrations in particular disrupt the proteins embedded in the membrane. Above a certain concentration, alcohol is lethal to cells

(hence its use as an antiseptic) because it has the ability to denature proteins. Hydrogen bonding occurs between amide groups in secondary structure and between side chains in tertiary structure of a protein. These bonds are disrupted when many alcohol molecules exist around proteins, since the intramolecular hydrogen bonds are replaced by new bonds between the alcohol molecule and protein side chains. Once the membrane is disrupted and the same effect applies to the tonoplast of the vacuole storing betacyaninit can no longer hold things inside the cell (or inside the vacuole) and betacyanin will diffuse according to its concentration gradient into the extracellular matrix.

EVALUATION Limitation and improvement After cutting the beetroot sections, they should be washed with distilled water. This is because there might be some leakage or the plasma membrane had broken. That is why there is red colouring in the water for temperatures which should not have affected the plasma membrane such as the 0C water. Therefore, it is advisable to wash off the excess red dye on the beetroot section first before putting them into respective boiling tubes.

Besides that, when removing the beetroot sections from the boiling tubes, we must make sure that we do not squeeze them so that there would not be excess dye inside the water. This could make the results not reliable. In order to get the best and accurate result, the beetroot sections must be remove with care and not to squeeze them. If they are very hard to be removed out from the boiling tube, a suitable instrument should be use instead such as a longer forceps.

Validity and reability

For ensure the validity of the results, the spectrophotometers reading was taken thr ice per every water temperature. Obtaining average reading will minimize random errors, thus ensure the validity and reability of the data.

Extra care was taken when beetroot cutting session took place so that there is less chance of the beetroot being cut vertically and it was ensured that all beetroot pieces was cut in same manner to make sure the surface area of those sections are the same. This guarantee the validity and reability of data. The test tubes also shaked twice for each evenly before putting into spectrophotometer. This means each test tubes was equal in the number of shakes therefore making the results more accurate. This means the result is more reliable and valid.

SAFETY PRECAUTIONS

In order to avoid any accident or injury during the experiment in laboratory, the precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory when conducting an experiment in the lab at all times to protect the skin from any harmful or irritating substances and spillages. This is to ensure that no beetroot juiced is spilled to our skin as it will stain very badly. Furthermore, the glassware such as measuring cylinder, boiling tubes, and cuvette should be handled with full care because they are fragile. Not only that, sharp objects such as knife and cork borer must be used properly to prevent any cuts or injuries. Care needs to be taken when working with the hot water baths. When taking measurements form the apparatus, make sure to avoid any human error such as parallax error. It is very important to take the reading of absorbance at least three times to get a more accurate, valid and reliable result. Besides that, we must make sure that we wipe the smooth sides of the cuvettes with tissue so that there would not be any errors that could make the results inaccurate and unreliable. Conclusion As the temperature increases, the spectrophotometer reading of the solution also increases. This indicates that the permeability of membrane increases too. Therefore, the higher the temperature is, the greater the permeability of beetroot cell surface membrane. The hypothesis is accepted.