|MadSci Network: Cell Biology|
Dear Alla, pH affects membranes by affecting the proteins that make up about 70% of most cell membranes (less in inactive cells, such as red blood cells, more in others, such as those of mitochondria). proteins are made of amino- acids and each amino-acid has a variable number of nitrogen and oxygen atoms in it. These can form HYDROGEN bonds with the many hydrogen atoms found in the molecule. The clever thing is, that the protein folds up to ensure that the MAXIMUM number of these hydrogen bonds is made. When the pH of a solution changes, the position of some of these hydrogen atoms also changes. This is because amino-acids are AMPHOTERIC, and tend to stabilise pH. Thus, they can lose an H+ ion at the COOH [or 'acid'] part of the molecule at higher pHs, or gain an H+ ion at the NH2 [or 'amino'] end of the molecule at lower pHs. This, in turn, causes the overall shape of the protein to change with pH. This is the reason why most enzymes (which need a precisely-shaped 'ACTIVE SITE') can only work well at a certain pH. Unlike heat, the denaturing of a protein by changing pH is (normally) REVERSIBLE. The dye in beetroot (betalain - see notes below) diffuses out of the cell when the membrane proteins are damged. Changing the pH is just one way of doing this - others include the use of heat or organic solvents such as ethanol or detergents. NOTES Follow: BEETROOT PIGMENTS - and membranes – These pigments are betalain pigments (not, as often thought, anthocyanins), which they replace in some organisms. They are named after the Beet family of plants (Beta) but are also found in fungi (Fly Agaric - the red, spotted one!). In petals they presumably attract pollinating insects and may be present in seeds/fruits to encourage birds to eat them and so disperse the seeds. Man has selected for colour in beetroot, both because it is more attractive but also because it may well be linked to genes for flavour too. There is no indication that they have any protective function (e.g. against UV light or insect/fungal/viral attack). Unlike anthocyanins, they are not pH indicators – their colour is stable over a wide range of pH. They are oxidised over time (going brown) and this may be prevented by 0.1% ascorbic acid ( = Vit.C); they are sometimes used as food colourants. They are found in the vacuole and thus are used as markers for scientists who wish to extract intact vacuoles from plants for research. To extract the pigment, the membranes must be disrupted. This can be done by heat shock, by detergents or by solvents (e.g. ethanol or acidified methanol). Thin slices have a larger surface area and so leak more pigment; freezing the beetroot first bursts the cell membranes and kills the cells, thus allowing the pigment to be extracted much more quickly. Effect of Heat: When you heat a beetroot, you disrupt the cell membranes. A biological membrane is made of a so-called phospholipid bilayer. These are formed because the phospholipids that make it up have a polar "water- loving" (hydrophyllic) head and a “water-hating” (hydrophobic) tail. The tails pack together, exposing only the polar heads to the water. The most effective way of doing this is to create two blankets one atop the other, with the fatty acid tails towards each other. This is the phospholipid bilayer. In a cell they form sacks. One goes all around the cell (the plasma membrane), others may form vacuoles (such as the tonoplast). Yet others may be like stacks of half empty bags (the endothelial reticulum, which is also continuous with the nuclear envelope. In these lipid seas, there will be a number of proteins in various degrees of submersion. Some span all the bilayer, thus being exposed on both sides. Others just drift on either of its surfaces. Typically, you will find that about 70% of a cell membrane is protein. The water around and within the compartments formed by the phospholipid bilayers is also crammed with protein (= cytoplasm). So what happens when you heat this? When you heat something you give it energy. Molecules start to spin and vibrate faster. The water will expand too. This will have a disruptive effect on any membrane in its way. To make things worse, lipids become more fluid as temperature goes up (think of what happens when you heat butter) so the membranes become more fragile. Proteins are remarkable machines: they're formed of coiled and folded strings of amino-acids, held together by hydrogen bonds and disulphide bridges. If you heat them too much, they will untangle and break apart (vibrations again). When this happens to the proteins spanning a lipid membrane, they will form holes that will destroy the delicate structure. Now, any pigments in the innermost compartment will spill out. Half-life: The half-life of beetroot pigment is 413 mins at 250C but only 83.5 mins at 600C. These values are doubled in 0.1% ascorbic acid. Metal ions speed up the breakdown – iron is particularly effective. They are stable between pH 4.0 and 7.0 – indeed, at high temperatures they are most stable in a pH between 4.0 and 5.0 – and most fruits and vegetables are acidic! Effect of organic solvents If you want to dissolve lipid-embedded pigments, place a beetroot in an organic solvent such as acetone and see what you get. You break down the structure between the phospholipids, not the phospholipids themselves that much. The proteins on the other hand, are truly destroyed. The basic structure of the tonoplast is the same as the plasma membrane (as described above). In this respect, they're similar, though with a higher proportion of protein in the plasma membrane than in the tonoplast. (why?) PIGMENTS IN PLANTS 1. The anthocyanins are common plant pigments. They are water- soluble glycosides with some or all of the sugar groups removed. The colour comes from a positive charge distributed over the chemical ring system. The colours of the charged anthocyanin pigments are dependent on the pH of the intracellular medium containing these pigments. 2. Many leaves frequently develop red coloration during development, at maturity, and during senescence. Most plants produce anthocyanins (usually cyanidin glycosides) as the basis of this colour, but members of the Caryophyllales produce nitrogenous pigments, betacyanins. 3. The betacyanin pigment of beet roots is normally sequestered in the vacuole and, by means of of the beet root. Of course if the beet root is cut cells are sliced open and the pigment spills out, but if the membrane is altered (phospholipid bilayer + proteins) more subtly leakage (diffusion) of betacyanin is induced. Betalains: What are betalains? Betalains are alkaloid pigments that are found in some families of plants belonging to the order Caryophyllales, but in no other plants. Little is known about the role of betalains. Betalains are not found in plants containing anthocyanin pigments – structurally they are unrelated They have also been found in some fungi e.g. Fly Agaric They can be divided into betacyanins and betaxanthins based upon their molecular structure. Betacyanins generally appear red to red violet in colour - they absorb in the 535-550nm range - hence our choice of filter in the colorimeter) Betaxanthins generally appear yellow in colour (absorb in the 475-480nm range) They cause colour in both flowers, fruits and sometimes vegetative organs They are found in the vacuole and they are water-soluble. Beetroot contains 2 Betacyanins - Betanin and a derivative Commercial uses: Beetroot pigment is used commercially as a food dye. It changes colour when heated so can only be used in ice-cream, sweets and other confectionary, but it is both cheap and has no known allergic side-effects. Beetroot itself, of course, is a common salad ingredient – when cooked, vinegar is added to the water to lower the pH. If you read all the above notes, you will see why! Ó IHW October 2003 ADVICE ON WRITING UP A BEETROOT MEMBRANR PRACTICAL (FOR THE UK!!) BEETROOT COURSEWORK - temperature or ethanol concentration - PLANNING: The independent variable is the factor that you control. Thus you need to include full details of how you set about ensuring that the values you state are as accurate and reliable as possible. So: Temperature – use water-baths and measure the actual temperature allow enough time for the ‘ingredients’ to reach the indicated temperature – state in minutes how long. NB ‘Room Temperature’ does not exist in AS coursework – but you can use a water-bath with tap-water, by all means….. Volume - what volumes of liquids are you going to use? Why? How are you going to measure them? Why? Beetroot - How much are you going to use? Why? How are you going to cut them? Why? Is the beetroot uniform? How are you going to randomise your beetroot disks? What about genetic / growing / storage variations in the beetroot? Reaction vessels - What are you going to react the beetroot in? Why? Time - How long are you going to react the beetroot for? Why did you choose this time? How are you going to measure it? How are you going to ensure each replicate is the same? Range - what range of readings are you going to take? How did you decide on this? Will it be sufficient for a reliable result? Concentration - Range sufficient? Why chose this? Control - This will, of course, be part of the range of results taken, and so no separate control will be needed. The dependent variable is the one you measure. So: Absorbance - What are you going to measure this with? Why did you decide to use a Harris Digital Colorimeter, set up using filter 5 (yellow- green, peak absorbance at 550 nm?). How to you propose to calibrate it? What is the volume you use in each cuvette? How will you measure this? Replicates - How many readings are you going to generate per set of results? Clearly, you realise that the minimum is going to be 5. How many replicates are you intending to use? Can you combine your results with any other groups? Are you going to average them? Why? DIAGRAM: Have you done one? In pencil? Labelled? COLLECTION OF DATA Do ensure that you have collected your data on a decent bit of paper! These results need to be included in your final submission (as an appendix), as well as the neat, fully titled, word-processed table(s) in the ‘Results’ section of your report. Ensure your results are taken to an appropriate degree of accuracy and that you have enough in each set (min 5) and enough repetitions (min 3). You can pool results with others and average them, without fear, providing you state that they are pooled data. GRAPHING DATA: Titled. Lines labelled. Axes right way round – independent variable on the X axis, and fully labelled -with units! Most of graph paper area used – don’t plot on computer – the exam board don’t like them! Line of best fit optional; ‘joining up the dots’ = compulsory! Take care not to extrapolate back to the origin, unless you are certain that that is correct (it usually is not!) Error bars might be a worthwhile inclusion – if a histogram/bar chart is used. Calculations. As well as a bit of averaging, you might well be able to calculate a rate or two – anything (simple!) like this helps to give an air of authority to your conclusions. INTERPRETING Describe the pattern of your results – using actual numbers! This seems pointless and even offensive, given that the examiner can read and interpret the graph as well as you can. But do it! Conclusions need to be detailed. This means that you have to give an interpretation of your results. In this case, the purple pigment (probably betanin) is found in the vacuole in the centre of the cell. It is water soluble. It is surrounded by both the vacuole and the cell membranes. The cellulose cell wall on the outside of the cell is fully permeable and so no barrier to the egress (= release) of the pigment. The pigment comes out because the two membranes are damaged. How? Membranes contain c.30% phospholipid (soluble in ethanol) and c.70% protein (denatured by high temps). So your account should explain what happens. The pigment is heat labile i.e. is denatured by heat – turning to 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 the numbers decrease. How does the pigment leave the cell (and why?)? The concentration outside the cell is low – so diffusion will be important. What factors affect the rate of diffusion? Did you leave the beetroot in the experiment for the optimum time? What would happen with (much) longer and shorter times? [See handout on Beetroot pigments] ANALYSING The main source of error in any biological experiment is usually the natural variation of living things. What did you do to ensure that this variation was minimised? The apparatus is more than accurate enough – but what about the disks themselves? ‘Experimental error’ counts for nothing – unless you detail the cause of the error and (better) indicate how the experiment could have been improved to reduce/eliminate the error that you have identified. Clearly, even with pooled class results only a limited range of results were abtained; what should be done to ensure that the results were reliable, repeatable and applicable to other situations? Simply repeating the experiment with the same apparatus and the same range of results will improve the reliability but not the accuracy. For that, the experiment must be modified – perhaps by using different apparatus (which we might not have – such as a more accurate colorimeter, which measures light over a much narrower range of wavelengths) Your anomalous results (or the class average’s) must be indicated on the graph(s) then seek to explain them – i.e. what is the most probable cause(s) of these results? Once again ‘Because that idiot XXXX did it’ won’t get any credit at all! Ó IHW October 2003
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