Life on Earth Plant Practical 2 - Seed Protein Analysis Aim: To help you understand how and why plant seeds are used for protein production Why? Because of the importance of amino acid production in plants to the living world and its implications for animal nutrition Introduction The seeds of many species are used by humans in a variety of ways. Over 55% of human nutritional needs are supplied directly by seeds, and this figure is even higher when seed products such as vegetable oils and margarine are also included. Seeds are also extremely important in animal feeds. Furthermore, seed products are used industrially; seed oils are used as lubricants, in chemical syntheses and nowadays even as fuel for advanced combustion engines. During seed development, low molecular weight metabolites formed via photosynthesis and nitrogen metabolism, such as sucrose and amino-acids, are imported into the developing seeds and converted into storage lipids and polymerised into starch and storage proteins. It is because of these storage products that certain seed plants have been domesticated. These plants are very important in human nutrition. Understanding something about seed proteins should deepen your understanding of human nutrition especially the production of essential amino acids. In this practical the protein content of the seeds of a variety of plants will be determined using the Bradford protein assay. Proteins represent the most abundant nitrogen store in seeds and are usually deposited in discrete membrane-bound organelles called protein bodies. The Species These are examples of the crop species whose seeds we have used in the lab. Have a look at them whilst you are centrifuging your extracts. To find out more about these species visit the websites of the research institutes of the Consultative Group on International Agricultural Research (CGIAR http://www.cgiar.org) which are dedicated to the various crops. The CGIAR is a very large research organisation dedicated to improving the food crops of the world, with particular emphasis on providing nutritionally balanced food for all. It has 16 very large research institutes around the world. For general information about human nutrition worldwide some of the pages of the Food and Agriculture Organisation of the UN are useful, try: http://www.fao.org/ Peanut (Arachis hypogea): This is a native of South America and was not known elsewhere until the discovery of the New World. It is often used with maize flour in staple diets in South America. The USA is now the world's biggest producer (about 120 million tonnes each year in US - a lot of peanuts!). ICRISAT is the CGIAR centre with responsibility for peanuts (also called groundnuts). If you explore their pages there's information about peanuts. http://www.icrisat.org/ Peas (Pisum sativum): These are native to the Mediterranean basin and were spread round Europe by the Romans. They are classically accompanied by wheat products in many European staple diets. Chickpeas (Cicer arietinum): These are native to the near east and perhaps northern India. Chickpeas dishes are very often accompanied by wheat products in staple diets of the near east and India. ICRISAT is the CGIAR centre with responsibility for chickpeas. If you explore their pages there's quite a bit of relevant info. http://www.icrisat.org/ Soya Beans (Glycine max): Native to China where they are often used to accompany rice dishes. The USA is now the world's biggest producer. Maize (Zea mays): Native to S. America where the Aztecs were responsible for its domestication. Classically accompanied by peanuts, beans or chocolate food stuffs (which are all native to S. America) and was introduced to the rest of the world after the discovery of the New World). Cimmyt is the CGIAR centre with responsibility for maize. Explore its website and note that a lot of its work is on improving maize protein content! http://www.cimmyt.org/ Wheat (Triticum aestivum): Domesticated in the near east (now, with gene mapping, domestication has been located to eastern Turkey about 6000BC) and initially used with chickpeas and lentils (which are all native to the near east) but now used with beans in S. America, and many pulses in India for example. Cimmyt also carries out research on wheat. http://www.cimmyt.org/ Rice (Oryza sativa): Domesticated in China and possibly India. Classically accompanied by soya products in China and chickpeas/lentils in India. About 450 million tonnes on world markets each year - this is a lot considering most rice is eaten by the person who grew it and doesn't even get on to world markets! Sorghum (Sorghum bicolor): Sorghum was domesticated in Africa but is now grown around the world. It is the most drought tolerant of the major cereals and is the most important cereal in much of the semi-arid tropics. It is one of the ICRISAT mandate crops and you can find out more about it from the ICRISAT website http://www.icrisat.org/ 2 Protein extraction method • • • • • You are supplied with seeds of a species of legume or cereal in a glass boiling tube. Exactly 1 gram dry weight has been measured for you then soaked overnight. Pour off the excess water from the seeds using the gauze supplied. Using a 10 mL glass pipette and pi pump add 8mL of Tris extraction buffer (50 mM Tris- HCl, pH 7.5) and then grind the seeds using the Ultra Turrax homogeniser. Transfer all the liquid and ground material to a 15 mL plastic centrifuge tube. You have a few extra mL to play with so use extra Tris buffer to rinse out all the bits from the boiling tube and into the centrifuge tube. Label your centrifuge tube with your name (not on the lid) and take it to the centrifuge where it will be spun at full speed for 10 minutes. Retrieve your centrifuge tube then measure and record the volume (V) of the supernatant (the liquid left above the solid pellet) by pouring it into a 25 mL measuring cylinder. This supernatant contains your extracted protein, so don't throw it away but, pour it into a small conical flask. Plant cells typically contain many compounds, such as lipids, that interfere with protein quantification assays. Consequently, the proteins in your extract will need to be precipitated to separate them from these compounds prior to assay. • • • • Using a micropipette fitted with a blue tip, set at 1000 microlitres (1 mL) transfer 1 mL of the extract to a micro-centrifuge tube (it's the small transparent tube with a snap lid attached). Change the volume setting of your micropipette accordingly and add 0.35 mL of 20% (w/v) trichloracetic acid (TCA), close the lid tightly and mix well by inverting the tube a few times. Label the lid of the tube then stand the tube in an ice bath for at least 10 min. Remember TCA is harmful. After 10 minutes, take your tube to the micro centrifuge and spin at 1500 × g for 10 min. This time the protein is in the pellet at the bottom, so discard the supernatant and re-suspend the pellet in 1mL of 1% (w/v) TCA. You will need to resuspend the pellet completely by stirring with a glass pipette with a squeezy bulb attached. Centrifuge again for 10 minutes and discard the supernatant. Transfer the pellet into a glass boiling tube using 5 mL of Tris extraction buffer. In order to resuspend it completely, use the Turrax homogeniser again. However, to avoid cross contamination, ensure that it is completely clean and dry before inserting into your sample. Protein determination This method is based on protein binding to the dye Coomasie Brilliant Blue G250 (Bradford, 1976). Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding Analytical Biochemistry 72(1–2), pp. 248–254. 3 Standard Curve: A standard curve must be constructed before the protein content of your extracts can be determined. This can be done at any time during the practical class. You will add different volumes of a pre-determined protein standard to deionised water, giving you concentrations from zero to 40 μg mL-1. You will then add Bradford Reagent to develop a colour whose strength is related to the concentration of protein. To do this, to six glass test tubes add the following using the micropipettes: Reagent Tube Tube Tube Tube Tube Tube 1 2 3 4 5 6 Vol. 40 μg mL-1 BSA* (mL) 0 0.1 0.25 0.5 0.75 1.0 Vol. deionised H2O (mL) 1.0 0.9 0.75 0.5 0.25 0 Final conc. BSA (μg mL-1) 0 4 10 20 30 40 *BSA is Bovine Serum Albumin - a reference protein from cow serum Add 2 mL of Bradford reagent (see below) to each of the tubes and mix well. Allow the blue colour to develop for 10 minutes and then determine the absorbance of the mixture from each tube at 595nm. How to use the Spectrophotometer: • • • • • ● Turn on and let the spectrophotometer warm up for 30 minutes Select absorbance function (ABS) using left and right arrows. Adjust wavelength to 595 nm using up and down arrows Lift lid, insert the blank (your deionized water with Bradford Reagent - Tube 1 mixture) in a 3mL cuvette (ensure clear window of the cuvette is in the correct orientation), close the lid and press CAL Spectrophotometer will calibrate to zero Remove blank and insert samples in 3mL cuvettes to be read Do not press CAL again or your results will be wrong Plotting the standard curve Use μg protein mL-1 on the horizontal x-axis (independent variable) and absorbance at 595nm on the vertical y-axis (dependent variable) and plot a graph with a line of best fit. This is not 'join the dots' but a prediction of what you think the underlying relationship to be. It will not be linear, so draw a smooth curve. Measuring Protein Concentration in Your Extract Your sample extracts will probably require considerable dilution before their protein content can be determined using the Bradford reagent and your standard curve. The dilution factor necessary (D) can only be worked out empirically, but we will suggest a starting dilution depending on which species' seeds you have extracted. When you have made a suitable dilution: • • Transfer 1.0 mL of your diluted seed protein extract to a 3 mL cuvette. Add 2.0 mL of Bradford Reagent and mix well. After 10 minutes determine the absorbance at 595 nm using the same spectrophotometer as you used to make your standard curve. The Bradford Protein Reagent: This is already made up. Briefly: 100mg of Coomasie Brilliant Blue G-250 were dissolved in 50 mL of 95% (v/v) ethanol. 100 mL of 85% (w/v) phosphoric acid were then added and the solution diluted to 1 litre with distilled water. The solution was left overnight and filtered to remove un-dissolved solids. Results Plot the standard curve and use it to determine the protein concentration in your diluted samples. From these data, calculate the protein concentration in the TCA-precipitated sample and then in the initial extract. Finally, calculate the protein content of the seed tissue in mg g¹. This is how to do the calculation: From the absorbance you obtained from your Bradford's reading of your diluted protein extract read off its protein concentration from the standard curve = X μg mL-1 Multiply by your dilution factor (D) = DX (μg mL-1) Multiply by 5 (because you also diluted the samples by a factor of 5 at the TCA stage) = 5DX (μg mL-1) Multiply by the volume of the initial extract (V) in mL = V5DX (µg) Divide by the weight of seed tissue extracted (W) in g = V5DX (µg g‍¹) W Enter your data on the class sheet in mg g‍1 (i.e. divide by 1000) and think about any differences there are between the seed protein contents of the different species. Keep your Bradford's standard curve. It will be useful as a guide when you come to do the coursework. 5