Recipes Isolation, Crystallization and Purification of Excelsin from Brazil Nuts Isolation and Crystallization of Ovalbumin Isolation and Crystallization of Phaseolin from Lima Beans Definitions   Isolation, Crystallization and Purification of Excelsin from Brazil Nuts (globulin) Ground Brazil nuts can be extracted with 5% aqueous NaCl at 50° to 60°C, filtered, then dialyzed against distilled water to obtain crystals. The following procedures can assist you in this process: KEYWORDS: Clarify Decant Dialyze Denature Dialysis Membrane or Tubing Filtrate Precipitate Supernatant Concentration Seed Pellet MATERIALS: Funnel Thermometer Brazil Nuts(raw) Centrifuge Tubes Dialysis Membrane 500ml and 150ml beaker Styrofoam Table Salt Glass Rod Cheesecloth or Glass Wool Pipette or Medicine Dropper EQUIPMENT: Heating Plate Blender or Coffee Grinder Small Centrifuge (up to 3000rpm) METHODS: I. Isolation of Excelsin Record the weight of ~8-9 raw nuts. Grind nuts (preferably organically grown). 1 nut = 2.8 grams. Measure 50mls of 5% NaCl solution into a 150 ml beaker. Record. Create a water bath for NaCl/protein mixture by placing a flat piece of styrofoam into the bottom of the 500ml beaker. Then add approximately 50 ml of water into the 500ml beaker. Place the smaller beaker in the larger beaker and pre-heat to 50°C. Add ground nuts to chloride solution. Heat mixture at 50°C to 60°C for 20 minutes, stirring occasionally.( Be careful not to stir vigorously or to heat protein above the listed temperatures because you may possibly denature the protein). (Option 1 ) -For classrooms without the use of a centrifuge. Place 1-2 layers of glass wool (or 10 layers of cheesecloth) in a funnel and place over a beaker. Pour mixture through glass wool (Caution: Glass wool should only be handled when wearing gloves). Measure and record amount of filtrate. Note: Filtrate is opaque in color. *Skip steps 5-7 and go directly to the crystallization method in section II. (Option 2) -For classrooms with a centrifuge Pour filtrate into 2 centrifuge tubes and weigh to make sure tubes are of equal weight. Record. Centrifuge for ~20 min. at 3000 rpm. Pipette or decant off the aqueous portion (located above pellet and just below fat). Discard pellet and oil. Centrifuge supernatant to clarify solution further. Supernatant is now a clear brown or brownish yellowish solution. If solution is not clear, centrifuge until it becomes clear. II. Crystallization of Excelsin Soak the dialysis membrane (small molecular weight, preferably below 8000) in water for 5 min or longer. Take the membrane out of beaker and tie a knot in one end. Refer to pages on handling of dialysis tubing. Measure and record volume of the extracted solution from above and pour into the membrane and tie other end. 3) Dialyze protein against a 1% NaCl solution. This volume should be at least three times the measured volume of the extracted solution. Crystals should appear 4-10 hours later. (If crystals do not appear at 1% NaCl, lower NaCl concentration to 0%) III. Recrystallization and Purification of Excelsin Cut one end of bag and decant solution off of crystals. Add just enough 5% NaCl solution (1-3mls) to get crystals to go back into solution. Either retie bag or seal open end with a clamp. Place bag into a fresh solution of 1% NaCl and let dialyze overnight until crystals reappear. By repeating this step a protein can usually be rendered essentially pure. To obtain larger crystals one should slowly decrease the concentration of NaCl in solution. For example, instead of going directly to 1% NaCl, start with 4.5% and decrease in increments of 0.5%. Also, one could seed the solution with some of the other crystals.   Size Number Morphology Crystal       TOP Isolation and Crystallization of Ovalbumin   KEYWORDS: Clarify Decant Dialyze Denature Pellet Filtrate Precipitate Supernatant Aqueous MATERIALS: Funnel Vinegar Glass Wool Centrifuge Tubes Dialysis Membrane Litmus Paper Glass Rod or Stir Bar 500ml and 150ml beaker Cheesecloth or Glass Wool Pipette or Medicine Dropper EQUIPMENT: Small Centrifuge (up to 3000rpm) METHODS: I. Isolation of ovalbumin from chicken egg whites Crack and separate six fresh eggs (preferably not more than four weeks old). May be done by puncturing a hole in the top of each egg and draining out the egg white. Save, measure, and record volume of the whites and discard the yolks. Once measured, transfer egg whites to a container which is at least 10 times the measured volume. Break up membranes of the egg whites by stirring gently for at least 20 minutes with a stir bar. Note: Another option for breaking membranes is by pressing or squeezing them through cheesecloth. 5) While continuing to stir mixture, slowly add an equal volume of saturated solution of ammonium sulfate (SAS). Mix after each addition of SAS. Record the total volume egg whites and ammonium sulfate. Calculate and add SAS which equals a tenth of the recorded total volume. Stir for 3-5 minutes. (Option 1 ) -For classrooms without the use of a centrifuge. Place 3- 5 layers cheesecloth in a funnel and place over a beaker. Filter through 3 single layers of cheesecloth, using a large funnel. To clarify further, filter using a layer or two of *glass wool. The yellow liquid is retained, measured, and recorded; all other material is discarded. Should be filtered until clear. * (Caution: Glass wool should only be handled when wearing gloves). From this point you may proceed to directly to the crystallization method in section II. (Option 2) - For classrooms with a centrifuge Pour filtrate into 2 centrifuge tubes and weigh to make sure tubes are of equal weight. Centrifuge for ~20 min. at 3000 rpm. Pipette or decant off the aqueous portion. Discard pellet. Centrifuge supernatant to clarify solution further. Supernatant is now a yellowish solution. If solution is not clear, centrifuge until it becomes clear. II. Crystallization of ovalbumin Using litmus paper, measure and record the pH of the current egg white solution. While stirring, slowly add vinegar until the pH is about 4.6. Next add a couple of milliliters of SAS to aid crystallization. Cover beaker and allow to crystallize overnight. Use a microscope to examine the precipitate. Thin needles should be observed.   Size Number Morphology Crystal       TOP Isolation and crystallization of phaseolin from lima beans KEYWORDS: Clarify Decant Dialyze Denature Slurry Filtrate Precipitate Supernatant MATERIALS: Funnel Vinegar Toluene Glass Wool Centrifuge Tubes Dialysis Membrane Citrate Buffer Glass Rod or Stir Bar 500ml and 150ml beaker Cheesecloth or Glass Wool Pipette or Medicine Dropper I. Isolation of Phaseolin Grind approximately 1 cup of large white lima beans into a fine meal. Weigh and record. Make a slurry by adding 1.5 times the amount of distilled water per part of bean meal. (Important not to add more than 1.5 times dH20). 3) Add three drops of toluene. It will act as a preservative for the protein. (Optional) Boil dialysis tubing for 10 minutes. Tie one end of the tubing and pour the bean meal slurry into the bag. Tie other end of tubing and allow to dialyze against 3500mL of distilled water for 48 hours. Change the distilled water every 24 hours. After dialyzing, pour slurry into a beaker until ready for crystallization. Slurry will keep for approximately 2 weeks at 4 to 7°C. II. Crystallization of Phaseolin Agitate mixture so that it is completely mixed. Then pour 1/2 of the mixture into a beaker and add an equal volume of citrate buffer (pH of 6.02). Stir mixture at 10 min. intervals for 40 min. Place other half of slurry in cold room. Pass mixture through 3 layers of glass wool. Then allow filtrate to stand overnight at 4°C. Spheroidal crystals will appear in 8 to 18 hours. Other proteins to try - suggestions for some similar seed storage proteins Albumins- are a class of simple, water-soluble proteins that can be coagulated by heat and are found in egg white, blood serum, milk, and many other animal and plant juices and tissues. Albumins are widely distributed in plant and animal tissues, e.g., ovalbumin of egg, lactalbumin of milk, and leucosin of wheat. Some contain carbohydrates. Albumins cling to various substances in the blood, e.g., AMINO ACIDS, and thus play a role in their transport. Albumins and other blood proteins aid in regulating the distribution of water in the body. Albumins are also used in textile printing, the fixation of dyes, sugar refining, and other important processes. Globulins- are any of a class of proteins found extensively in blood plasma, milk, muscle, and plant seeds that are insoluble in pure water, soluble in dilute salt solution, and coagulable by heat. The distinction between globulins and other types of proteins which is founded on the solubility in dilute salt solutions is one of the most convenient means of grouping a large part of the known proteins obtained from seeds. (pg. 18, The Vegetable Proteins, Thomas Osborne, Longmans, Green and Co.; Paternoster Row, London, New York Toronto, Bombay, Calcutta, and Madras) Industrially, crystallization is often used as a means of producing high purity product from impure solutions. Note that the proteins in the above recipe's are all crystallized from very impure solutions. Major problem with proteins is that any one is often a trace component in the solution - i.e., a difficulty in getting the solution concentration high enough to enable one to achieve supersaturation. Most proteins in the cookbook selected to be at high conc. in the starting material. Preliminary precipitation, followed by a crystallization of the concentrated redissolved precipitate, may work for lower concentration starting materials.   Size Number Morphology Crystal       Basic experiments to try with the crystals once obtained - dissolve and recrystallize - do they get bigger? - concentration effects on # and size of crystals grown - temperature effects - precipitant effects - pH effects -all on crystal size, #, morphology.   Concentration Effects pH Effects Temperature Effects Precipitant Effects Size         Number         Morphology         TOP DEFINITIONS Aqueous - Relating to, similar to, containing, or dissolved in water; made from by or with water Clarify - To make clear by removing impurities or solid matter. Concentration - The amount of a specified substance in a unit amount of another substance measure of the relative proportions of two or more quantities in a mixture (see COMPOUND). Concentrations may be expressed in a number of ways. The simplest is in terms of a component's percentage by weight or volume. Mixtures of solids or liquids are frequently specified by weight-percentage concentrations, whereas mixtures of gases are usually specified by volume percentages. Very low concentrations, such as those of various substances in the atmosphere, are expressed in parts per million (ppm). The molarity of a SOLUTION is the number of MOLES of solute per liter of solution. The molality of a solution is the number of moles of solute per 1,000 grams of solvent. The mole fraction of a solution is the ratio of moles of solute to the total number of moles in the solution.1 Denature - To change or alter the natural structure or qualities of (a protein), as with heat, alkali, or acid, so that some of its original properties, especially its biological activity, are diminished or eliminated. Dialysis - In chemistry, transfer of dissolved solids (solute) across a semipermeable membrane, which permits or hinders diffusion of molecules according to their size. Dialysis is frequently used to separate different components of a solution. For example, in artificial kidney machines hemodialysis is used to purify the blood of persons whose KIDNEYS have ceased to function. In the machine, blood is circulated on one side of a semipermeable membrane, while dialysis fluid&emdash;containing substances necessary to the body and closely matching the chemical composition of the blood&emdash;is circulated on the other side. Metabolic waste products, such as urea, diffuse through the membrane into the dialysis fluid and are discarded, while the diffusion of substances necessary to the body is prevented. In peritoneal hemodialysis, the dialysis fluid is introduced into the abdominal cavity. Waste products leach from the blood vessels into the fluid, which is later drained from the patient. Filtrate - Material, liquid, that has passed through a filter. Morphology - Shape of crystal; e.g., needlelike, hexagonal. Pellet - A small, solid or densely packed mass, as of food, wax, or medicine. Precipitate - A solid obtained by precipitation, i.e., the separation of a substance from a SUSPENSION, or SOLUTION. In a suspension, such as sand in water, the solid spontaneously precipitates (settles out) on standing. A solute may be precipitated by evaporation or by the addition of a compound that reacts with the solute to form an insoluble precipitate. In each case, the precipitate formed may settle out spontaneously or may be collected by filtration or centrifugation. Slurry - A thin mixture of a liquid, especially water, and any of several finely divided substances, such as cement, plaster of Paris, or clay particles. Solution - A homogeneous or uniform mixture of two or more substances, which may be solids, liquids, gases, or a combination of these. Supernatant - The clear fluid above a sediment or precipitate.