Salting in, salting out, and dialysis of proteins

Transcription

1 Physical Biochemistry Lab Experiment no. 5: Salting in, salting out, and dialysis of proteins Objective: To learn the techniques of isolation of proteins on basis of their solubility in water. Introduction and principle: Proteins are the most abundant biological macromolecules occurring in all cells and all parts of cells. Our understanding of protein structure and function has been derived from the study of many individual proteins. To study a protein in detail, the researcher must be able to separate it from other proteins and must have the techniques to determine its properties. The necessary method comes from protein chemistry, a discipline as old as biochemistry itself and one that retains a central position in biochemical research. A pure preparation is essential before a protein's properties and activities can be determined. Given that cells contain thousands of different kinds of proteins, how can one protein be purified? Methods for separating proteins take advantage of properties that vary from one protein to the next. The source of a protein is generally tissues or microbial cells. The first step in any protein purification procedure is to break open these cells, releasing their proteins into a solution called a crude extract. Once the extract is ready, various methods are available for purifying one or more of the protein it contains. Commonly, the extract is subjected to treatments that separate the proteins into different fractions based on a property such as size or charge, a process refered to as fractionation. Early fractionation steps in purification utilize differences in protein solubility. Because a protein contains multiple charged groups, its solubility depends on the concentrations of dissolved salts, the polarity of the solvent, the ph, and the temperature. Some or all of these variables can be manipulated to selectively precipitate certain proteins while others remain soluble. The solubility of a protein at low ion concentrations increases as salt is added, a phenomenon called "salting in". The additional ions shield the protein's multiple ionic charges, thereby weakening the attractive forces between individual protein molecules (such forces can lead to aggregation and precipitation). However, as more salt is added, particularly with sulfate salts, the solubility of protein again decreases. This "salting out" effect is primarily a result of the competition between the added salt ions and the other dissolved solutes (protein molecules) for molecules of solvent (water). At very high salt concentrations, so many of the added ions are solvated that there is significantly less bulk solvent available to dissolve other substances, including proteins. Page 1 of 5

2 Since proteins precipitate at different salt concentrations, salting out is the basis of one of most commonly used protein purification procedures. Adjusting the salt concentration in a solution containing a mixture of proteins to just below the precipitation point of the protein to be purified eliminates many unwanted proteins from the solution. Then, after removing the precipitated proteins by filtration or centrifugation, the salt concentration of the remaining solution is increased to precipitate the desired protein. The precipitation of desired protein is then dissolved in water to make a solution of this protein. This procedure results in a significant purification and concentration of large quantities of protein. Ammonium sulfate, (NH 4 ) 2 SO 4, is the most commonly used salt for salting out proteins because its large solubility in water, its relative freedom from temperature effects, and it has no harmful effects on most of the proteins. The most effective ph region for salting out of the desired protein is at its isoelectric point because the protein is least soluble when its net charge is zero. Effect of salt concentration on protein solubility Page 2 of 5

3 A solution containing the protein of interest often must be further altered before subsequent purification steps are possible. Dialysis is one of the common operations in biochemistry to separate dissolved molecules by passing through a semi-permeable membrane according to their molecular dimensions. Semi-permeable membrane is containing pores of less than macromolecular dimensions. These pores allow small molecules, such as those of solvents, salts, and small metabolites, to diffuse across the membrane but block the passage of larger molecules. Cellophane (cellulose acetate) is the most commonly used dialysis material although many other substances such as nitrocellulose and collodion are similarity employed. So, dialysis is a method in which an aqueous solution containing both macromolecules and very small molecules which are placed in a dialysis bag which is in tern placed in a large container of a given buffer or distilled water. Thus small solute molecules freely pass through the membrane, and after several hours of stirring the equilibrium will reach (the concentration inside and outside the bag are the same). Thus, at equilibrium the concentration of small molecules outside and inside the bag is the same while the macromolecules remain inside the bag. During dialysis the external fluid should be changed in order to reach the required composition inside the dialysis bag. There are three factors that affecting the rate of dialysis: the first is the concentration differences of that molecules between the internal and external solution (which is the driving force for the movement of the molecules). The second is mixing on both sides of dialysis membrane will increase the rate of movement prevent the small particles on the side of low concentration. The third is dialyzable particles size versus pore size of the membrane, substances that are very much smaller than the pore size will reach equilibrium faster than substances that are only slightly smaller than the pores. The main point to be noted is that there is a rapid initial drop in dialysis process followed by a slow approach to equilibrium. In this experiment myoglobin will isolated from skeletal muscle by salting out technique which discards up to 75% of the crude proteins in the protein purification process. The isoelectric point of the myoglobin is 7 and it is precipitate at salt (ammonium sulfate) concentration of 55%. The ammonium sulfate used in salting out procedure will removed by dialysis "desalting". Page 3 of 5

4 Materials and apparatus: 1. Skeletal muscle 2. Distilled water 3. Ammonium hydroxide solution (2M) 4. Ammonium sulfate salt (solid) 5. Balance 6. Waring blender 7. Centrifuge tubes 8. Pasteur pipette 9. Centrifuge 10. ph meter 11. Measuring cylinder (10 ml) 12. Beaker (50 ml) 13. Dialysis bag 14. Dialysis tank Method: 1) Cut skeletal muscle (100g) into small pieces and homogenize for 10 minutes in 100 ml of distilled water at room temperature in a waring blender. 2) Divide the homogenate in 5 equal parts in centrifuge tube. 3) Allow the homogenate to stand for 20 min. 4) Centrifuge at 2000 rpm for 10 min. at 4 C. 5) Discard the residue and adjust the ph of the supernatant to 7 by the addition of (NH 4 ) 2 SO 4 (2M). 6) Measure the volume of the sample (supernatant). Page 4 of 5

5 7) Calculate the required of ammonium sulfate salt needed to saturate the solution 35% using ammonium sulfate nomogram. 8) Add the required salt to the solution slowly with small quantities and mix well continuously after each addition. 9) After the addition is completed and the salt is completely dissolved, centrifuge at 3500 rpm for 10 min. 10) Discard the pellet and measure the volume of the supernatant. 11) Calculate the required salt in gram needed to saturate the solution 55% using ammonium sulfate nomogram. 12) Add the required salt to the solution slowly with small quantities and mix well continuously after each addition. 13) Centrifuge for 10 min. at 3500 rpm. 14) Discard the supernatant and dissolve the pellet in 10 ml dis. water and place it in dialysis bag overnight. Questions: 1) What do you understand by the terms: a. Salting in b. Salting out c. Dialysis 2) Why ammonium sulfate is commonly used for "salting out" process? * Figure 5-5 obtained from Fundamentals of Biochemistry life at the molecular level by Voet & Voet * Figure 5-14 obtained from Biochemistry by Voet & Voet Page 5 of 5