【正文】 inSolvent Interaction The solubility of a protein reflects a delicate balance between different energetic interactions, both internally within the protein and between the protein and the surrounding solvent. Consequently, the protein‘s solvent or thermal environment could affect both its solubility and structure. As we have seen, extreme changes can lead to denaturation. In this part, we will, for the most part, be concerned with conditions under which the native structure is maintained. Changes in protein solubility that do not destroy the molecule‘s strucutral integrity can occur in several ways. A. Minimum in solubility occurs at the isoelectric point Proteins typically have on their surfaces charged amino acid chains that undergo energetically favorable polar interactions with the surrounding water. The total charge on the protein is the sum of the sidechain charges. However, the actual charge on the weakly acidic and basic sidechain groups also depends on the solution pH. The decrease in solubility at the isoelectric pH reflects the fact that the individual protein molecules, which would all have a similar charge at pH values away from their isoelectric points, cease to repel each other. B. Salting in and salting out Proteins also show a variation in solubility that depends on the concentration of salts in the solution. These frequently plex effects may involve specific interactions between charged side chains and solution ion, or, particularly at high salt concentrations, may reflect more prehensive changes in the solvent properties. Figure and Saltingin effect: The effect of salts such as sodium chloride on increasing the solubility of globulins is often referred to as salting in. The salting in effect is related to the nonspecific effect the salt has on increasing the ionic strength of the solution. The higher the ionic strength, the smaller are the interactions between charged groups on the same or different proteins. Saltingout effect: The effect of salt such as ammonium sulfate on decreasing the solubility of proteins is refered to as salting out, which occurs with salts that effectively pete with the protein for available water molecules. In this case the protein molecules tend to associate with each other because at high salt concentrations, proteinprotein interactions bee energetically more favorable than proteinsolvent interactions. Each protein has a characteristic saltingout point, and we can exploit this fact to make protein separations in crude extracts. Several Methods are Available for Determina tion of Gross Size and Shape Several methods are available for determining the size and shape of protein molecules in solution. A. Sedimentation rates is a function of size and shape Information concerning the molecular weight of a protein can be obtained by observing its behavior in an intense centrifugal field. To get a qualitative understanding of how this method works, we must first recognize that protein molecules are generally slightly denser than water. However, the molecules in a protein solution seldom settle out in the earth‘s gravitational field(1?g) because they are constantly being stirred up by collisions with surrounding solvent molecules. Nevertheless, protein molecules in solution can be made to settle if they are subjected to very high centrifugal force field(~100000?g), such as can be attained in an ultracentrifuge. The protein molecules slowly migrate toward the bottom of the centrifuge tube at a rate that proportional to their molecular weight. B. Gelexclusive chromatography gives a measure of size Gelexclusive chromatography is used for size estimation as well as protein purification. This popular technique exploits the availability of both natural polysaccharide and synthetic polymers that can be formed into beads with varying pore sizes, depending on the extent of crosslinking between polymer chains. Figure and Electrophoretic Methods are the Best Way to Analyze Mixtures Electrophoresis is one of the most monly used techniques in biochemistry. Electrophoresis is very much like sedimentation, since in both cases a force gradient leads to protein transport in the direction of the force. In the case of sedimentation the force is gravity, so the rate of migration depends on the effective mass of the particle. In electrophoresis the force is the electrical potential, E, so the rate of migration depends on the charge on the molecule, rather than its mass. (Figure and ). (1) SDSPAGE (2) IEFSDSPAGE Methods for Protein Purification Before we can fully characterize a protein, we must purify it from a natural source. Once the decision has been made to purify a particular protein, several factors must be weighed. . how much material is needed? What level of purity is required? The starting material should be readily available and should contain the desired protein in relative abundance. If the protein is part of a large structure, such as nucleus, the mitochondria, or the ribosome, then it is advisable to isolate the large structure first from a crude cell extract. Purification must usually be performed in a series of steps, using different techniques at each step. Some purification techniques are more useful when handling large amounts of material, whereas others work best on small amounts. A purification procedure is arranged so that the techniques that are best for work with large amounts are during early steps in the overall purification. The suitability of each purification step is evaluated in terms of the amount of purification achieved by that step and the percent recovery of the desired protein. Combining t