This technique is a modification of electrophoresis, in which proteins are separated by on a gel matrix which has a pH gradient across it. Proteins migrate to the location where the pH equals their isoelectric point and then stop moving because they are no longer charged. This methods has one of the highest resolutions of all techniques used to separate proteins. Gels are available that cover a narrow pH range (2-3 units) or a broad pH range (3-10 units) and one should therefore select a gel which is most suitable for the proteins being separated.
Electrophoresis is often used to determine the protein composition of food products. The protein is extracted from the food into solution, which is then separated using electrophoresis. SDS-PAGE is used to determine the molecular weight of a protein by measuring m, and then comparing it with a calibration curve produced using proteins of known molecular weight: a plot of log (molecular weight) against relative mobility is usually linear. Denaturing electrophoresis is more useful for determining molecular weights than non-denaturing electrophoresis, because the friction to movement does not depend on the shape or original charge of the protein molecules.
In electrophoresis proteins are separated primarily on their molecular weight. Proteins are denatured prior to analysis by mixing them with mercaptoethanol, which breaks down disulfide bonds, and (SDS, which is an anionic surfactant that hydrophobically binds to protein molecules and causes them to unfold because of the repulsion between negatively charged surfactant head-groups. Each protein molecule binds approximately the same amount of SDS per unit length. Hence, the charge per unit length and the molecular conformation is approximately similar for all proteins. As proteins travel through a gel network they are primarily separated on the basis of their molecular weight because their movement depends on the size of the protein molecule relative to the size of the pores in the gel: smaller proteins moving more rapidly through the matrix than larger molecules. This type of electrophoresis is commonly called or
In a small study (six subjects with type 1 diabetes), Gray et al.35 reported on the rate of restoration of euglycemia after treatment of hypoglycemia with either identical amounts of carbohydrate (15 g) or carbohydrate supplemented with protein (14 g) and the subsequent development of hypoglycemia.
To determine how far proteins have moved a is added to the protein solution, bromophenol blue. This dye is a small charged molecule that migrates ahead of the proteins. After the electrophoresis is completed the proteins are made visible by treating the gel with a protein dye such as Coomassie Brilliant Blue or silver stain. The relative mobility of each protein band is calculated:
The majority of protein is digested, and the amino acids not used for gut fuel are metabolized in the intestinal mucosal cells and transported by the portal vein to the liver for protein synthesis or gluconeogenesis.12 In the liver, nonessential amino acids are largely deaminated, and the amino group (nitrogen) removed is converted into urea for excretion in the urine.13 It has been shown that in subjects without and with mild type 2 diabetes, ~5070% of a 50-g protein meal is accounted for over an 8-hour period by deamination in the liver and intestine and synthesis to urea.14 It has been assumed that the remaining carbon skeletons from the nonessential amino acids are available for glucose synthesis, which would then enter into the general circulation.
The (pI) of a protein is the pH where the net charge on the protein is zero. Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion keeping them apart. Proteins have different isoelectric points because of their different amino acid sequences (relative numbers of anionic and cationic groups), and thus they can be separated by adjusting the pH of a solution. When the pH is adjusted to the pI of a particular protein it precipitates leaving the other proteins in solution.
With insulin deficiency, the oxidation of branched chain amino acids in muscle and uptake of alanine (the principle glycogenic amino acid) by the liver is accelerated, resulting in increased gluconeogenesis and augmented protein catabolism.18 The accompanying rise in glucose levels is most likely due to an increased conversion of ingested protein into glucose and to a decreased glucose removal rate.
The solubility of a protein depends on the of the solution that surrounds it because this alters the magnitude of the electrostatic interactions between charged groups. As the dielectric constant of a solution decreases the magnitude of the electrostatic interactions between charged species increases. This tends to decrease the solubility of proteins in solution because they are less ionized, and therefore the electrostatic repulsion between them is not sufficient to prevent them from aggregating. The dielectric constant of aqueous solutions can be lowered by adding water-soluble organic solvents, such as ethanol or acetone. The amount of organic solvent required to cause precipitation depends on the protein and therefore proteins can be separated on this basis. The optimum quantity of organic solvent required to precipitate a protein varies from about 5 to 60%. Solvent fractionation is usually performed at 0oC or below to prevent protein denaturation caused by temperature increases that occur when organic solvents are mixed with water.
In subjects with diabetes who had insulin withheld for 24 hours, there was a three- to fourfold increase in liver glucose output after protein ingestion.19 However, in the presence of insulin, alanine uptake by the liver is virtually zero,20 and hepatic glucose production falls by 85%.21 Indirectly then, insulin could reduce gluconeogenesis in the liver by decreasing the amino acid substrate supply.
Proteins are precipitated from aqueous solutions when the salt concentration exceeds a critical level, which is known as , because all the water is "bound" to the salts, and is therefore not available to hydrate the proteins. Ammonium sulfate [(NH4)2SO4] is commonly used because it has a high water-solubility, although other neutral salts may also be used, NaCl or KCl. Generally a two-step procedure is used to maximize the separation efficiency. In the first step, the salt is added at a concentration just below that necessary to precipitate out the protein of interest. The solution is then centrifuged to remove any proteins that are less soluble than the protein of interest. The salt concentration is then increased to a point just above that required to cause precipitation of the protein. This precipitates out the protein of interest (which can be separated by centrifugation), but leaves more soluble proteins in solution. The main problem with this method is that large concentrations of salt contaminate the solution, which must be removed before the protein can be resolubilzed, by dialysis or ultrafiltration.
Many proteins are denatured and precipitate from solution when heated above a certain temperature or by adjusting a solution to highly acid or basic pHs. Proteins that are stable at high temperature or at extremes of pH are most easily separated by this technique because contaminating proteins can be precipitated while the protein of interest remains in solution.