Denaturing precipitation

Chloroform/methanol precipitation Wessel and Flugge (1984) dilute watery protein solutions (volume of 10 to 150 pi) in Eppendorf tubes with methanol and precipitate the proteins with chloroform. Addition of water separates the water/methanol/chloroform solution into two phases. The precipitated proteins collect in the interphase. Test volumes of 0.2 to 2 ml can also be processed with Corex glass tubes. 

Advantages In spite of its complexity, the method works reliably also for low protein amounts (20 ng) and in the presence of detergents or high salt concentrations. The rest of the chloroform/methanol/water mixture can be removed easily and quickly on the speed-vac (see Section 1.5.3). This (largely invisible) pellet contains the dry protein, largely free of residues from precipitation agent or buffer components. 

Problems When you take off the upper (watery) phase, the protein easily goes down the drain. Chloroform is a liver poison (vent hood ). 

Alternatives to Wessel and Flugge (1984) are precipitation with 10% trichloroacetic acid in the presence of yeast RNA as a carrier following Polachek and Cabib (1981) or acetone precipitation, as follows. The watery sample is mixed with four parts acetone and cooled for 1 h to -20° C. Then the precipitated proteins are extracted by centrifugation. Finally, proteins in solutions of higher concentration ( 0.1 mg/ml) can also be precipitated with perchloric acid, trichloroacetic acid, or by applying heat. Rests of trichloroacetic acid in the pellet are removed by repeated rinsing with ice-cold 80% acetone. 

Polachek, I., and Cabib, E. (1981). A Simple Procedure for Protein Determination by the Lowry Method in Dilute Solutions and in the Presence of Interfering Substances, Anal. Biochem. 117 311-314. 

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Physical changes include denaturation, precipitation, adsorption, and aggregation (with like molecules or with excipients).4... 

Because the polymer used in this experiment is a protein (albumin), precipitation denatures the polymer causing permanent changes in its secondary, tertiary, and quaternary structure. Therefore the precipitated polymer cannot redissolve. The denaturing/precipitation occurs because the three-dimensional shape of albumin is sensitive to temperature and to the nature of its environment (stable in water but not in other solvents). 

The action of detergents on proteins depends on the conditions of study and may result in denaturation, precipitation, complex formation, or even catalyzed hydrolysis at acid pH. It is frequently difficult to differentiate some of these phenomena even in systems containing purified proteins. The action of detergents on biological systems is accordingly more complex, and explanation of phenomena such as the antibacterial potency of detergents must await elucidation of their interaction with components of the bacterial cell and surface, notably the proteins. 

Soybean Protein Isolates. Soybean protein isolates, having a protein content of >90 wt%, are the only vegetable proteins that are widely used in imitation dairy products (1). Most isolates are derived from isoelectric precipitation, so that the soybean protein isolates have properties that are similar to those of casein. They are insoluble at thek isoelectric point, have a relatively high proportion of hydrophobic amino acid residues, and are calcium-sensitive. They differ from casein in that they are heat-denaturable and thus heat-labile. The proteins have relatively good nutritional properties and have been increasingly used as a principal source of protein. A main deterrent to use has been the beany flavor associated with the product. Use is expected to increase in part because of lower cost as compared to caseinates. There has been much research to develop improved soybean protein isolates. 

Purification. Enzyme purity, expressed in terms of the percent active enzyme protein of total protein, is primarily achieved by the strain selection and fermentation method. In some cases, however, removal of nonactive protein by purification is necessary. The key purification method is selective precipitation of the product or impurities by addition of salt, eg, sodium sulfate, or solvent, eg, ethanol or acetone by heat denaturation or by isoelectric precipitation, ie, pH adjustments. Methods have been introduced to produce crystalline enzyme preparations (24). 

Formulations. Any formulation is a compromise between the previously mentioned requirements. For example, the fermentation broth may contain enzyme-stabilizing substances, but the appHcation of the enzyme or precipitation problems in the formulation may demand a high degree of purification that eliminates the stabilizers. Alternatively, the pH necessary for good microbial or physical stabiHty may differ from the pH that gives optimum enzyme stabiHty, or a preservative that is effective at the optimum pH for enzyme stabiHty may have a denaturing effect on the enzyme. 

Product recoveiy from reversed micellar solutions can often be attained by simple back extrac tion, by contacting with an aqueous solution having salt concentration and pH that disfavors protein solu-bihzation, but this is not always a reliable method. Addition of cosolvents such as ethyl acetate or alcohols can lead to a disruption of the micelles and expulsion of the protein species, but this may also lead to protein denaturation. These additives must be removed by distillation, for example, to enable reconstitution of the micellar phase. Temperature increases can similarly lead to product release as a concentrated aqueous solution. Removal of the water from the reversed micelles by molecular sieves or sihca gel has also been found to cause a precipitation of the protein from the organic phase. 

The crude ketal from the Birch reduction is dissolved in a mixture of 700 ml ethyl acetate, 1260 ml absolute ethanol and 31.5 ml water. To this solution is added 198 ml of 0.01 Mp-toluenesulfonic acid in absolute ethanol. (Methanol cannot be substituted for the ethanol nor can denatured ethanol containing methanol be used. In the presence of methanol, the diethyl ketal forms the mixed methyl ethyl ketal at C-17 and this mixed ketal hydrolyzes at a much slower rate than does the diethyl ketal.) The mixture is stirred at room temperature under nitrogen for 10 min and 56 ml of 10% potassium bicarbonate solution is added to neutralize the toluenesulfonic acid. The organic solvents are removed in a rotary vacuum evaporator and water is added as the organic solvents distill. When all of the organic solvents have been distilled, the granular precipitate of 1,4-dihydroestrone 3- methyl ether is collected on a filter and washed well with cold water. The solid is sucked dry and is dissolved in 800 ml of methyl ethyl ketone. To this solution is added 1600 ml of 1 1 methanol-water mixture and the resulting mixture is cooled in an ice bath for 1 hr. The solid is collected, rinsed with cold methanol-water (1 1), air-dried, and finally dried in a vacuum oven at 60° yield, 71.5 g (81 % based on estrone methyl ether actually carried into the Birch reduction as the ketal) mp 139-141°, reported mp 141-141.5°. The material has an enol ether assay of 99%, a residual aromatics content of 0.6% and a 19-norandrost-5(10)-ene-3,17-dione content of 0.5% (from hydrolysis of the 3-enol ether). It contains less than 0.1 % of 17-ol and only a trace of ketal formed by addition of ethanol to the 3-enol ether. 

Water soluble protein with a relative molecular mass of ca. 32600, which particularly contains copper and zinc bound like chelate (ca. 4 gram atoms) and has superoxide-dismutase-activity. It is isolated from bovine liver or from hemolyzed, plasma free erythrocytes obtained from bovine blood. Purification by manyfold fractionated precipitation and solvolyse methods and definitive separation of the residual foreign proteins by denaturizing heating of the orgotein concentrate in buffer solution to ca. 65-70 C and gel filtration and/or dialysis. 

The shaking of protein solutions may lead to aggregation and precipitation as a result of several mechanisms, such as air oxidation, denaturation at the interface, adsorption to the vessel, or mechanical stress. These possibilities were systematically examined for solutions of human fibroblast interferon [50]. In this example, mechanical stress was identified as the causative factor in the inactivation. The proposed mechanism of inactivation by mechanical stress was through orientation of the asymmetrical protein in the... 

Salting-out salts (Na2S04, NaCl, MgS04) Surface tension increase Weak binding Stabilize globular proteins and precipitants of native and denatured proteins... 

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