Proteins denatured, removal

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. 

Ion exchange resins are also useful for demineralising biochemical preparations such as proteins. Removal of metal ions from protein solutions using polystyrene-based resins, however, may lead to protein denaturation. This difficulty may be avoided by using a weakly acidic cation exchanger such as Bio-Rex 70. 

After two fractionations between 0.33 and 0.6 saturated ammonium sulfate, several precipitations at 0.6 saturation were made throughout a period of two or three days, during which the preparation was stored in the refrigerator. Any denatured, insoluble proteins were removed by centrifugation, and a solution of the desired activity was prepared by dilution with buffer or distilled water. 

Whey protein concentrates (WPC) are produced by a variety of processing treatments to remove both lactose and minerals (20) as indicated in Figure 5. Even though it would be highly desireable to remove most of the lactose and minerals in these processes, it is not practical from an economic standpoint and thus most of these products only range in protein content from 35 to 50 %.The major objective of most of these processes is to produce a WPC with minimal protein denaturation in order to obtain a product with maximum protein solubility and functionality. However, from a practical consideration this objective is not readily obtainable, and thus most WPC products commercially available exhibit variable whey protein denaturation and functionality (20). 

Addition of CaCl2 to about 0.2 M causes aggregation of the casein such that it can be readily removed by low-speed centrifugation. If calcium is added at 90°C, the casein forms coarse aggregates which precipitate readily. This principle is used in the commercial production of some casein co-precipitates in which the whey proteins, denatured on heating milk at 90°C for 10 min, co-precipitate with the casein. Such products have a very high ash content. 

Throughout the purification process we must have a convenient means of assaying for the desired protein, so we can know the extent to which it is being enriched relative to the other proteins in the starting material. In addition, a major concern in protein purification is stability. Once the protein is removed from its normal habitat, it becomes susceptible to a variety of denaturation and degradation reactions. Specific inhibitors are sometimes added to minimize attack by proteases on the desired protein. During purification it is usual to carry out all operations at 5°C or below. This temperature control minimizes protease degradation problems and decreases the chances of denaturation. 

As mentioned earlier, proteins can be removed by ultrafiltration through a very fine membrane filter. Ultracentrifugation at high speeds can also be used to separate proteins from smaller molecules based on size differences. The most commonly used protein removal techniques for HPLC involve protein denaturation. Heating denatures most proteins. If the compounds to be separated are temperature resistant, the crude mixture remaining can be boiled and then filtered or centrifuged. Particulates and denatured protein are removed together. 

Purified COX-2 (0.79 nmol) is treated with 1.0 mol equivalent of inhibitor and the mixture is incubated for 60 min at room temperature. The remaining activity at this time is 4% that of a vehicle-treated control. The sample is then divided in two and the protein denaturated by treatment with four volumes of ethyl acetate/methanol/1 M citric acid (30 4 1). After extraction and centrifugation (10000 g for 5 min), the organic layer is removed and the extraction repeated. The two organic layers are combined and dried under N2. The extract is dissolved in 10 pi of HPLC solvent mixture consisting of water/acetonitrile/acetic acid (50 41 0.1) and 50 pi are injected onto a Novapak C-18 column (3.9 x 150 mm) and developed at 1 ml/min. The inhibitor is detected by absorption at 260 nm and eluted with a retention time of 6.6 min in this system. Control experiments for inhibitor recovery are performed with incubation of the inhibitor in the absence of enzyme and processing of the samples in an identical fashion before quantitation by HPLC. 

In a typical procedure the drag to be investigated is spiked to serum or plasma in a concentration of 25 pM and subsequently incubated at 37 °C over a time of up to several hours (typically 5 min to 1 h). After addition of acetonitrile, denatured proteins are removed and the supernatant is analyzed appropriately. 

A trial was made to take a look at the valence of iron in adrenodoxin (29) using 3 moles of p-chloromercuribenzoate (PCMB) per gram atom of iron (less than saturated level of PCMB), all of the iron could be extracted by 5% trichloroacetic acid solution as ferric iron, which produces a ferrous-o-phenanthroline complex only by the addition of ascorbate as reductant. In the absence of the mercurial, some 50% or more of the iron atoms in the protein can be removed in the ferrous state. This result indicates that the acid extraction causes intramolecular reduction of the protein-bound iron. As shown in Fig. 10, 5.7 M urea as a protein denaturant can slowly bleach the visible absorption under aerobic conditions. About 10% of the residual absorption remains at 414 mp after the reaction is completed. In the presence of both urea and o-phenanthroline, all of the iron present in adrenodoxin reacts with o-phenanthroline to produce the ferrous complex under aerobic conditions. Similar experiments under anaerobic conditions reveal that the... 

Prepare the standard and unknown protein samples for analysis by mixing 10 p of each with 5 pi of 4X sample buffer in microcentrifuge tubes, mixing gently, and heating at 100°C for 5 min in a boiling water bath. This step is to ensure that all of the proteins in the sample are completely denatured. Remove the samples from the water bath and allow them to cool to room temperature. 

The reaction mixture contained 80 /xL of 130 mM Hepes-67 mM Tris buffer (pH 7.4) 10 ju,L each (to give final concentration of 1 mM) of NAD, thiamine pyrophosphate, coenzyme A, MgCl2, and dithiothreitol 20 /xL of tissue extract or enzyme source, and 30 /xL of bovine serum albumin (1 mg). The reaction was started by adding 20 fiL of a-ketoglutarate to give a final concentration of 10 mM After incubation at 30°C for 1, 5, or 20 minutes for purified enzyme from bovine heart, brain, or liver mitochondria, or platelet homogenates, the reaction was stopped by addition of 20 /xL of 60% perchloric acid and the denatured protein was removed by centrifugation. A 10 /xL aliquot was used for HPLC analysis. 

The reaction mixture consisted of 0.9 mL mitochondrial suspension (0.05-4 mg protein) and 31.5 /xL 30 mM L-dihydroorotic acid. Reactions were terminated after 5 to 60 minutes by addition of 180 /xL 16% trichloroacetic acid and chilling on ice for 20 minutes. Denatured proteins were removed by centrifugation, and the supernates neutralized with alanine-Freon before analysis by HPLC. The assay was linear with protein in the range of 0.05 to 2 mg protein/mL, and with time up to at least 60 minutes. 

The reaction mixture contained 50 /xM guanine, 50 fiM hypoxanthine, 100 /xM PRibPP, and 1 mM MgCh in potassium phosphate (pH 7.4). The reaction was initiated by the addition of the HGPRTase activity. An intervals the reaction was terminated by heating in a boiling water bath for 1 minute. Denatured protein was removed by centrifugation, and the sample was purified... 

It has been shown that by treatment with ammonium sulfate at acid pH, flavin can be partially removed from the enzyme (318). Addition of FAD, but not FMN, reactivated the enzyme. Zinc is not removed under these conditions, and its addition is not required for reactivation. The metal appears to be very tightly bound to the enzyme (312) its removal without protein denaturation has not been achieved. 

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