Proteins removal

It is important to notice that the united-atom simplification cannot be applied to functional hydrogens which are involved in the formation of a hydrogen hond or a salt bridge. This would destroy interactions important for the structural integrity of the protein. Removing the hydrogen at the u-carbon of the peptide backbone is also dangerous, because it prevents racemization of the amino acid. 

The normal paraffins produced are raw materials for the manufacture of biodegradable detergents, plasticizers, alcohols, and synthetic proteins. Removal of the / -paraffins upgrades gasoline by improving the octane rating. 

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. 

This precipitation process can be carried out rather cleverly on the surface of a reverse phase. If the protein solution is brought into contact with a reversed phase, and the protein has dispersive groups that allow dispersive interactions with the bonded phase, a layer of protein will be adsorbed onto the surface. This is similar to the adsorption of a long chain alcohol on the surface of a reverse phase according to the Langmuir Adsorption Isotherm which has been discussed in an earlier chapter. Now the surface will be covered by a relatively small amount of protein. If, however, the salt concentration is now increased, then the protein already on the surface acts as deposition or seeding sites for the rest of the protein. Removal of the reverse phase will separate the protein from the bulk matrix and the original protein can be recovered from the reverse phase by a separate procedure. 

From animal tissue, especially bovine lung and liver (e. g. autolysis of comminuted tissue parts, heating with ammonium sulfate in alkaline solution, filtration and acidification yield heparin as complex with protein, removal of fat with alcohol and treatment with trypsine for the purpose of decomposition of proteins, precipitation with alcohol and various purification methods). 

Polymer Labs. PLRP-S Gradient A 0.001 M oxalic acid, 0.5% formic acid + 3% THF in water, B = THF Positive ESF MS/MS OTC, CTC, TC, DC, and its 4-epimer in pig tissues Extraction with sodium succinate solution, protein removal with TCA, SPE cleanup on polymeric RP column DL = 0.5-4.2 ng/g [57]... 

The most efficient precipitants for protein removal were zinc sulfate, acetonitrile, and trichloroacetic acid (at 2 1 volume of precipitant to plasma, the protein removal values were, respectively, 96, 92, and 91% with <1% RSD for n = 5). 

Sloan JL, Grubb BR, Mager S (2003) Expression of the amino acid transporter ATB 0 + in lung possible role in luminal protein removal. Am J Physiol... 

This enzyme (RNase A) is a single chain protein of 124 amino acid residues, cross-linked by four intrachain disulfide bonds. Limited proteolysis of the enzyme cuts a single peptide bond between residues 20 and 21 (Richards and Vithayathil, 1959). The derived protein, RNase S, retains enzymic activity although the N-terminal peptide of 20 amino acids (S-peptide) is no longer covalently attached to the balance of the molecule (S-protein). Removal of S-peptide from... 

Protein removed by filtration using Centricon-10 membrane. 

Polishing. This last process step prepares the product for final formulation or for actual sale. It is designed to remove any aggregated protein, remove residual chromatographic eluent(s), and place the product into a specific solvent. These requirements are admirably served by gel filtration. At this point, the sample volume is small and the product fraction to be applied is fairly clean. The gel and column equipment requirements are now within reason and, the clean samples result in much longer gel life. 

Protons that could logically be involved in a membrane Bohr effect are those present on imidazole rings coordinated to Fe or Cu in redox proteins. Removal of an electron from the metal ion could be accompanied by displacement of electrons within the imidazole, within a peptide group that is hydrogen-bonded to an imidazole, or within some other acidic group. A hypothetical example is illustrated in Eq. 18-12 in which a carboxyl group loses a proton when "handed" a second. If the transiently enolized peptide linkage formed in... 

Carboxypeptidase action at 25° on S-protein removes Val 124 very rapidly with no effect on the RNA activity regenerated with added S-peptide 90). Further digestion removed Ser 123 with an activity drop to 45%, but the peptide-protein binding constant changed very little. More... 

The effect on chromatography is to complicate the separation greatly. If we consider a reverse-phase separation, the first thing we notice is an almost irreversible binding of protein to the column. Even after protein removal, we find polar peaks, which overload the early part of the chromatogram and tail into the compounds of interest. The components that are more nonpolar than our compounds of interest adhere to the column and must be washed off before the next injection. To ensure polar elution before our target compounds and nonpolar removal afterwards, we are almost forced to run solvent gradients. 

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. 

As a comparison, the methods of high-abundance protein removal (specific depletion or immunodepletion) to suppress the signal obscuration due to very concentrated species subtract also associated proteins. However it engenders a very important problem related to the dilution effect of depletion the rare species that would need to be concentrated to be detected are here diluted, thus aggravating the difficulty for their detection. 

Prior to metabolomic analysis, sample treatment is typically needed, as CSF contains approximately 0.3 mg/mL protein (114) that may hinder metabolite analysis. Consequently, CSF sample treatment is essentially directed to protein removal by means of organic solvent addition (84,88) or by ultrafiltration (85,89,90). The final metabolic extract composition will depend in a great extent on the sample treatment (115), and it will be selected mostly regarding the metabolomic approach and the analytical technique that will be afterward applied. 

Poison C et al (2003) Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 785 263-275... 

A final example of totally automated HPLC (although it isn t for pesticides) will demonstrate how many different unit operations can be done in a single system to take the tedium out of repetitive analyses. The drug analyzer depicted in Figure 14 was designed to determine therapeutic levels of theophylline in human serum (8). The sampler (in the center) aspirates 50 uL of serum into the analytical cartridge, then to the EDM, and finally to the LC module. The following series of operations takes place at the rate of 20 samples per hour without operator intervention unmeasured, untreated sample is aspirated, diluted with buffer, and mixed with an internal standard the system then precipitates the protein, removes the particulates, extracts the analyte (and internal standard) into... 

Often coconut milk or coconut water is supplied. The latter is obtained as the whey when coconut milk is autoclaved and the precipitated protein removed by filtration. Apart from poorly defined nutrients, coconut milk provides the growth factor, ldnetin, but it is preferable to supply this factor separately and avoid the use of the coconut milk, which varies greatly from batch to batch. 

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