Protein precipitation, basic proteins

Relative extraction efficiencies of polar polymeric neutral, cation, and anion exchange sorbents (HLB, MCX, and MAX) for 11 beta antagonists and 6 beta agonists in human whole blood were probed.109 Initial characterization of MCX and MAX for acidic and basic load conditions, respectively, showed that both the agonists and antagonists were well retained on MCX, while they were recovered from MAX in the wash with either methanol or 2% ammonia in methanol (see Table 1.6). Blood samples were treated with ethanol containing 10% zinc sulfate to precipitate proteins and the supernatants loaded in 2% aqueous ammonium hydroxide onto the sorbents. After a 30% methanol and 2% aqueous ammonia wash, the analytes were eluted with methanol (HLB), 2% ammonia in methanol (MCX), or 2% formic acid in methanol (MAX). The best recoveries were observed with MCX under aqueous conditions or blood supernatant (after protein precipitation) spiked sample load conditions (see Table 1.7). Ion suppression studies by post-column infusion showed no suppression for propranolol and terbutaline with MCX, while HLB and MAX exhibited suppression (see Figure 1.6). 

Unlike clenbuterol, salbutamol is a difficult compound to analyze due to its particular chemical attributes. It is a basic compound subjected to protein binding poor recoveries are obtained especially when protein precipitation techniques are used to prepare the extracts (145). In addition, salbutamol is charged at all pH values and does not readily lend itself to simple, specific back-extracting procedures. This severely restricts the options of sample cleanup. However, a Subtilisin protease digestion step followed by acid clarification and solid-phase extraction has been suggested (146) as an adequate extraction and cleanup procedure prior to the end-point determination of salbutamol by an enzyme immunoassay (139) based on the cross-reactivity of anticlenbuterol antibodies. 

The uses of constant-current coulometry for the determination of drugs in biological fluids are few, basically due to sensitivity restriction. Monforte and Purdy [46] have reported an assay for two allylic barbituric acid derivatives, sodium seconal and sodium sandoptal, with electrogenerated bromine as the titrant and biamperometry for endpoint detection. Quantitative bromination required an excess of bromine hence back titration with standard arsenite was performed. The assay required the formation of a protein-free filtrate of serum with tungstic acid, extraction into chloroform, and sample cleanup by back extraction, followed by coulometric titration with electrogenerated bromine. The protein precipitation step resulted in losses of compound due to coprecipitation. The recoveries of sodium seconal and sodium sandoptal carried through the serum assay were approximately 81 and 88%, respectively. Samples in the concentration range 7.5-50 pg/mL serum were analyzed by this procedure. 

Covalent bonds between oxidized phenolics and proteins were shown to be more likely to form at high pH since phenolics are more readily oxidized at high pH. This was particularly true with basic proteins and less so with acidic proteins.124163164 Because precipitation of phlorotannin-protein complexes was independent of temperature, hydrophobic forces were not considered likely to be important in their formation.124 164 These studies suggest that, although phlorotannin structure is important, at least part of the variation in sensitivity to dietary tannins is due to variation in herbivore gut chemistry. 

Acidic dialysis provides tremendous purification from bacterial proteins, most of which precipitate at a pH lower than 6. However, not every recombinant protein remains soluble at acidic pH either. To test the solubility of your protein under these conditions, after at least 18-20 h of basic dialysis, transfer a small aliquot of the protein into a separate dialysis bag and put it in a precooled acidic dialysis buffer for 4-6 h, just long enough for bacterial proteins to form visible precipitation. Once the precipitate is formed, separate it by centrifugation in a table-top microcentrifuge for 5-10 min at 23,500 g. Analyze the presence of your protein in the supernatant and in the pellet by SDS-PAGE or Western blotting. If your protein is found in the supernatant, you can transfer the dialysis bag with the bulk of protein from basic dialysis conditions to acidic dialysis. 

Histones. Basic proteins, as defined by their high content of lysine and arginine. Soluble in water and precipitated by ammonia. 

Highly concentrated protein solutions, thin protein films, or precipitate suspensions in various solvents can be used for Fourier transform infrared spectroscopy (FTIR). Great care should be taken in interpreting deconvoluted spectra the basic data is still only one curve. Using IR difference spectra avoids the possible errors of deconvolution, but the protein environment during the measurement must be absolutely controlled to obtain meaningful data. 

The importance of zinc ions for stabilizing insulin preparations has been known since the first reported crystallization of insulin in the presenoe of zino ions in 1934 (36). Suspensions of zinc insulin were used at that time. Presently, all pharmaceutical preparations are either solutions of zino insulin or suspensions of insoluble forms of zino insulin. A longer-acting and more stable form of insulin is protamine zinc insulin, which is prepared by precipitating insulin in the presence of zinc ions and protamine, a basic protein. This precipitate is known to contain two zinc ions per insulin hexamer. A somewhat shorter-acting and more useful preparation is neutral protamine Hagedorn (NPH) insulin, which includes m-cresol... 

Several extraction techniques have been reported in the literature for the analysis of sulfonamides. Because of their polar nature, sulfonamides are readily extracted by organic solvents ° ° the most commonly used are acetonitrile.Other organic solvents used for analyte extraction and protein precipitation include dichloromethane, " acetone, ethanol, chloroform, and ethyl acetate, " which are often used either alone or in conjunction with one another. Other techniques used for protein precipitation include the use of acids such as perchloric or formic and the use of basic buffers such as potassium hydrogen phosphate and ammonium sulfate. In the case of honey, the use of acids such as trichloroacetic, " " hydrochloric, and phosphoric is necessary for hydrolysis, releasing carbohydrate-bound sulfonamide residues. Other extraction techniques reported in the literature include the use of pressurized liquid extractions, " matrix solid-phase dispersion, and magnetic molec-ularly imprinted polymers. Of additional note, several authors have observed that analyte recoveries were largely... 

Protamine sulfate is a strongly basic protein (white powder, see pK) used to precipitate nucleic acids from cmde protein extracts. It dissolves to the extent of 1.25% in H2O. It is freely soluble in hot H2O but separates as an oil on cooling. It has been purified by chromatography on an IRA-400 ion-exchange resin in the SO form and is washed with dilute H2SO4. Eluates are freeze-dried under high vacuum below 20°. This method is used to convert proteamine and protamine hydrochloride to the sulfate. [UV Rasmussen Hoppe Seyler s Z Physiol Chem 224 97 1934, Ando Sawada J Biochem (Tokyo) 49 252 1961, Felix Hashimoto Hoppe Seyler s Z Physiol Chem 330 205 1963.]... 

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