Protein denatured, retention

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. 

Solubility is a critical functional characteristic because many functional properties depend on the capacity of proteins to go into solution initially, e.g. gelation, emulsification, foam formation. Data on solubility of a protein under a variety of environmental conditions (pH, ionic strength, temperature) are useful diagnostically in providing information on prior treatment of a protein (i.e. if denaturation has occurred) and as indices of the potential applications of the protein, e.g. a protein with poor solubility is of little use in foams). Determination of solubility is the first test in evaluation of the potential functional properties of proteins and retention of solubility is a useful criterion when selecting methods for isolating and refining protein preparations (1). Several researchers have reported on the solubility of extracted microbial proteins (69,82,83,84). In many instances yeast proteins demonstrate very inferior solubility properties below pH 7.5 because of denaturation. 

Because the influence of drying parameters is not the same for all materials, optimal drying conditions vary depending on the final objective volatile retention, preservation of enzymatic activity and avoidance of protein denaturation, fat oxidation or crystallisation. Furthermore, some interactive influences may appear between components, an effect that is positive for protection of labile compounds by a network of polymers as polysaccharides, gums, proteins (Dumoulin and Bimbenet 1998). The phenomena may vary between centre and surface of drops, with some possible segregation by internal movement in the drop. 

Equation (59) allows us to pieJict the effect of protein denaturation on retention (and separation) in SEC Equally important, this relationship... 

The methacrylic backbone structure makes the spherical Toyopearl particles rigid, which in turn allows linear pressure flow curves up to nearly 120 psi (<10 bar), as seen in Fig. 4.45. Toyopearl HW resins are highly resistant to chemical and microbial attack and are stable over a wide pH range (pH 2-12 for operation, and from pH 1 to 13 for routine cleaning and sanitization). Toyopearl HW resins are compatible with solvents such as methanol, ethanol, acetone, isopropanol, -propanol, and chloroform. Toyopearl HW media have been used with harsh denaturants such as guanidine chloride, sodium dodecyl sulfate, and urea with no loss of efficiency or resolution (40). Studies in which Toyopearl HW media were exposed to 50% trifluoroacetic acid at 40°C for 4 weeks revealed no change in the retention of various proteins. Similarly, the repeated exposure of Toyopearl HW-55S to 0.1 N NaOH did not change retention times or efficiencies for marker compounds (41). 

Its solubility characteristics in aqueous systems are such that retention of toxicity to insects by dissolved crystal protein is always suspect, and loss of activity on dissolution owing to denaturation is often observed. The protein is soluble only in relatively strong aqueous alkali. Thus, it has been variously reported to be soluble in 0.01N- to 0.05N sodium hydroxide (1) and alkali at pH 10.5 in the presence of thioglycollate (35) we have also observed its solubility in alkali at pH 9.5 in the presence of urea and potassium boro hydride. One difference between the characteristic proteins produced by various strains of crystalliferous bacilli is observed in the degree of alka-... 

Protein applications are extremely sensitive to solvent pH, salt concentration, and small molecular weight additives such as trifluoroacetic acid (TFA), which affect solute equilibria. These effects are known and depending on the specific application, proteins are often run under denaturing conditions, which offer vastly different retention conditions than nondenaturing conditions. 

Hydrophobic interactions are very sensitive to temperature. Retention of most proteins increases with temperature, but for some the opposite is true, and the magnitude of response is highly individual in any case.34-36 If you elevate temperature sufficiently (56°C and above) you may begin to denature proteins in the sample. This may expose more hydrophobic sites and alter selectivity to a greater degree. Whether or not you exploit temperature as a selectivity factor, good temperature control is essential for assay reproducibility. 

The packing material first described for direct injection of biological samples was prepared by simply saturating the accessible adsorption sites of a Cis reversed-phase silica with human plasma proteins (105). After saturation, the human plasma proteins were denatured at the external surface, and their native conformation was destroyed. With this treatment, the proteins formed a hydrophilic layer with weak ion-exchange properties, which provided protection from contact with the sample proteins, whereas the alkyl ligands inside the pores remained unchanged and thus served for analyte retention. The retention behavior of the saturated phase did not alter with this treatment, but the efficiency was reduced dramatically. Such protein-coated columns have shown a lifetime of several months (106). 

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