Precipitation points, determining protein

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 current method for the hyalurcnidase assay described in the United States Pharmacopeia (USP) [132] is based on the inability of hydrolyzed potassium hyalurooate to form a complex precipitate with proteins from added serum, reflected in a decreased turbidity of the reaction mixture (measured after 30 min). The method is, from the enzymological point of view, not well defined since it does not actually evaluate the kinetics of the hydrolysis of the substrate. An assay with end-point determination is only valid if the reaction rate does not change during this reaction time. We found that only with the two lowest test concentration (0=15 nnH 0=3 PJ) was this condition fulfilled, while with the three higher test concentrations the reaction is not linear. Commercially available hy aluronates can be contaminated with chondroitln sulfates. They are more acidic than... 

For a given protein, the distribution curves for different values of Co are all determined by the same differential curve. Co merely determines the point of entry to the curve. Taking the case of two solutions of carboxy-myoglobin, one containing 30 gm per liter and the other being a tenfold dilution of this, we read off from Fig. 5 the respective salt concentrations for first precipitation (points A and B) and transfer them to Fig. 6. The two distribution curves of the precipitation will now be portions A to C and B to C of the differential curve in Fig. 6 for the stronger and weaker solutions respectively. The two peaks represented by the areas AA C and BB C are of course of different sizes because of the different amounts of protein taken, but if they are normalized by expressing the amounts as... 

It is to be noted that the point of zero net proton charge can be determined only for proteins which can be deionized without precipitation or other change. The point would also have little significance (and probably could not in any event be determined unequivocally) if the pH lies in a region where the titration curve is not behaving reversibly. 

The simplest technique used to grow protein crystals is the batch method in which the protein is mixed with salts or other precipitants to achieve supersaturation (Fig. 2), and the vessel is sealed and set aside until crystals appear. Frequently, the supersaturation point required to induce nucleation is empirically determined by observing the onset of transient turbidity as powdered salt is progressively added to the solution. Crystals of hen egg white lysozyme used for most systematic studies of protein crystallization are grown by batch methods (Blundell and Johnson, 1976). Mouse pancreatic ribonuclease (Perry and Palmer, 1988) and the biotin operon repressor (Brennan et al., 1989) represent recent examples of use of the batch method. 

For preparative purposes, it is perhaps more convenient to start from pancreas acetone powder (pancreatin). Ninty-five per cent pure bovine procarboxypeptidase A has been obtained by ammonium sulfate fractionation of pancreatin extracts and isoelectric precipitations (47). When the proteins precipitated by 0.39 saturated ammonium sulfate are chromatographed on DEAE-cellulose in a concentration gradient of pH 8.0 phosphate buffer, the two last and most acidic peaks contain procarboxypeptidase A in an electrophoretically homogeneous form (48). The molecular weight of the protein determined by light scattering and sedimentation-diffusion is 94-96,000. Its isoelectric point in univalent buffers of 0.2 ionic strength is below 4.5. 

The stability of latex is due to a thin layer of proteins on particles, which acts as a colloid stabilizer. Natural rubber is practically obtained by the precipitation and drying of the latex. The precipitation is done with acids (acetic acid is commonly used for this purpose) when the isoelectric point of the protecting protein is reached (pH 4.6). The macromolecules have a MW between 5 10 to 3 10 Dalton and contain between 600 to 50,000 units of isopentene. Due to the double bond, both cis and trans isomers are possible for the monomer units. It was determined that natural rubber is an isotactic polymer formed exclusively from cis units and has the following (idealized) structure (in reality the polymer is not perfectly planar) ... 

It is a common feature of the numerous methods of separating globin from heme or hematin that one component is removed from the mixture by adsorption, as on kieselguhr (hematin) or precipitation, as by acetone (globin). It is, in principle, impossible to determine the amounts of split products present in labile equilibrium by removal of one component, since removal of one product of the dissociation is bound to result in a shift of the equilibrium point in the direction of increased dissociation. In addition, when the medium is changed, as by addition of acetone, profound changes must occur in the acidic dissociations in the protein which affect the reaction (see Section II of this review). Both of these effects would... 

Strains that produced each of the five serotypes known at the time were obtained and those expressing the most toxin were selected for use in further study, although most of the research involved serotype A.2 Culture conditions required to produce maximal levels of toxin were established, and techniques appropriate for purification and concentration were perfected. An important advance was crystallization of the toxin. The preferred method of toxin purification involved an initial acid precipitation from the culture supernatants, followed by redissolving the toxin in an aqueous buffer. At that point, the addition of ammonium sulfate produced toxin in a form called crystalline. To a protein chemist of today, this term means a highly purified protein that may be suitable for three-dimensional structure determination. The... 

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