Point proteins

Just as individual amino acids have isoelectric points, proteins have an overall p/ because of the acidic or basic amino acids they may contain. The enzyme lysozyme, for instance, has a preponderance of basic amino acids and thus has a high isoelectric point (p/= 11.0). Pepsin, however, has a preponderance of acidic amino acids and a low- isoelectric point pi 1.0). Not surprisingly, the solubilities and properties of proteins with different pi s are strongly affected by the pH of the medium. Solubility- is usually lowest at the isoelectric point, where the protein has no net charge, and is higher both above and below the pi, where the protein is charged. 

The dye-binding properties of extracts from cheese have been investigated as an index of ripening (Basch et ah, 1989 Kroger and Weaver, 1979). These methods are based on the principle that at a pFI below the isoelectric point, proteins have net positive charge and bind with anionic dyes such as amido black and acid orange 12. Protein-dye complexes are removed by filtration/centrifugation and the reduction in color is proportional to the protein concentration. 

Standard discontinuous alkaline buffer system for polyacrylamide-gei electrophoresis of low-isoelectric-point proteins... 

Use of Complex Standards for Cell Proteins and Two-Dimensional Electrophoresis. One of the more widely-used cell lines chosen as a reference standard is the lymphoblastoid cell line GM607, derived from a normal individual and available from the Human Genetic Mutant Cell Depository, Camden, NJ 08103. This cell line may be grown in defined media, labeled with a radioactive tracer, and reproducibly separated in a 2-DE system. Heat shock proteins may readily be isolated and visualized from this cell line, as shown by Anderson et al. (42). For serum, a reference preparation for serum proteins is available as a certified reference material prepared and assayed by the College of American Pathologists (CAP) and by the U. S. Centers for Disease Control. A widely available human serum standard is that provided by the National Bureau of Standards as SRM 909. If sufficient interest from the user community is evident, a full electrophoretic characterization of this material can be included in the documentation. If the amount of selected standard proteins loaded on a gel is known, "relative" quantification of similar proteins could be obtained. In addition, the National Bureau of Standards could serve as an impartial evaluator of potential national standards (e.g. molecular weight standards, "tie-point" proteins, and Isoelectric point standards) to assess suitability and stability. 

Overview - Regulatory proteins such as cyclins (see here), which are essential in certain parts of the cell cycle and deleterious in others, must be eliminated at some point. Proteins that have become damaged must also be removed. 

Female Spfague-Dawicy rats were treated orally with two 0.25 UD i doses ai 0 and 21 hr with pe.stieide.< and killed al the 24-hr lime point. Protein kinase C activity was monitored using a kit from Upstate Biotechnology. Values wilii nonidcntical. superscripts are significantly different (p < 0.05). 

CIP of Protein A-Sorbents Stability of the expensive Protein A-resins is a cmcial point. Protein A in its native form has only a limited stability towards caustic conditions. Therefore, careful CIP procedures have to be followed. 

The Waters Protein columns contain a high performance proprietary packing designed especially for SEC of proteins which have acidic to mildly basic Isoelectric points proteins of higher Isoelectric points are apparently adsorbed to the packing. 

Early work by protein chemists referred to in earlier chapters had revealed that the properties of proteins in solution can be substantially changed by addition of ionic surfactants (see also Ref. 113). In outline (1) Below their isoelectric point, proteins complex strongly with anionic surfactants, generally forming precipitates that can usually be solubilized on... 

The degree of ionization depends on the pH of the solution. An excess of H+ ions (low pH) retards dissociation of the acidic groups at high pH, the basic groups remain uncharged. At a certain pH, the number of positive and negative charges will be exactly equal. This particular pH is called the isoelectric point (pl). At the isoelectric point, proteins are found to be least soluble and can be precipitated most easily. 

One of the most important methods is salting out with a neutral salt such as ammonium sulfate, sodium sulfate, or magnesium sulfate, since highly concentrated solutions of these salts can be prepared. The salt content of a protein solution is increased stepwise, and after each addition the precipitated protein is centrifuged off. The pH may be varied to allow multiple fractionation (around their isoelectric points proteins become least soluble). Another method is precipitation with organic solvents (alcohol, acetone) according to E. Cohn. This must be carried out under refrigeration (cf. Chapt. IV-9). 

---