Isoelectric pH of Proteins

Righetti, P. G., Isoelectric focusing of proteins in conventional and immobilized pH gradients, in Protein Structure A Practical Approach, Creighton, T. E., Ed., IRL Press, New York, 1989, 23. 

Milk catalase is a haem protein with a molecular weight of 200 kDa, and an isoelectric pH of 5.5 it is stable between pH 5 and 10 but rapidly loses activity outside this range. Heating at 70°C for 1 h causes complete inactivation. Like other catalases, it is strongly inhibited by Hg2+, Fe2+, Cu2+, Sn2+, CN- and NOJ. 

The isoelectric pH of immunoglobulins (approx 8.6) is higher than the majority of the other serum proteins. As a consequence, they will be among the few proteins that will not bind to anion exchange media at neutral pH, providing a rapid an effective means of purification, particularly if combined with precipitation methods such as ammonium sulphate fractionation. 

Clearly, the results of osmotic pressure measurements on solutions of charged colloidal particles, such as proteins, will be invalid unless precautions are taken either to eliminate or to correct for this Donnan effect. Working at the isoelectric pH of the protein will eliminate the Donnan effect but will probably introduce new errors due to coagulation of the protein. Working with a moderately large salt concentration and a small protein concentration will make the... 

Ferrozine 3-(2-pyridyl)-5,6-bis(4-phenylsulphonic acid)-1,2,4-triaz-ine (Fig. 4.3) reacts with iron in the iron(II) ferrous state to form an intense pink complex which can be measured spectrophotometrically (lmax = 562 nm). The assay can be performed at pH 1.6 to eliminate interference from turbidity at pH values close to the isoelectric points of proteins which may be present in biological samples under investigation, e.g., serum, and in some instances to decrease interference in the assay from copper (see below). In determinations in which such problems are not considerations, the assay may be carried out at pH 7.4. (This might be more relevant for the assessment of membrane-bound iron under in vivo conditions.)... 

In practice, the aqueous solution of proteins to be separated is injected by a syringe into the central section of the polyampholyte gel specimen. Then, the DC electric field is switched on, which induces the formation of a hnear pH gradient along the gel sample. In its turn, the generated pH gradient promotes the migration of protein molecules until they are gradually locahzed at their isoelectric pH. Protein molecules will be separated and concentrated if the isoelectric pH of the proteins coincides with appropriate pH zones of the amphoteric gel. 

Thus, the equations of isotherms (30) and (31) can be used to determine the isoelectric points of proteins by the dependence of their sorption on pH. This approach is realized in Refs. [41,42]. However, it should be noted that this determination is only possible for a low affinity of protein molecules to surface sites. As Figs. 12 and 13 show, for a high affinity (a low Ka) a significant sorption is observed also at pH>pI for cation exchangers and at pH[Pg.721]

Further evidence for the occurrence of a diester linkage in this protein is reflected in the electrophoretic behavior. Pepsin still moves anodically in 0.1 N hydrochloric acid, pH 1.08. On the other hand, the phosphorus-free pepsin is positively charged at this pH. It has an isoelectric pH of 1.7. As shown in Fig. 6, the mobilities of these two proteins differ by a... 

As natural milk proteins have acidic isoelectric points, they have low solubility at acid pH and this compromises many of their functional properties in acidic environments. Esterification of proteins increases the net negative charge and raises the isoelectric point of proteins, making them more functional at acidic pHs. 

The precipitation and fractionation of soy protein using CO2 in the pressure range Q.1. 7 MPa was also examined (111,112). Pressurized CO2 allowed better control of the solution pH than the addition of mineral acids and prevented local pH overshoots, which can result in poor quality precipitates. Protein precipitates formed by isoelectric precipitation using CO2 were spherical, whereas those formed using sulfuric acid were irregular (111). The decrease of solution pH to a value close to the isoelectric pH of the proteins was the primary cause of protein precipitation. Volatile acid precipitation is unlike conventional GAS, in which pressures of 5-10 MPa are required to achieve precipitation from an expanded organic solution. 

As with emulsion formation, the maximal absorption of proteins has been reported to occur near the isoelectric point [6,18,47], The effects of pH on the properties of proteins at emulsion surfaces, noted previously, probably also apply to the behavior of proteins in films. While foams formed near the isoelectric point of proteins tend to be more stable than those formed at other pH values, it is often possible to obtain higher overruns away from the isoelectric point [48]. The largest effect of pH on foam stability can probably be explained by the more viscoelastic nature of the films formed in this pH region... 

Density materials used are practically identical with those used on the analytical scale, e.g., sucrose, and less frequently glycerol, sorbitol, ethylene glycol, ficoll and dextran. It should be noted that for the isoelectric fractionation of proteins with p7 values beyond 8.0 other density material than sucrose should be used (sucrose tends to be dissociated at high pH) (e.g., glycerol) (Fig. 6.29). 

Shimao, K., Computer simulation of electrophoretic transport. II. Modified general procedure and application to isoelectric focusing of proteins using immobilised pH gradient, Jap. J. Electrophor., 38, 221, 1994. 

Dissolution the creation of an aqueous solution containing two different polymers, generally a protein and a polysaccharide, usually at a temperature above the gelling point and pH that is above the isoelectric point of protein. 

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