Isoionic point, protein

Since the binding of extraneous ions considerably alters the value of lEP of an aminoacid or protein, this point is not a constant. The term isoionic point (IP) is used to designate the pH value of a pure protein in salt-free water. The direct determination of this constant is difficult and because many proteins are insoluble in the absence of salts. The isoionic point is usually determined indirectly, that is, by measuring the lEP at different concentrations of the neutral salts and extrapolating to zero concentration The value of the isoionic point may differ from lEP by more than a pH unit (Haurowitz 1963). 

For a protein, the isoionic pH is the pH of a solution of pure protein with no other ions except H+ and OH Proteins are usually isolated in a chaiged form together with counterions such as Na+, NH4, or C1. When the protein is subjected to intensive dialysis (Demonstration 27-1) against pure water, the pH in the protein compartment approaches the isoionic point if the counterions are free to pass through the semipermeable dialysis membrane that retains the protein. The isoelectric point is the pH at which the protein has no net charge. Box 10-2 tells how proteins can be separated on the basis of their different isoelectric points. 

Acid-base titration of the enzyme ribonuclease. The isoionic point is the pH of the pure protein with no ions present except H+ and OH. The isoelectric point is the pH at which the average charge on the protein is 0. [C. I Tanford and J. D. Hauenstein, Hydrogen Ion Equilibria of Ribonuclease." J. Am. Chem. Soc. 1956, 78.5287.]... 

The opening page of this chapter shows the titration curve for an enzyme. Is the average charge of the protein positive, negative, or neutral at its isoionic point How do you know ... 

The isoionic points of a number of the milk proteins are given in Table 3.5. 

Protein Approx % of skimmilk protein Number of components 5 Isoionic point Molecular weight0... 

The distinction between the isoelectric and isoionic states of a protein was first made in a classic paper by S0rensen et at. (1926). Three definitions of the isoionic point were proposed, one of these being the stoichiometrically defined point which we have called the point of zero net proton charge. The other tw o were operational definitions (summarized by Linderstr0m-Lang and Nielsen, 1959). The term isoionic point, as used here, corresponds to one of these two operational definitions, chosen because it always permits calculation of the point of zero net proton charge, which is the only parameter of real interest in the analysis of titration curves. The same choice has been made by Scatchard and Black (1949). 

Speakman and Hirst (1933) and Speakman and Stott (1934) consider that an isoelectric region rather than an isoelectric point should be recognized similar results are observed with silk (Howitt, 1946). Lemin and Vickerstaff (1946) claim to have determined the isoionic point of wool by measurement of the pH at which addition of salt does not affect the pH. They obtained a value of 6.2 which is approximately the midpoint of the isoelectric region. It is not possible to determine the isoionic point of a protein by measurements made, as in the present instance, at constant salt concentration. The true value could be obtained by carrjdng out the... 

Myosin is another protein to which the theory of Linderstr0m-Lang in its present form is not applicable, since in myosin the ratio of molecular length to width is 100/1—far from the sphericity on which the theory is based. Thus experimental values of the parameter w cannot be easily interpreted quantitatively. Myosin is soluble in the presence of salt on the alkaline side of its isoionic point only, and thus should behave as a soluble protein above pH 5.7 to 5.8 and as an insoluble one below this. Mihdlyi (1950) has studied the effect of salt on the titration of myosin and reports that its insolubility in acid in the presence of greater than 0.05 M KCl does not affect the reversibility of the titration nor are there any obvious discontinuities in his titration curves, shown in Fig. 4. The data for basic solutions appear to be affected by salt very much as those of other soluble proteins, and reach an apparent limiting curve at a fairly low ionic strength (0.15). In acid solution where the protein is insoluble, however, the effect of salt closely resembles that for wool, except that the displacements of the parallel central portions of the curves are somew hat less than for wool, consistent with a lower affinity of myosin for chloride ion. The slopes of these portions of the curves are within 10 % of those observed for... 

Proteins differ so very greatly in their tendencies to bind ions of either charge type, however, that exceptions occur in the vicinity of the usual isoionic point. 

Molar ratio of anhydride to protein e-NH, groups modified %) Isoionic point Relative mobility Molar extinction coefficient at 280 nm, x 10 ... 

The pH at which a protein or particle has an equivalent number of total positive and negative charges as determined by proton exchange is the isoionic point. The pH at which a protein or a particle does not migrate in an electric field is called the isoelectric point. The isoionic point is a whole fiber property of hair and is reflected in the equilibrium acid-base properties of the total fiber the isoelectric point is related to the acid-base properties of the fiber surface. 

If the only ions present in this solution are H+, OH- and protein ions, it is clear that the mean net charge on the protein cannot be exactly zero unless the isoionic point happens to coincide with the pjj of neutrality. Thus, if the pH of the electrodialyzed solution is 5, we have [H+] = io-6M, [OH-] negligible, and the protein must carry a small negative charge to balance the excess of H+ over OH- ions. For a serum albumin solution, concentration yg./l. (io-4M) this requires thatZj, for the electrodialyzed solution, be —0.1 (= — io-3/io-4), instead of zero. The difference is well within the usual experimental error, but it can be corrected for if necessary. The correction obviously becomes more important, the more acid the isoionic point of the protein and the more dilute the protein solution. 

The same holds true (with a negligible simplification) for the acidic amino acids, whereas the isoionic point for the basic amino acids is equal to KpA 2 + P s)- The isoelectric point of an amino acid is defined as the pH value at which the amino acid will move neither towards the positive nor negative pole when subjected to an electric current. The isoelectric points may depend on the presence of other ions but for all practical purposes the isoelectric point and isoionic point may be considered identical for amino acids (except when complexes between amino acids and metal ions are involved (see Section IV,G)). For proteins there may be substantial differences. The isoelectric point is not equal to the pH value of a solution of the amino acid in water, the latter being somewhere between the isoelectric point and pH 7. The difference will normally not be great, but neutral amino acids have a very small buffering capacity at the isoelectric point and the pH values found in their aqueous solutions are therefore variable. 

The isoionic point, for a protein solution not too low in concentration, is experimentally defined as that pH which does not change when a small amount of pure protein is added to the solution (46). This definition also applies to a protein which has been focused isoelectrically (7,26). If more protein is added to the zone, the pH does not change. The buffering at the site of focusing is considered to be completely dominated by the... 

The isoelectric pH of a protein is the pH at which the protein is immobile in an electric field. At this pH, the protein exists as a zwitterion having equal number of positive and negative charges, the net charge being zero. The Isoionic point of a protein is defined as the pH at which the total number of H taken up by a protein is equal to the total number of H dissociated from it. Isolonic and Isoelectric point of a protein will essentially be the same if the protein does not bind ions other than H". Since this is an ideal condition and not usually obtainable, the Isoionlc and isoelectric points of a given protein differ. 

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