Isoelectric overall charge

Isoelectric points are useful concepts for the separation and purification of amino acids and proteins using electrophoresis. Under the influence of an electric field, compounds migrate according to their overall charge. As we have just seen for amino acids, this very much depends upon the pH of the solution. At the isoelectric point, there will be no net charge, and, therefore, no migration towards either anode or cathode. 

The pH at which an amino acid, a peptide, or a protein has zero overall charge after summing the contributions of all the charges is called the isoelectric point (pi). 

Figure 4.2. Model polypeptide of 20 amino acids. The three-letter and one-letter abbreviations for amino acids are shown. Ionizable amino acids (-R) are shown as positively or negatively charged in bold. Note that the amino-terminal and carboxy-terminal ends are charged. All charges contribute to an overall charge state of the protein at a specific pH. The pi (isoelectric point) is the pH at which a proteins carries no net charge. The pi is helpful in classifying proteins as acidic, neutral, or basic. The addition of posttranslational modifications can also add charges to proteins and greatly affect the pi value.

Lastly, when working with macromolecules, the isoelectric point (pi) of the protein must be assessed to determine if the pH of the solution will have a large or small effect on the overall charge on the molecule. If the pH of the solution is far from the pi, the difference in the apparent pH and the actual pH may not have a large effect on the stability or conformation of the protein. 

Subsequently Prince, Cogdell and Crofts also examined the reaction between Rp. sphaeroides reaction centers and mammalian cytochrome c as well as native cytochrome isolated from the same cells. They confirmed the electrostatic nature of the interaction between the Rp. sphaeroides reaction centers and mammalian cytochrome c. However, unlike the Rp. sphaeroides reaction center/mammalian cytochrome c system, the reaction with cytochrome isolated from Rp. sphaeroides is independent of pH between pH 4 and 10, and the effect of changing ionic strength on reaction rate is negligible at all pH values. The pH-independent behavior in this case has been attributed to the fact that the reaction-center complex and cytochrome have essentially the same isoelectric points, and thus similar overall charges at all pHs. 

The effect of pH on proteins, (a) This protein has an overall charge of 2+. When a base is added, some of the protonated amino groups lose their protons. Now the protein is isoelectric it has an equal number of positive and negative charges, (b) This protein has an overall charge of 2-. As acid is added, some of the carboxylate groups are protonated. The result is that the protein becomes isoelectric. 

At the isoelectric point, ionized amino acids have an overall charge of zero. 

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. 

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