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Charge properties of proteins and peptides
Protein and peptide net charges depend on their contents of charged amino acids. These carry weak acidic or amino groups and the net charge is therefore a function of pH.
At low pH values the dissociation of the acidic groups is suppressed and they loose their negative charges in accordance with their respective pKa values (see table to the right!). Amino groups on the other hand are protonated and positively charged at low pH values.
At high pH values the acidic groups dissociate and become negatively charged while amino groups loose a proton and become neutral.
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Below a certain pH value the protein obtains a positive net charge.
Above this pH value the protein obtains a negative net charge.
At a certain intermediate pH value, the number of positive charges equals that of the negative ones, making the net charge zero. This pH value is called the isoelectric point (pI) and is specific for each individual protein.
Titration curves (Fig 5.1) reflect the dependence of net charge on pH and can be of great help in finding the optimal pH for an IEX separation of proteins or peptides.
Fig 5.1. The titration curve reflects how the net charge of a protein or peptide varies with pH.
Electrofocusing in gels produces stable pH gradients that can be used to investigate protein titration curves. After having created the pH gradient, normal electrophoresis is run at right angles to the gradient. The sample is applied in the middle of the gel so that it can migrate in either direction depending on net charge. Figure 5.2 shows the result obtained when a meat extract was analyzed in this way. It demonstrates the variation of charge properties between the different proteins of this sample.
Fig 5.2. Electrophoretic titration curves. |
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