Protein characterization

Size and charge

To determine both the size and charge characteristics of the protein and, indeed, of the contaminating proteins, we must use electropboretic separation methods. Although preparative electrophoresis techniques have been developed, these can be costly, time-consuming and difficult to use on any large-scale purification. The value of electrophoresis lies in its ability to provide highly resolved, analytical separations of complex protein mixtures.

Many electrophoretic media are available; however, polyacrylamide gels are by far the most commonly used for high-resolution protein separations. These gels are easily adjusted, controlled and reproduced, easily stored, and can be scanned if required.

Native polyacrylamide gel electrophoresis (PAGE) is most suitable for studying the composition and structure of native proteins, as both their conformation and biological activity will remain intact during the analysis. However, it is often difficult to find standard proteins which resemble the shape, partial specific volume and degree of hydration of the native protein under investigation. Consequently, SDS-PAGE (sodium dodecyl sulfate) is easier and in most cases more reliable than native PAGE for molecular weight determinations.

In SDS-PAGE, proteins are first treated with SDS under reducing conditions at an elevated temperature. This treatment denatures the proteins, causing them to unfold and assume a rod-like structure coated with SDS molecules. SDS binds to protein in a constant weight ratio and provides a net negative charge. Thus when applied to the polyacrylamide gel in an electric field, all the SDS complexes will migrate at the same rate until the sieving properties of the gel take over. The outcome is a separation based on size. The molecular weight of the proteins can be calculated by comparing their electrophoretic mobility with that of protein standards of known molecular weight.

Isoelectric focusing (IEF) is a high-resolution electrophoretic technique for separating proteins on the basis of their isoelectric points (pi) and, as a consequence, can be used to determine the range of isoelectric points of proteins in a mixture. Homogeneous gels are prepared containing carrier ampholytes, such as Pharmalyte®. Under an electric field, the ampholytes create a stable, linear pH gradient across the gel (the pH range being dependent upon the ampholytes selected) and proteins will then migrate, essentially unhindered by the gel, to a point in the pH gradient that corresponds to their pi. Markers indicate the pH positions across the gels.

Electrophoretic titration curve analysis is a two-dimensional technique for analyzing protein charge characteristics. In the first dimension, the pH gradient is generated, as in IEF. The gel is then rotated through 90° and the sample applied perpendicular to the pH gradient across the middle of the gel. The proteins become positively or negatively charged, depending on their pi, and will migrate toward the anode or cathode. The rate of their migration will depend on the magnitude of their charge.

From the titration curves, we can predict optimal conditions for further separation by chromatography, in particular ion exchange chromatography as shown in Figure 1. All of these electrophoretic techniques can be run on any horizontal electrophoresis system and it is advisable to use these powerful analytical tools not only for initial sample characterization but also for continual evaluation of all stages of purification. Considerable time and effort can be saved by using precast gels on dedicated equipment, such as PhastSystem™, which enables analyses to be performed in under 30 minutes.

Biospecificity and hydrophobicity

The biospecificity of the protein is likely to be known only if precise details of its activity and structure are known beforehand. The hydrophobicity of the protein is less easily defined. All proteins are, to some extent, hydrophobic, and it is only the difference in their relative hydrophobicities which makes selection

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