A stable pH gradient is established in the gel after application of an electric field.

Protein solution is added and electric field is reapplied.

After staining, proteins are shown to be distributed along pH gradient according to their pi values.

FIGURE 3-21 Isoelectric focusing. This technique separates proteins according to their isoelectric points. A stable pH gradient is established in the gel by the addition of appropriate ampholytes. A protein mixture is placed in a well on the gel. With an applied electric field, proteins enter the gel and migrate until each reaches a pH equivalent to its pI. Remember that when pH = pI, the net charge of a protein is zero.

electrophoresis permits the resolution of complex mixtures of proteins (Fig. 3-22). This is a more sensitive analytical method than either electrophoretic method alone. Two-dimensional electrophoresis separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but different molecular weights.

Unseparated Proteins Can Be Quantified

To purify a protein, it is essential to have a way of detecting and quantifying that protein in the presence of many other proteins at each stage of the procedure. Often, purification must proceed in the absence of any information about the size and physical properties of the protein or about the fraction of the total protein mass it represents in the extract. For proteins that are enzymes, the amount in a given solution or tissue extract can be measured, or assayed, in terms of the catalytic effect the enzyme produces—that is, the increase in the rate at which its substrate is converted to reaction products when the enzyme is present. For this purpose one must know (1) the overall equation of the reaction catalyzed, (2) an analytical procedure for determining the disappearance of the substrate or the appearance of a reaction product, (3) whether the enzyme requires co-factors such as metal ions or coenzymes, (4) the dependence of the enzyme activity on substrate concentration, (5) the optimum pH, and (6) a temperature zone in which the enzyme is stable and has high activity. Enzymes are usually assayed at their optimum pH and at some convenient temperature within the range

25 to 38 °C. Also, very high substrate concentrations are generally used so that the initial reaction rate, measured experimentally, is proportional to enzyme concentration (Chapter 6).

By international agreement, 1.0 unit of enzyme activity is defined as the amount of enzyme causing transformation of 1.0 ^mol of substrate per minute at 25 °C under optimal conditions of measurement. The term activity refers to the total units of enzyme in a solution. The specific activity is the number of enzyme units per milligram of total protein (Fig. 3-23). The specific activity is a measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure (Table 3-5).

TABLE 3-6 The Isoelectric Points

of Some Proteins

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