Working with Proteins: -Proteins Can Be Separated and Characterized by Electrophoresis

Another important technique for the separation of proteins is based on the migration of charged proteins in an electric field, a process called electrophoresis. These procedures are not generally used to purify proteins in large amounts, because simpler alternatives are usually available and electrophoretic methods often adversely affect the structure and thus the function of proteins. Electrophoresis is, however, especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of different proteins in a mixture or the degree of purity of a particular protein preparation. Also, electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer polyacrylamide (Fig. 3–19). The polyacrylamide gel acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. Migration may also be affected by protein shape. In electrophoresis, the force moving the macromolecule is the electrical potential, E. The electrophoretic mobility of the molecule, μ, is the ratio of the velocity of the particle molecule, V, to the electrical potential. Electro phoretic mobility is also equal to the net charge of the molecule, Z, divided by the frictional coefficient, f, which reflects in part a protein’s shape. Thus:

The migration of a protein in a gel during electrophoresis is therefore a function of its size and its shape. An electrophoretic method commonly employed for estimation of purity and molecular weight makes use of the detergent sodium dodecyl sulfate (SDS).

SDS binds to most proteins in amounts roughly proportional to the molecular weight of the protein, about one molecule of SDS for every two amino acid residues. The bound SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificant and conferring on each protein a similar charge-to-mass ratio. In addition, the native conformation of a protein is altered when SDS is bound, and most proteins assume a similar shape. Electrophoresis in the presence of SDS therefore separates proteins almost exclusively on the basis of mass (molecular weight), with smaller polypep tides migrating more rapidly. After electrophoresis, the proteins are visualized by adding a dye such as Coomassie blue, which binds to proteins but not to the gel itself (Fig. 3–19b). Thus, a researcher can monitor the progress of a protein purification procedure as the number of protein bands visible on the gel decreases after each new fractionation step. When compared with the positions to which proteins of known molecular weight migrate in the gel, the position of an unidentified protein can provide an excellent measure of its molecular weight (Fig. 3–20). If the protein has two or more different subunits, the subunits will generally be separated by the SDS treatment and a separate band will ap pear for each.

FIGURE 3–19 Electrophoresis. (a)Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection currents caused by small temperature gradients, as well as protein movements other than those induced by the electric field. (b)Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to the proteins but not to the gel itself. Each band on the gel represents a different protein (or protein subunit); smaller proteins move through the gel more rapidly than larger proteins and therefore are found nearer the bottom of the gel. This gel illustrates the purification of the enzyme RNA polymerase from E. coli. The first lane shows the proteins present in the crude cellular extract. Successive lanes (left to right) show the proteins present after each purification step. The purified protein contains four subunits, as seen in the last lane on the right.

Isoelectric focusing is a procedure used to determine the isoelectric point (pI) of a protein (Fig. 3–21). A pH gradient is established by allowing a mix ture of low molecular weight organic acids and bases (ampholytes; p. 81) to distribute themselves in an electric field generated across the gel. When a protein mix ture is applied, each protein migrates until it reaches the pH that matches its pI (Table 3–6). Proteins with different isoelectric points are thus distributed differently throughout the gel. Combining isoelectric focusing and SDS electrophoresis sequentially in a process called two-dimensional 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.

FIGURE 3–20 Estimating the molecular weight of a protein. The electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight, Mr. (a)Standard proteins of known molecular weight are subjected to electrophoresis (lane 1). These marker proteins can be used to estimate the molecular weight of an unknown protein (lane 2). (b)A plot of log Mr of the marker proteins versus relative migration during electrophoresis is linear, which allows the molecular weight of the unknown protein to be read from the graph.

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.

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مواضيع ذات صلة

The Covalent Structure of Proteins:-Large Proteins Must Be Sequenced in Smaller Segments

The Covalent Structure of Proteins:- Short Polypeptides Are Sequenced Using Automated Procedures

The Covalent Structure of Proteins:-The Amino Acid Sequences of Millions of Proteins Have Been Determined

The Covalent Structure of Proteins:-The Function of a Protein Depends on Its Amino Acid Sequence

Working with Proteins:- Unseparated Proteins Can Be Quantified

Working with Proteins:- Proteins Can Be Separated and Purified

Peptides and Proteins:- Some Proteins Contain Chemical Groups Other Than Amino Acids

Peptides and Proteins: - Polypeptides Have Characteristic Amino Acid Compositions

Peptides and Proteins: -Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes

Peptides and Proteins: -Peptides Can Be Distinguished by Their Ionization Behavior

Peptides and Proteins: - Peptides Are Chains of Amino Acids

Amino Acids: -Amino Acids Differ in Their Acid-Base Properties