Electrophoresis
المؤلف:
Peter Atkins، Julio de Paula
المصدر:
ATKINS PHYSICAL CHEMISTRY
الجزء والصفحة:
ص663-665
2025-12-17
58
Electrophoresis
Many macromolecules, such as DNA, are charged and move in response to an electric field. This motion is called electrophoresis. Electrophoretic mobility is a result of a constant drift speed, s, reached by an ion when the driving force zeE (where, as usual, ze is the net charge and Eis the field strength) is matched by the frictional force fs. The drift speed (which is treated in detail in Section 21.7) is then: s=
There fore, the mobility of a macromolecule in an electric field depends on its net charge, size (and hence molar mass), and shape. The latter two factors are implied by the dependence of s on f. The drift speeds attained by polymers in traditional electrophoresis methods are rather low; as a result, several hours are often necessary to effect good separation of complex mixtures. According to eqn 19.21, one way to increase the drift speed is to increase the electric field strength. However, there are limits to this strategy because very large electric fields can heat the large surfaces of an electrophoresis apparatus unevenly, leading to a non-uniform distribution of electrophoretic mobilities and poor separation. In capillary electrophoresis, the sample is dispersed in a medium (such as methyl cellulose) and held in a thin glass or plastic tube with diameters ranging from 20 to 100µm. The small size of the apparatus makes it easy to dissipate heat when large electric fields are applied. Excellent separations may be effected in minutes rather than hours. Each polymer fraction emerging from the capillary can be characterized fur ther by other techniques, such as MALDI-TOF.
Advances in biotechnology are linked strongly to the development of physical tech niques. The ongoing effort to characterize the entire genetic material, or genome,of organisms as simple as bacteria and as complex as Homo sapiens will lead to important new insights into the molecular mechanisms of disease, primarily through the discov ery of previously unknown proteins encoded by the deoxyribonucleic acid (DNA) in genes. However, decoding genomic DNA will not always lead to accurate predictions of the amino acids present in biologically active proteins. Many proteins undergo chemical modification, such as cleavage into smaller proteins, after being synthesized in the cell. Moreover, it is known that one piece of DNA may encode more than one active protein. It follows that it is also important to describe the proteome, the full complement of functional proteins of an organism, by characterizing directly the pro teins after they have been synthesized and processed in the cell. The procedures of genomics and proteomics, the analysis of the genome and pro teome, of complex organisms are time-consuming because of the very large number of molecules that must be characterized. For example, the human genome contains about 30 000 genes and the number of active proteins is likely to be much larger. Success in the characterization of the genome and proteome of any organism will depend on the deployment of very rapid techniques for the determination of the order in which molecular building blocks are linked covalently in DNA and proteins. An important tool in genomics and proteomics is gel electrophoresis, in which biopolymers are separated on a slab of a porous gel, a semirigid dispersion of a solid in a liquid. Because the molecules must pass through the pores in the gel, the larger the macromolecule the less mobile it is in the electric field and, conversely, the smaller the macromolecule the more swiftly it moves through the pores. In this way, gel electrophoresis allows for the separation of components of a mixture according to their molar masses. Two common gel materials for the study of proteins and nucleic acids are agarose and cross-linked polyacrylamide. Agarose has large pores and is better suited for the study of large macromolecules, such as DNA and enzyme complexes. Polyacrylamide gels with varying pore sizes can be made by changing the concentration of acrylamide in the polymerization solution. In general, smaller pores form as the concentration of acrylamide is increased, making possible the separation of relatively small macromolecules by polyacrylamide gel electrophoresis (PAGE). The separation of very large pieces of DNA, such as chromosomes, by conventional gel electrophoresis is not effective, making the analysis of genomic material rather difficult. Double-stranded DNA molecules are thin enough to pass through gel pores, but long and flexible DNA coils can become trapped in the pores and the result is impaired mobility along the direction of the applied electric field. This problem can be avoided with pulsed-field electrophoresis, in which a brief burst of the electric f ield is applied first along one direction and then along a perpendicular direction. In response to the switching back and forth between field directions, the DNA coils writhe about and eventually pass through the gel pores. In this way, the mobility of the macromolecule can be related to its molar mass. We have seen that charge also determines the drift speed. For example, proteins of the same size but different net charge travel along the slab at different speeds. One way to avoid this problem and to achieve separation by molar mass is to denature the proteins in a controlled way. Sodium dodecyl sulfate is an anionic detergent that is very useful in this respect: it denatures proteins, whatever their initial shapes, into rods by forming a complex with them. Moreover, most protein molecules bind a constant number of ions, so the net charge per protein is well regulated. Under these conditions, different proteins in a mixture may be separated according to size only. The molar mass of each constituent protein is estimated by comparing its mobility in its rod-like complex form with that of a standard sample of known molar mass. How ever, molar masses obtained by this method, often referred to as SDS-PAGE when polyacrylamide gels are used, are not as accurate as those obtained by MALDI-TOF or ultracentrifugation. Another technique that deals with the effect of charge on drift speed takes advantage of the fact that the overall charge of proteins and other biopolymers depends on the pH of the medium. For instance, in acidic environments protons attach to basic groups and the net charge is positive; in basic media the net charge is negative as a result of proton loss. At the isoelectric point, the pH is such that there is no net charge on the biopolymer. Consequently, the drift speed of a biopolymer depends on the pH of the medium, with s = 0 at the isoelectric point (Fig. 19.8). Isoelectric focusing is an electrophoresis method that exploits the dependence of drift speed on pH. In this technique, a mixture of proteins is dispersed in a medium with a pH gradient along the direction of an applied electric field. Each protein in the mixture will stop moving at a position in the gradient where the pH is equal to the isoelectric point. In this manner, the protein mixture can be separated into its components. The separation of complicated mixtures of macromolecules may be difficult by SDS-PAGE or isoelectric focusing alone. However, the two techniques can be combined in two-dimensional (2D) electrophoresis. In a typical experiment, a protein mixture is separated first by isoelectric focusing, yielding a pattern of bands in a gel slab such as the one shown in Fig. 19.9a. To improve the separation of closely spaced bands, the first slab is attached to a second slab and SDS-PAGE is performed with the electric field being applied in a direction that is perpendicular to the direction in which isoelectric focusing was performed. The macromolecules separate according to their molar masses along this second dimension of the experiment, and the result is that spots are spread widely over the surface of the slab, leading to enhanced separation of the mixture’s components (Fig. 19.9b).
Fig. 19.8 The plot of drift speed of the protein bovine serum albumin in water against pH. The isoelectric point of the macromolecule corresponds to the pH at which the drift speed in the presence of an electric field is zero.
Fig. 19.9 The experimental steps taken during separation of a mixture of biopolymers by two-dimensional electrophoresis. (a) Isoelectric focusing is performed on a thin gel slab, resulting in separation along the vertical direction of the illustration. (b) The first slab is attached to a second, larger slab and SDS-PAGE is performed with the electric field oriented in the horizontal direction of the illustration, resulting in further separation by molar mass. The dashed horizontal lines show how the bands in the two-dimensional gel correspond to the bands in the gel on which isoelectric focusing was performed.
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