Methods in biochemistry

Centrifugation and electrophoresis

An important tool in biochemical research is the centrifuge, which through rapid spinning imposes high centrifugal forces on suspended particles, or even molecules in solution, and causes separations of such matter on the basis of differences in weight. Thus, red cells may be separated from plasma of blood, nuclei from mitochondria in cell homogenates, and one protein from another in complex mixtures. Proteins are separated by ultracentrifugation—very high speed spinning; with appropriate photography of the protein layers as they form in the centrifugal field, it is possible to determine the molecular weights of proteins.

Another property of biological molecules that has been exploited for separation and analysis is their electrical charge. Amino acids and proteins possess net positive or negative charges according to the acidity of the solution in which they are dissolved. In an electric field, such molecules adopt different rates of migration toward positively (anode) or negatively (cathode) charged poles and permit separation. Such separations can be effected in solutions or when the proteins saturate a stationary medium such as cellulose (filter paper), starch, or acrylamide gels. By appropriate color reactions of the proteins and scanning of color intensities, a number of proteins in a mixture may be measured. Separate proteins may be isolated and identified by electrophoresis, and the purity of a given protein may be determined. (Electrophoresis of human hemoglobin revealed the abnormal hemoglobin in sickle-cell anemia, the first definitive example of a “molecular disease.”)

Chromatography and isotopes

The different solubilities of substances in aqueous and organic solvents provide another basis for analysis. In its earlier form, a separation was conducted in complex apparatus by partition of substances in various solvents. A simplified form of the same principle evolved as ‘‘paper chromatography,” in which small amounts of substances could be separated on filter paper and identified by appropriate color reactions. In contrast to electrophoresis, this method has been applied to a wide variety of biological compounds and has contributed enormously to research in biochemistry.

The general principle has been extended from filter paper strips to columns of other relatively inert media, permitting larger scale separation and identification of closely related biological substances. Particularly noteworthy has been the separation of amino acids by chromatography in columns of ion-exchange resins, permitting the determination of exact amino acid composition of proteins. Following such determination, other techniques of organic chemistry have been used to elucidate the actual sequence of amino acids in complex proteins. Another technique of column chromatography is based on the relative rates of penetration of molecules into beads of a complex carbohydrate according to size of the molecules. Larger molecules are excluded relative to smaller molecules and emerge first from a column of such beads. This technique not only permits separation of biological substances but also provides estimates of molecular weights.

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A variety of other physical techniques, such as nuclear magnetic resonance, electron spin spectroscopy, circular dichroism, and X-ray crystallography, have become prominent tools in revealing the relation of chemical structure to biological function.