Detection And Purification Of Biochemical Compounds

The tremendous amount that is known about biochemistry may seem somewhat mysterious without an appreciation for the methods used to gain it. A brief description of some of the techniques used to purify and detect biochemical compounds may help with understanding how this body of knowledge came about.

Thin-layer chromatography is done with paper or a glass plate coated with silica powder or other adsorbent material. A solution containing a mixture of biochemical compounds, ranging from amino acids and nucleic acids to polypeptides, is placed in a spot near one corner. One edge is then placed in contact with a solvent, which carries the compounds along the plate as it is drawn up by capillary action. Compounds with various physicochemical properties move at different speeds. This spreads the compounds out in a line along one edge. That edge is then placed in a different solvent, which moves the compounds across the plate in the other direction. This distributes the individual compounds from the mixture across the two dimensions of the plate (Figure 3.15). The resulting spots can be made visible by chemical treatment or can be removed for further experiments.

If the molecules are electrically charged, such as amino acids and polypeptides at the appropriate pH, one of the solvent steps can be replaced with an electrostatic field to move the compounds. This is called electrophoresis.

Large molecules and subcellular particles can be separated by sedimentation. This is done in an ultracentrifuge, which creates accelerations up to 400,000g by spinning as fast as 75,000 rpm. Molar masses of large macromolecules can be determined from their sedimentation rate. Particle size is often given in terms of its settling velocity in a centrifuge, measured in Svedberg units.

Immunoassay uses antibodies to form a precipitate with specific compounds. Antibodies are special proteins produced by the body to bind with foreign substances so that they can be made harmless. Each antibody is highly specific, binding only to a single substance and binding extremely tightly in what is called a lock-and-key relationship. Molecular biology techniques have enabled the production of large quantities of antibodies of a specific type, called monoclonal antibodies. They are used for research purposes as well as to detect specific hormones in pregnancy tests and tests for prostate cancer. Immunoassay is a highly sensitive and selective detection method. Its use has been extended to organic pollutants and even to heavy metals.

The power of the methods described above is often increased by the use of radioiso-

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