Hormones: Diverse Structures for Diverse Functions:- The Discovery and Purification of Hormones Requires a Bioassay
How is a hormone discovered and isolated? First, re searchers find that a physiological process in one tissue depends on a signal that originates in another tissue. Insulin, for example, was first recognized as a substance that is produced in the pancreas and affects the volume and composition of urine (Box 23–1). Once a physiological effect of the putative hormone is discovered, a quantitative bioassay for the hormone can be developed. In the case of insulin, the assay consisted of injecting extracts of pancreas (a crude source of insulin) into experimental animals deficient in insulin, then quantifying the resulting changes in glucose concentration in blood and urine. To isolate a hormone, the biochemist fractionates extracts containing the putative hormone, with the same techniques used to purify other biomolecules (solvent fractionation, chromatography, and electrophoresis), and then assays each fraction for hormone activity. Once the chem ical has been purified, its composition and structure can be determined. This protocol for hormone characterization is deceptively simple. Hormones are extremely potent and are produced in very small amounts. Obtaining sufficient hormone to allow its chemical characterization often in volves biochemical isolations on a heroic scale. When Roger Guillemin and Andrew Schally independently purified and characterized thyrotropin-releasing hormone (TRH) from the hypothalamus, Schally’s group processed about 20 tons of hypothalamus from nearly two million sheep, and Guillemin’s group extracted the hypothalamus from about a million pigs! TRH proved to be a simple derivative of the tripeptide Glu–His–Pro (Fig. 1). Once the structure of the hormone was known, it could be chemically synthesized in large quantities for use in physiological and biochemical studies. For their work on hypothalamic hormones, Schally and Guillemin shared the Nobel Prize in Physiology or Medicine in 1977, along with Rosalyn Yalow, who (with Solomon A. Berson) developed the extraordinarily sensitive radioimmunoassay (RIA) for peptide hormones and used it to study hormone action. RIA revolutionized hormone research by making possible the rapid, quantitative, and specific measurement of hormones in minute amounts. Hormone-specific antibodies are the key to the radioimmunoassay. Purified hormone, injected into rabbits, elicits antibodies that bind to that hormone with very high affinity and specificity. When a constant amount of isolated antibody is incubated with a fixed amount of the radioactively labeled hormone, a certain fraction of the radioactive hormone binds to the antibody (Fig. 2). If, in addition to the radiolabeled hormone, unlabeled hormone is also present, the unlabeled hormone competes with and displaces some of the labeled hormone from its binding site on the antibody. This binding competition can be quantified by reference to a standard curve obtained with known amounts of unlabeled hormone. The degree to which labeled hormone is displaced from antibody is a measure of the amount of unlabeled hormone in a sample of blood or tissue extract. By using very highly radioactive hormone, re searchers can make the assay sensitive to picograms of hormone. A newer variation of this technique, enzyme linked immunosorbent assay (ELISA), is illustrated in Figure 5–28b.
FIGURE 1 The structure of thyrotropin-releasing hormone (TRH). Purified (by heroic efforts) from extracts of hypothalamus, TRH proved to be a derivative of the tripeptide Glu–His–Pro. The side-chain carboxyl group of the amino-terminal Glu forms an amide (red bond) with the residue’s -amino group, creating pyroglutamate, and the carboxyl group of the carboxyl-terminal Pro is converted to an amide (red ONH2). Such modifications are common among the small pep tide hormones. In a typical protein of Mr ~50, the charges on the amino- and carboxyl-terminal groups contribute relatively little to the overall charge on the protein, but in a tripeptide these two charges dominate the properties of the peptide. Formation of the amide derivatives removes these charges.
FIGURE 2 Radioimmunoassay (RIA). (a) A low concentration of radiolabeled hormone (red) is incubated with 1 a fixed amount of antibody specific for that hormone or 2 a fixed amount of antibody and various concentrations of unlabeled hormone (blue). In the latter case, unlabeled hormone competes with labeled hormone for binding to the antibody; the amount of labeled hormone bound varies inversely with the concentration of unlabeled hormone present. (b) A radioimmunoassay for adrenocorticotropic hormone (ACTH). A standard curve of the ratio [bound] to [unbound radiolabeled ACTH] vs. [unlabeled ACTH added] is constructed and used to determine the amount of (unlabeled) ACTH in an unknown sample. If an aliquot containing an unknown quantity of unlabeled hormone gives, say, a value of 0.4 for the ratio [bound]/[unbound] (see arrow), the aliquot must contain about 20 pg of ACTH.
