Radioisotope Labelling

Radioactive isotopes were much used in biosynthetic studies, but stable isotopes are more used now because they can avoid the difficult degradation steps required with radio-labels, as will be illustrated below. Although some common compounds, e.g. 14C-labelled glucose can be purchased, frequently isotopically enriched compounds have to be synthesized from low molecular mass compounds such as 14C02, 3H20, K14CN, or CH335SH and the synthesis of the desired labelled compounds alone may be a major research task.

Compounds can be either uniformly labelled or specifically labelled at known atoms. By growing a plant or green alga in the presence of 14C02, uniformly labelled glucose can be obtained, in which all the 12C carbon atoms have an equal probability of being replaced by 14C. More usual is to use a specifically labelled compound, such as [2-14C]-acetic acid, in which labelled atoms are found only in the methyl group. Specific labelling is used in experiments in which a biosynthetic route is suggested, a labelled compound is fed to the system and a plan of degradation of the product compound is made that will show that the radio-labelled atoms are in the expected places. For this, all the common degradative reactions of organic chemistry can be used, e.g. ozonolysis, decarboxylation, double bond cleavage, etc. and the Barbier-Wieland degradation of fatty acids, by which one carbon atom at a time is removed from the chain.

The amount of radio-labelled compound obtained at the end of the experiment may be so little that more 'cold' or unlabelled final compound may have to be added to have enough to manipulate. In the studies of the biosynthesis of cholesterol (Chapter 7) unlabelled cholesterol for dilution is available in abundance. That is rarely so with insect substances, and a sample of the 'cold' target compound may have to be synthesized too.

Sometimes it is useful to watch for the appearance of radioactivity in a particular gland or in a compound to obtain information about the sequence of events in biosynthesis. A hormone or pheromone may only be formed at a certain stage of development, or a gland may be activated. A technique not much used but potentially valuable is to take sections through an insect after it has imbibed a radioactive compound and expose the sections on a photographic plate to see in which organs the activity accumulates (whole body autoradiography).

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