Methodology

BY S. A. BROWN

1 Introduction

The relatively short history of modern biosynthetic experimentation, which has spanned little more than two decades, has suggested the need for a chapter on methodology to introduce the first volume of this series. Because of the nature of the material, coverage has not been restricted to the recent literature, and a comprehensive coverage has not been attempted. Instead, some general principles and their adaptations, of value throughout the field, will be discussed, and in a very selective way examples will be drawn from a variety of areas to serve as illustrations of the various techniques to be considered.

Space limitations have forced a number of areas in which important bio-synthetic work is in progress to be omitted from detailed consideration. Conspicuous among these is that of proteins and nucleic acids. While much of what follows is germane to this line of research, many of the specialized techniques employed lack application elsewhere, and to attempt to treat them in detail would compel omission of material of much wider application which should attract a broader interest among readers of this Report. It has also been regretfully decided to omit all but incidental mention of enzymological techniques, which are the ultimate tools of biosynthetic investigation. This is only justifiable on the basis that a great deal of easily accessible reference material is already available covering this very extensive field, especially as it relates to animal and microbial enzymes. In addition, other chapters in this volume may be consulted for references to specific enzymological investigations on particular classes of compound. Compilations dealing with the aspects chosen for discussion here are much less readily available, and it is hoped that a treatment of them will satisfy the greater need. While examples to be given will deal specifically with a microbial, plant, or animal system, many of the methods are equally applicable, with appropriate modifications, to any biological system. Much of the emphasis will be on isotopic tracer methods, which are now so universally employed that it is becoming uncommon to see an experimental publication on biosynthesis in which some use of tracers is not reported.

2 Some General Considerations

Definition of Biosynthesis. — While the roots of the term 'biosynthesis' would suggest its application to the formation of any substance in a living organism, it will be used here only in the more restricted anabolic sense. Acetylcoenzyme A could be regarded as being biosynthesized from fatty acids by β-oxidation or from hexose by glycolysis and oxidative decarboxylation, but these processes are exergonic and t heir prime biochemical function is to yield energy for ATP synthesis. Biosynthesis in the sense to be used in this chapter comprises the elaboration of molecules from less complex precursors by endergonic reactions.

Approaches to the Study of Biosynthesis. — Before considering detailed methods of biosynthetic investigation we might look briefly at the general approaches available at the present time, with a view to seeing a broad picture of the overall investigative procedure. Some of them will be examined more extensively in later sections. Preliminary clues to the nature of biosynthetic sequences can sometimes be gained by chemical analysis. The pattern of structurally related compounds existing in a species, either at one stage of development or over a period of time, can serve as a basis for hypotheses about biosynthetic pathways. This approach has been most widely developed by Robinson, and also by Geissman and Hinreiner. Some recent examples have concerned pathways by which sesquiterpene lactones are elaborated in species of Artemisia. It is a truism that the early compounds in any metabolic sequence are formed first chronologically, and in theory sequential analysis should detect the order in which they appear. But the pathway may branch, with simultaneous formation of structurally related compounds, and in practice the successive steps in the sequence often proceed with such rapidity that it is impossible to distinguish the order of synthesis of individual substances. In spite of these drawbacks the method has yielded important information, as will be seen in the later discussion.

Information on the pattern of certain related compounds in an organism obtained by sequential analysis or simply by analysis at one point in time can, with some intuition, be used to select compounds for testing as possible precursors. In modern practice this almost invariably means the use of isotopic labelling for tracer studies. Such wide ranges of nuclides and synthetic techniques are now available that virtually any atom of most precu rsor molecules can be specifically labelled, although generally labelled com pounds are suitable for some purposes. Most labelled atoms are radioactive, with 14C and 3H dominating the field, but the worker who has to label oxygen or nitrogen must be content with stable nuclides, at considerable sacrifice of sensitivity. A stable isotope of carbon, 13C, has acquired more biosynthetic significance in recent years with the development of techniques to locate its position in a molecule without degradative reactions; this will be discussed in detail later (p. 15).

Studies of biosynthesis with isotopic tracers fall into two general categories, concerned respectively with pathways and reaction mechanisms. It is only natural that the former have greatly predominated, since one must establish the identity of at least some of the intermediates in a reaction sequence before mechanisms of the conversions can be seriously considered. Labelling requirements for the study of pathways are much less stringent than for research on mechanisms. Whereas in the latter case one must introduce the label into a specific position, often stereospecifically, much information about pathways has been gained from generally labelled compounds (usually containing 14C), and considerations of convenience often dictate the labelling position even in chemically synthesized compounds.

Precursors and intermediates can be identified with a good degree of confidence in intact organisms, and considerable information on mechanisms is also obtainable by this approach. But, generally speaking, the ideal systematic approach would be identification of intermediates in a pathway with tracers, followed by the purification of the enzymes mediating the individual steps, and finally the use of these enzymes in detailed study of the reaction mechanisms with the aid of position-specific or stereospecific labelling. This last step, of course, is no different in principle from the study of chemical reaction mechanisms in general. In practice, many short cuts have been taken, and decades before the widespread use of tracer techniques vast amounts of data were...