CHAPTER 1
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 accumulating about biosynthetic reactions from enzymological studies alone. It is precisely because enzymological techniques are so well established in the literature that their consideration in this chapter would be redundant. But it is still true (and this fact is not always fully appreciated by those trained in classical biochemistry) that application of tracer methodology can greatly narrow the search in preparation for the more definitive experiments of the enzymologist.
A final technique available to the student of biosynthesis, although it is almost entirely restricted to microbial investigations, is the use of auxotrophic mutants. This method offers the opportunity to establish a compound as an obligatory intermediate — a member of a path that is the only one by which an organism can synthesize a given product from given source materials — with the greatest degree of probability. A powerful investigative tool, it can be used alone or in combination with tracer and enzyme techniques to provide valuable data on pathways, and it will be discussed more extensively in a later section (p. 29).
Limitations on Biosynthetic Studies. — A qualification which must apply to conclusions from all biosynthetic studies to date was pointed out nearly two decades ago by Adelberg. It relates to the possibility that an intermediate on a biosynthetic pathway may be readily interconvertible with another metabolite not on the direct pathway, as exemplified by C and F in the sequence:
[ILLUSTRATION OMITTED]
The only known way to resolve this general problem is to establish that a single enzyme catalyses the conversion of B to X and another single enzyme that of X to D, where X is the suspected intermediate. If this can be done, then X must occupy the position C, and not F.
The difficulty here concerns the criteria for proving beyond question that a preparation contains only one enzyme. Enzyme homogeneity has been established with a very high degree of probability in many instances, particularly where crystalline preparations have been obtained, but with increasing uncertainty about purity there is a parallel uncertainty whether a compound has been positively identified as a metabolic intermediate. It is thus a tenable position to maintain that no com pound has been so identified with absolute finality. The problem of whether citric acid is in the main tricarboxylic acid cycle or is formed in a side reaction was a notable example of the uncertainties that can arise.
It seems unlikely that such reversible interconversions are the rule, or even common, in metabolism. But we must always bear in mind that data from biosynthetic studies are currently subject to misinterpretation in the sense just discussed, especially in plants, where relatively little work has been done with purified enzymes.
3 Sequential Analysis
Successful application of this technique is largely dependent upon growth that is slow enough so that the periods during which successive intermediates form can be differentiated. Such slow growth, and attendant slower metabolism, is more likely to be found in multicellular organisms than in microbes, with their rapid reproduction rates, and experiments with higher plants have, in fact, yielded clues about pathways.
Investigations of the relationships among the alkaloids of Conium maculatum were carried out by Fairbairn and others,6 in which analyses were done by paper chromatography, first at weekly intervals and later at intervals of one day, four hours, and two hours. The peak in the total content per fruit of γ-coniceine, now known to be (1), occurred one week earlier than that of coniine (2), an observation consistent with derivation of the latter alkaloid from the former. However, the very rapid changes in the levels of both compounds revealed by the shorter-term experiments showed that the picture is much more complex than had been believed, and raised the possibility of a reconversion of coniine into γ-coniceine as part of some oxidation-reduction process.
Kasprzyk and Fonberg-Broczek estimated triterpenic monols and diols of Calendula officinalis from the seedling stage to that of seed production using thin-layer chromatography and a colorimetric test. They observed that the monols taraxasterol (3; R = H) and ψ-taraxasterol (4; R = H) were formed 3 — 4 days earlier than the diols arnidiol (3: R = OH) and faradiol (4:R = OH), with significant synthesis of the monols being detectable in the leaves surrounding the flower bud before any trace of diol could be found. The synthesis of monols was also found to cease in the flowers before that of the diols. Both facts are, or course, indicative of diol formation through hydroxylation of the corresponding monol.
In other cases the approach fails as a result of too rapid a synthesis, which does not permit the sequence to be deduced. Yet even in microbial species it is sometimes possible to obtain meaningful results by the sequential analysis approach. Bu'Lock and his associates, for example, have shown that in well-phased cultures of Penicillium urticae, stages in the biosynthetic route to patulin (5) from 6-methylsalicylic acid make their appearance successively, and to some extent separably.
Whatever the species, this technique is generally less useful than those which employ isotopic labelling, and in fact it may be stated that its chief function is to provide indications about pathways which can then be tested by the more rigorous labelling methods.
4 Isotopic Tracer Methods
In this section it will be assumed that the reader is familiar with the basic principles of radioactivity and isotopes. There are available a number of excellent monographs on the subject, such as those by Broda, Chase and Rabinowitz, Wang and Willis, Wolf, Comar, and Kamen. Although they are older works, the last two of these are of special value for their exhaustive discussions of isotopes available for biological research. More specialized publications which could be mentioned would include the monograph on tritium by Evans, the one on carbon-14 by Catch, and books on scintillation spectrometry by Birks and Schram.
It is not proposed in this chapter to undertake a review of methods used in the synthesis and degradation of radioactive compounds, which would require literally volumes and would encompass a significant portion of the field of organic reactions. In recent years the problem of radiochemical synthesis has receded in importance with the increasing variety of commercially available labelled compounds, and this trend will certainly continue. The treatise by Murray and Williams, while now somewhat outdated, is still a useful starting point for a literat ure search on radiochemical synthesis. A more recent but shorter monograph covering 14C is also available. One field in which commercial sources are of little help and where much synthetic expertise is still required is the stereospecific incorporation of tritium and deuterium. This approach has been extensively used in research on biosynthetic mechanisms, especially in steroids, and notably by Popjak, Cornforth, and their collaborators. Reactions useful in degradation are discussed in detail in a quite recent monograph by Simon and Floss.
The Time and the Place. — If meaningful results are to be obtained, a labelled compound administered to an organism obviously must not only penetrate to the synthetic site, but must do so at a time when the enzyme systems mediating the synthetic reactions are present and active. Some of the practical questions in getting a compound to the site of synthesis are considered in Section 7 of this chapter. Except in cell-free systems, the permeability barriers within and between cells are a cause of universal concern in biosynthetic studies, and in work with cell suspensions this problem and that of substrate solubility are the only ones likely to stand in the way. The inability of metabolically active phosphate esters to penetrate cell walls has long been known, and other compounds of such varying structures as mevalonate and acetate have also failed to cross-permeability barriers. Compartmentation of metabolic pools 26 can severely com plicate interpretation of results, and is thus important in the present context.
The highly developed circulatory system in higher animals makes systemic administration of labelled compounds relatively easy, and the problems encountered are usually manipulative, especially if one must employ small experimental animals. Injection into individual organs is also feasible (cf. Section 7, p. 33). In plants the difficulties are greater, except in the administration of carbon dioxide, which is rapidly absorbed by the photosynthetic organs. Some compounds given to plants in solution by injection, or through roots or cut stems, are poorly translocated, but it is nevertheless clear from the results of many experiments that most substances fed to plants in solution do reach synthetic sites at least in part, and are metabolized there if they can be utilized as precursors.
Because of these potential problems of permeability and, especially in plants, transport, negative results of tracer experiments have to be regarded with caution, particularly if the compound being tested is of a type known to encounter permeability barriers.
In initiating experiments on a previously untested system the investigator must establish at the outset that it can synthesize the compound being studied. To take examples that now seem obvious, one would attempt biosynthetic studies on phospholipids with brain rather than kidney tissue, and the administration of potential nicotine precursors to cut tobacco shoots is futile, since synthesis occurs only in the roots. Many further exam pies of such localizations in multicellular organisms are known.
