23 January 1965

Papers and Originals

Landmarks and Perspectives in Biochemical Research*

E. B. CHAIN,t D.PHIL., PH.D., F.R.S.

Brit. med. J., 1965, 1, 209-220

The science of biochemistry has become a magnificent structureof enormous dimensions, diversity, and complexity, with manythousands of people engaged in advancing it, with new avenuesopening up all the time, and new information pouring in atsuch a flood rate that to follow the literature even superficiallyis a hopeless task.

To-day's occasion seemed to me an excellent opportunity forstocktaking and survey, as well as to do a little crystal-gazinginto the future: to see which position we have actually reached,by what means we have got there, and where we are goinghence.The most appropriate way in which this task could be per-

formed was, I decided, to give you a broad panoramic reviewof the great landmarks of biochemical achievements, and toanalyse the most important features of the strategy and tacticsby which they were reached. Then, looking from the heightsof achievements into the distant horizon of the future, we can,I think, discern certain perspectives of paths to be explored andcan gather at least a vague conception of the exploratory kitwith which we shall have to equip ourselves in order topenetrate into the unknown regions.

Three Basic Approaches to Biochemical Research

Before starting with my task, allow me to make a fewgeneral remarks about biochemical research which I considerimportant and which, in any case, will serve to illustrate to youclearly my own attitude to this field of scientific activity.

It has been apparent to me for a long time-and becameincreasingly clear as I prepared this lecture-that successfulbiochemical research throughout the history of the subject hasinvolved the application of the following three basic lines ofapproach either singly or in combination:

1. The Recognition of the Indivisibility of BiochemistryBy this is meant that the choice of the biological system for

the study of a certain biochemical effect is determined primarilyby the criteria as to where this biochemical effect manifests itselfmost strongly and lends itself most readily to experimentation. Anyspecies, whether mammal, bird, mollusc, crustacea, insect, plant,mould, protozoon, bacterium, or alga, can serve the purpose. Veryoften the most important factor determining the success of bio-chemical research has been the choice of the most suitable bio-logical system. In particular, animal biochemistry and chemicalmicrobiology form one inseparable, closely interwoven unit.

2. The Importance of the Biological Approach to BiochemicalResearch

Many great biochemical discoveries had at their origin a biologicalobservation which subsequently, through the application of suitablebiochemical techniques, could be explained in chemical terms. The

sequence of events in these cases was always: biology first, chemistrysecond-not vice versa. The importance of the biological approachfor biochemical research cannot be overemphasized. Close contactand collaboration in a harmonious team between the biochemist andthe biologist in the different regions of the biological sciences-andthis includes, it should not be forgotten, clinical medicine-has beena characteristic feature of much successful biochemical research inthe past and will increasingly become such in the future.When the chemist in his approach to biochemical problems

neglects the biological aspects the work will usually remain dulland of limited general significance; when the biologist in hisobservations neglects the chemical aspect, the work will remaindescriptive and come to a dead end.

Through lack of collaboration between the biolog'. t and thechemist progress has been delayed, sometimes for years. Twoexamples of many that could be given will serve to illustrate thisassertion.

The first, Fleming's penicillin, is known to you all. Flemingreported its existence in 1929, but for lack of collaboration withchemists it lay dormant for almost ten years, until Florey andmyself decided in 1938 to undertake a reinvestigation of this sub-stance and, together with our collaborators, discovered in 1940its unique curative properties in experimental and clinical bacterialinfections.

The second example, of perhaps less spectacular practical butcertainly of no less fundamental importance, is the discovery ofthe transforming principle. In 1928 the London bacteriologistF. Griffith published a remarkable observation in the 7ournal ofHygiene: rabbits infected with a mixture of dead smooth virulentpneumococci of type III and living avirulent rough pneumococci oftype II unexpectedly succumbed to infections, and bacteriologicalexamination showed that the organism causing death of the animalswas the smooth virulent type III strain. There was obviouslysomething in the dead type III pneumococci which had caused thetransformation of the avirulent rough type II strain into the virulentsmooth type III strain. The morphological and biological propertiesof these strains were known to be genetically controlled, and forthis reason a transformation of one type into another by a chemicalsubstance was a most remarkable and entirely new phenomenonopening up revolutionary perspectives in the sciences of both geneticsand biochemistry. Yet again, because of lack of contact betweenthe biologist and the biochemist, nothing was done tofollow up, from the chemical point of view, this extraordinarilyinteresting and striking biological observation and to characterize theprinciple responsible for the transformation phenomenon, until 16years later Avery, McCarthy, and McLeod, of the RockefellerInstitute of New York, decided to reinvestigate it and succeededin isolating the active principle and identifying it as deoxyribo-nucleic acid, thus demonstrating for the first time the role ofdeoxyribonucleic acid in transmitting genetical information.

Research limited to investigations on the chemical structure ofcell constituents, irrespective of whether these are of low or highmolecular weight, once it becomes too remote from the living cell,ceases to be functional biochemistry, and becomes a different,

$Inaugural Lecture as Professor of Biochemistry at the Imperial Collegeof Science and Technology, University of London, given on 12May 1964.

t Professor of Biochemistry, Imperial College of Science and Technology,London.

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Biochemical Research-Chainthough of course not less important, field of science: natural-pro-ducts chemistry. Knowledge of the structure of a compound andits biologically active centres is often a useful help, but not neces-

sarily essential-and, in any case, never the only or even the mainpremise for the advancement of our understanding of the metabolicreactions in the living cell, which is the primary task of functionalbiochemistry. What I said about the relationship between biologyand biochemistry applies also to natural-products chemistry andbiochemistry: close collaboration of the biochemist with the natural-products chemist is the surest way to success.

3. The Development and Application of New TechniquesThe courageous development and application of both new ana-

lytical and preparative techniques, using the whole range of physico-chemical, physical, and engineering methods which progress in therespective fields of science, has made available irrespective of thecomplexity and cost of the apparatus requested, and with the onlycriterion of whether or not they will bring nearer the solution of aparticular important biochemical problem.The realization of the immense complexity of cell organization,

on the one hand, and on the other hand the recognition of thegreatness of the impact which almost every major biochemicaldiscovery has had on human health and welfare, and, in conse-quence, of our stakes in the advance of biochemical knowledge,justifies the use of almost any technique which will bring us nearerour goal of unravelling yet another of nature's great biochemicalsecrets. Good facilities for development of new instrumentationand preparative techniques must therefore form an essential featureof a modern Biochemical research laboratory.

Ten Major Landmarks in Biochemical ResearchI shall now proceed to illustrate the validity of these theses

which, for better or worse, are at the basis of the whole structureand organization of my new Institute, with some concreteexamples of important landmarks in biochemical research.The whole course of development of functional biochemistry

can, I believe, be mapped by ten major landmarks. These are:

1. The Pasteur era in which the endowment of micro-organisms with well-defined specific biochemical activitieswas discovered.

2. The discovery by Buchner that a cell-free yeast-pressjuice retained the fermenting capacity of the living cell.

3. The discovery by Harden and Young that Buchner'syeast-press juice needed a cofactor of low molecular weightfor its activity, in addition to high molecular proteins.

4. The recognition of the biochemical and medical signi-ficance of low molecular coenzymes.

5. The discovery of new cofactors through nutritional andclinical studies on man, animals, and micro-organisms, andof enzyme systems in cell-free extracts.

6. The discovery of the significance of cofactors forenzymic reactions.

7. The study of enzymic reactions leading-to the recogni-tion of metabolic pathways: (a) in the pre-isotope era, (b) bymethods involving heavy and radioactive isotope markers,(c) by genetical methods.

8. The discovery of the hormones and their significanceas regulators of metabolic reactions.

9. The discovery of the role of nucleic acids in proteinsynthesis.

10. The discovery of the antibiotics and their significanceas inhibitors of metabolic reactions.These ten landmarks could be a good synopsis of a com-

prehensive two-year course of biochemistry such as we intendto organize in the new Department, but in this inaugural lectureI must, of course, limit myself to a few brief comments.

BRITISHMEDICAL JOURNAL

1. The Pasteur EraFirst of all a few words about the Pasteur era, which marks

the beginning of modern functional biochemistry. The mainpurpose of my remarks is to convey to you something of myown feelings of boundless admiration for the immense contribu-tions towards the development of functional biochemistry whichwe owe to Pasteur, one of the greatest scientific geniuses of alltime, and the school he created.For those who frown from lofty heights on contacts between

the universities and industry as irreconcilable with and debasingthe great ideals of pure scientific research, it may be salutaryas well as instructive to remember that the impulse which ledPasteur to the line of research from which later a whole new

science-or perhaps it would be more correct to say several newsciences-was destined to emerge and which had vast theore-tical and practical repercussions, came from an industrialconsultant job which he was asked to undertake while he wasProfessor of Chemistry in Lille, for an industry with as hedon-istic an aim as that of wine production. Pasteur was a greatFrench patriot to whom the economic fortunes and prosperityof his country were at least as important as his scientificresearch. The wine industry was of great economic importancefor France. For this reason he felt that he could not refuse a

request from the wine producers of his region to take an interestin the wine fermentation process, then considered to be a purelychemical process, and to make suggestions, if possible, on anysteps which could be taken to avoid the occurrence of irregu-larities and aberrations in this process which they encounteredat unpredictable intervals and under ill-defined circumstances,and which cost them a great deal of trouble and heavy financiallosses.

As' you know, Pasteur discovered that the fermentation pro-cess was not purely chemical in nature, but was a biologicalphenomenon, caused by the action of micro-organisms; theregular fermentations were found to be brought about by certaintypes of yeast, the abnormal " pathological" ones by otheryeasts or bacteria. These observations were then extended fromthe diseases of wine and beer to diseases of animals and man,and led to the revolutionary results in public health, medicine,surgery, and agriculture with which we are all familiar. Pasteurand his school were thus the first to demonstrate that differentspecies of microbes were endowed with specific biochemicalactivities. They were shown to be capable of producing lowerand higher alcohols, lower and higher fatty acids, such as acetic,propionic, and butyric acid, hydroxy acids such as lactic andmalic acid, toxins responsible for the pathogenic action ofbacteria in man and animals, antigens producing specific anti-bodies (observations on which the science of immunology, bothin the biological and in the chemical aspects, is based). Theywere shown to display antagonistic properties, one speciesagainst another (which later were found to be due to the pro-duction of antimicrobial substances now known as antibiotics).Some species of micro-organisms were found to be capable ofliving and exerting their chemical function in the absenceof molecular oxygen, la vie sans oxygene, a startling discoveryat that time, and of great fundamental importance for bio-chemical research. The discoveries of Pasteur and his schoolwere extended by the great soil microbiologists of the end of thelast and the beginning of this century, among them particularlyprominent the great Russian soil microbiologists Winogradskyand Omeljanski and the Dutchman Beijering. We owe to thelatter the immensely important fundamental discovery of theprocess of nitrogen fixation by the root nodule bacteria ofleguminous plants, to the former the discovery of the nitrifyingbacteria which oxidize ammonia to nitrite and nitrate, and ofthe fact that these and other soil organisms are capable ofchemo-autotrophic existence-that is, that they can synthesizetheir cell material in the absence of light entirely from inorganicsubstances, using CO2 as their carbon source.

Altogether, it may be said that the discovery of the bio-chemical activities of micro-organisms by Pasteur and his

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school, supplemented by those of the soil microbiologists, whofollowed in their trail, has provided a reservoir of new factsand phenomena so rich and vast that even to-day biochemistsin many laboratories still draw freely and extensively on it as

one of the most important sources of research subjects.

2. Buchner's Discovery of the Fermenting Capacity of a

Cell-free Yeast-press Juice

The next important landmark in biochemical research isBuchner's discovery in 1896, one year after Pasteur's death, thata juice obtained from compressed yeast ground with sand andsubjected to the action of a hydraulic press, in which no

living cells were present, had retained the capacity of the livingyeast cell to perform all the reactions of alcoholic fermentations.The first consequence of this discovery was that Pasteur's theorypas de fermentation sans vie, which he defended tenaciouslyand successfully over 25 years against fierce opposition, hadthe fate of all theories: a limited lifetime ; it survived Pasteurby exactly one year. But the great importance of Buchner'sdiscovery for biochemistry lies in the fact that it renderedcomplex metabolic systems accessible to chemical experimentalstudy ; for this reason it had very great repercussions. It marksthe beginning of the modern biochemistry of intermediate meta-bolism, enzyme chemistry, electron transport, and energytransfer. It has kept biochemists busy for almost 80 years andstill continues to do so. Naturally Buchner's success withyeast-press juice of obtaining a complex metabolic system infully functioning state in the absence of any living cells led tothe extension of this method to other cell kinds, mammalian,plant, and microbial. The result has been that a whole array

of enzyme systems have thus become accessible for study, cover-

ing a gamut of increasing complexity, with the simple hydrolyticenzymes, the simple dehydrogenases and oxidases at one end ofthe scale, and on the other the very complex particulate mulzi-enzyme systems, bringing about complex reactions such as

oxidative phosphorylation, fat and steroid synthesis, purineand porphyrin synthesis, protein synthesis, nitrogen fixation,and photo-synthesis. In fact, the rate-determining factor forprogress in several important fields of intermediate metabolismhas been to find cell-free extracts retaining the metabolicfunction under study.The various particulate multi-enzyme systems can be

separated from the soluble enzymes and fractionated by differen-tial centrifugation in density gradients. In this way morpho-logically and biochemically well-characterized subcellularfractions have been obtained, such as nuclei, cell membranes,mitochondria, and microsomes ; for the separation of the latterhigh gravitational fields, reached by ultracentrifugation, are

required.The subcellular fractions exhibit a definite pattern of enzyme

action. Thus, the expressive concept of the "cytoskeleton"advanced by Peters in the 1920's to indicate that the cell interioris not just a homogeneous solution of enzymes, but has a definitestructural organization, has found consolidation by irrefutableexperimental evidence.The preparation of cell-free metabolically active extracts is

one of the typical specifically biochemical operations, andrequires great skill in the choice of the right organisms and theright conditions as well as luck. Bacteria have proved a

particularly important source for complex enzyme systems.

These are much more difficult to break up than yeast cells, andthe hydraulic press method was found to be inadequate forthis purpose. Several other tools for breaking up this kind ofcell had therefore to be developed, including treatment withultrasonic waves, treatment by high pressure and sudden release,squeezing through small apertures in special presses under highpressure, such as the Hughes and the Edebo press. Thisproblem of breaking up bacterial cells belongs to the area of

BRmsUMEDICAL JOURNAL 211

biochemical engineering with which our new Department willbe concerned, and has only partially been solved. It is stillonly possible to obtain relatively small quantities of bacterialcell extracts, and the design of continuous method for thispurpose which would enable people to obtain many litres ofsuch extracts is one of the important tasks of biochemicalengineering for the future.

3. Harden and Young's Discovery of Cozymase

The next important landmark in biochemical research onwhich I must comment is the discovery by Harden and Youngin 1905 that Buchner's yeast extract lost its fermenting powerafter dialysis and that this power could be restored by the addi-tion of a very small amount of boiled yeast extract. Thisdemonstrates that the fermentation system consisted not onlyof high molecular heat-labile enzymes as was believed until thattime, but also of a low molecular heat-stable cofactor whichwas termed cozymase. The importance of this discovery beyondthe limited field of fermentation chemistry became clear when itwas demonstrated by Meyerhof that the same factor was neces-sary for muscle glycolysis which he showed to occur in a

muscle-press juice prepared more or less following Buchner'smethod. The fact that the same factor was required as an

essential component of basic metabolic processes in a micro-organism, such as yeast, and in an animal tissue, was one of thefirst demonstrations of the close similarity of the elementarymetabolic processes in living matter.

The isolation and purification of the fermentation cofactorwith which the name of Hans von Euler is prominently con-nected and the elucidation of its structure took over 20 years,and necessitated the working up of many hundreds of kilogramsof yeast. It is the first example of what has become and stillremains one of the main pillars of biochemical research, therecognition and study of trace factors forming essentialcomponents of metabolic systems.

4. The Function of Cozymase in Cellular Oxidation. TheRespiratory Chain. Methodology

The full immense signifi ance of the yeast cofactor becameapparent only gradually over the course of years through twolines of research which at first seemed unconnected. On theone hand, it was soon demonstrated that the two enzymes

responsible for the terminal steps in the reaction sequence ofalcoholic fermentation and glycolysis-that is, the enzymes

reducing acetaldehyde to alcohol and pyruvic acid to lactate-required the presence of cozymase and were inactive in itsabsence. Thus the yeast fermentation cofactor was shown tobe involved in oxido-reduction reactions.The mechanism of biological oxidation, as the source of

energy for all the manifestations of life, has rightly formed thecentre of attention of biochemical research right from its verybeginnings. An army of biochemists has been occupied formany years with this fascinating complex problem, but the manwho has contributed more than anyone else towards unravellingit was Otto Warburg, one of the greatest giants of biochemicalresearch and, without any doubt, the greatest living biochemist-he has just completed 80 years-in full creative activity.Warburg conceived the idea that biological oxidation was a

process catalysed by heavy metals, in the way that the oxidationof some organic substances was known to be metal catalysed,and that in particular iron, and in some cases copper, playeda central role in this process.

I have already indicated that half the success in biochemicalresearch is to find a suitable biological system. Warburg, whocombined in one person the talents of an outstanding biologistwith those of a brilliant chemist, has demonstrated a particulargenius in this respect in all his work. The cell system which

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Biochemical Research-Chain

he selected for his first studies on cell oxidation was the sea-urchin egg, which after fertilization has a fairly high rate ofrespiration and because of its homogeneity and small size lendsitself readily for experimental investigations. Later he extendedhis studies on cellular respiration to animal cells, where heintroduced the technique of tissue slices, the use of the isolatedrat diaphragm and the Ehrlich ascites cancer cells, and to yeastand bacterial cells. In all these systems he demonstrated thatthe process of respiration proceeded by catalytic activation ofoxygen through an autoxidizable iron-containing enzyme towhich he gave the name "respiratory enzyme." The iron, hepostulated, was present in this enzyme in the form of aporphyrin complex of the haematin type.The process of oxido-reduction was, according to Warburg's

concepts, brought about by continuous valency changes of theiron, which yielded an electron to the oxygen by being oxidizedfrom the divalent to the trivalent stage, and subsequentlyaccepted an electron from the substance, being thus againreduced to the divalent stage ; the oxygen in this process wasthus reduced to hydrogen peroxide, which in turn washydrolysed to water and molecular oxygen by the enzymecatalase which also was shown to contain an iron porphyrincomplex as prosthetic group.

Warburg's concept of iron-catalysed oxygen activation asthe basis of cellular respiration was supported by superb experi-mental evidence and formulated in a series of papers with asimplicity, clarity, and logical strength which remind onestrongly of Pasteur's writings. It received further strongsupport from the isolation, purification, and extensive studyof a cellular haem pigment, which had been discovered manyyears earlier, in 1886, by McMunn and termed histohaematin,but whose observations had been forgotten. This haem pigment,renamed cytochrome c by Keilin, was shown by this investigatorto change its spectrum reversibly on reduction and oxidation.The structure of the haem part of cytochrome c as well as thecomplete structure of the peptide chain to which it is attachedis now known. It became clear, however-and Warburg wasthe first to point this out, against much opposition-that cvto-chrome c was not as readily autoxidizable as was Warburg'srespiratory enzyme and therefore not identical with it ; therespiratory enzyme was later shown to be a related compoundtermed cytochrome a. Subsequently several other cytochromeswere identified. The probable structure of the haem moiety ofcytochromes a, b, or c and the peptide chain immediatelyattached to the latter is shown in Fig. 1.

Despite the mass of irrefutable evidence which Warburgproduced, his ideas on the central function of iron in cellular

CHO CH3

H3C CH CH2. CH3N N

CH pFe( CH

N N

H3C CH CH,

higher CHfatty CH

(9) acid peptide chain

CH=CH2 CH3

H3C -CH CHHCH2N N

CH Fe( CH

N N

H3C CH CH3

ICH2 C, H2CH2 CH2

( b) COOH COOH

ARITIStlMEDICAL JOURNAL

respiration were vehemently attacked by nearly all the bio-chemical authorities on cellular respiration of the time, with aviolence, prejudice, and irrational dogmatism which is difficultto understand to-day, even in the light of the then existingevidence. The main argument of Warburg's adversaries was thedemonstration of the existence in all cells of iron-free enzymescapable of activating hydrogen in the substrates of oxidation,which led them to the conclusion that in cellular oxidationactivation of hydrogen was more important than activation ofmolecular oxygen.

It fell to Warburg to demonstrate that both hydrogen activa-tion and oxygen activation were equally important in cellularrespiration and were, in fact, inseparably linked with each otheras members of one chain of reactions, the respiratory chain.One of the crucial links between the two reactions was, asWarburg discovered, the fermentation cofactor of Harden andYoung and of von Euler.The laborious purification studies of this factor, as I have

mentioned above, continued over two decades. Structural in-vestigations on the most highly purified preparations obtainedhad shown that it was a nucleotide containing adenylic acidas one of its constituents, but the structure of the rest of themolecule remained obscure. Warburg, with his collaboratorChristian, succeeded, in one operation of typical Warburgianbrilliancy, not only in elucidating the complete structure of thecoenzyme but at the same time its biological functions. Hewas able to isolate from a highly purified preparation which heobtained from yeast by a new method of his own a few milli-grams of nicotinamide. This showed that the coenzyme wasa dinucleotide in which one of the mononucleotides wasadenylic acid and the other contained nicotinamide as base;both were linked in pyrophosphate linkage through their ter-minal phosphate groups. It is now termed nicotinamideadeninedinucleotide (NAD) (Fig. 2). Warburg recognized immediately

rCHHC fC-CONH2.I

Nicotinamide _______

mononucleotide 0 CH 0

-Op=H CH,

Pyrophosphate OH OH NH2linkage INC

-HO-P=O 411 ~C-'NHC 11

l ~~\NC- sCHN N

Adeno sine -5 O CH2 0phosphate \

C H H C

OH OH

FIG. 2.-Nicotinamide adenine dinuclcotide.

that this substance was a redox system and its capability to actas a cofactor for dehydrogenases resided in the nicotinamidegroup, which acted as electron and proton acceptor from sub-strate hydrogen, after activation by the respective substratedehydrogenases. It became subsequently clear that the hydrogenin the thus reduced form of NAD did not react directly withthe cvtochrome and molecular oxygen, but through the inter-

mediation of another redox system of diribonucleotide natureafter activation through a specific dehydrogenase, termed NADdehydrogenase. This diribonucleotide contained the alloxazineriboside riboflavin instead of the nicotinamide riboside moiety(Fig. 3). Riboflavin phosphate had previously been shown byTheorell to form part of the prosthetic group of anotherdehydrogenase, oxidizing glucose-6-phosphate to the lactone ofthe corresponding gluconic acid, an important enzyme dis-covered by Warburg and Christian and later shown by Dickens,in this country, to catalyse the basic reaction of an importantmetabolic pathway for the direct oxidation of glucose.

212 23 January 1965

R-Val-GIu(NH2)-Lys-Cys- Ala-Glu CNH2)- Cys- CO-CH-NH-Thr-Val-Glu-RS S CH2

CH.CH3 OH3 \

H3C OH CH ININ N H3 HC OCH

CHCH Fe

JLN N',

H3C \ CH / CH3

CH2 OH2CH2 CH2

(C) COOH COOH

FIG. l.-Cytochromes

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Electrons from reduced FAD were shown to react with thecytochrome system, again after activation of its hydrogensthrough specific enzymes, termed cytochrome reductases, andthe last member of the cytochrome system, cytochrome a orcytochrome oxidase, to pass on the electrons to molecularoxygen, reducing it to hydrogen peroxide.

NH2

OH OHCH2-O-P-O-P-OCH, |

HOCH 0 O H

HOCH

HOCH OH OH

CH2

CH3, 3 ) NH0

FIG. 3.-Riboflavin adenine dinucleotide.

Biological oxidation was thus shown to be a flow of electronsin steps across a series of redox systems of decreasing electro-negativity. This series of redox systems is known as the respira-tory chain, and this concept denotes one of the most importantlandmarks of biochemical research with wide ramifications. Theexact sequence of the redox system in the respiratory chain invarious cell types is still a subject of intensive investigations,and new members, such as coenzyme Q, non-haem iron, andcopper-containing redox systems, are being discovered. Fig. 4gives a picture of the respiratory chain in animal tissues, as itis proposed by some investigators in the light of the latestevidence. In anaerobic micro-organisms the final electronacceptor is not molecular oxygen but organic substances, or, inthe case of some of the autotrophic bacteria, inorganic salts,such as nitrate and sulphate, which are reduced to ammonia,or sulphur or sulphide respectively. The respiratory chain ex-

plained the toxic action of substances such as cyanide, azide,sulphide, hydroxyamine, etc., in consequence of their formationof a firm complex with the iron of the final link of the respira-tory chain, which is thus prevented from effecting the electron

Substrate -H2 dehydroqenuse 2H' + 2C+Substrate

C/ CONH2 CH2 CONH2 + H*I) 2H~++2E+ 'i~Cti (N

R(NAD) RACNADH)

R RI

+CONH2 H3C co NADH CONH2 H3C

2) H++ + J|C Is dehydroqenase +

HI~N.-

R CNADH) 6(FAD) R (NAD)

R. o OH

H3C NNHCO CHOyN.CH3 H3C 'Nco CH30 CH3

H3C N N C H30O CsoH8 H3C N C CH30O C5oHeH OCFADH) 0 (Coo) (FAD) OH (CoQ-

OH

CH30 + 2 Fe3

CH3,O 50H81 (ox.Cyt b)

OH(Coa-H2)

0

CH30 CH3

CH30O C5soH

C Coo)

5) Fe2 Cred.Cyt b) + Fe3 (ox. Cyt cl) Fe3 tox. Cyt b) +

6) Fe2(red. Cytc1) + Fe3 (ox.Cyt c ) Fe3 (OX.Cyt cl) +

7)F (red.Cyt c )+ Fe3(ox Ct a )

8) Fe2( red.Cyta ) CU2

2Fe2C red. C

Fe2 (red. C

Fe2Cred.Cy

Fe3 + Fe2(ox. Cyt c ) ( red.Cy

(ox.Cytx ). Cul

9) 2 Cu' +2H++02 HO-OH+2 Cu2

FIG. 4.-The respiratory chain.

BRITISHMEDICAL JOURNAL 213

transfer from substrate to oxygen so that the whole respiratorychain is put out of action.

I myself was able to show that the powerful neurotoxic effectof certain snake venoms was due to their action on the respira-tory chain. The link affected in this case was the nicotinamideadenine dinucleotide coenzyme, which, I showed, was rapidlyand completely broken down into two mononucleotides by anenzyme of nucleotidase nature contained in the venom.

The techniques for studying the phenomena connected withcellular respiration were nearly all introduced by Warburg. Notonly did he develop the manometric measurements to the fineart they have become-Warburg's manometric techniques havebecome an indispensable tool of every biochemical laboratory-but he was also responsible for the introduction of the spectro-photometric absorption techniques for studying the- kinetics ofthe oxidation-reduction reactions of the respiratory coenzymes.

These spectrophotometric methods, which include measure-ments in the visible and the ultra-violet range as well asfluorescent spectrophotometry, have become an indispensabletool in the biochemical laboratory. They have been used exten-sively not only for kinetic measurements of isolated oxidativeenzyme systems-and in this field the work of Hugo Theorellon substrate-enzyme relations in catalase, peroxidase, andalcohol dehydrogenase is an important landmark-but also forfollowing the kinetics of single components of the respiratorychain in intact cells and sub-cellular fractions, and this becamepossible through the application of modern electronic methodsby Britton Chance which rendered the spectrophotometric andfluorometric methods very much more sensitive.

I should like at this point to recall and pay tribute to thefundamental and pioneering investigations of Leonor Michaelisand Mansfield Clark on the thermodynamics of redox systemswhich have been of the greatest importance for the evolution ofour basic concepts on biological oxidation.

5. Discovery of Identity of Cofactors and Vitamins

We have seen that the study of the yeast cofactor of Hardenand Young and related substances led to the elucidation of the

mechanism of biological oxidation.Multi enyzme Interest in the study of enzymic cofactors

was very much stimulated and theirposition appeared in quite a differentlight when it was recognized that apartfrom their theoretical importance for theunderstanding of basic metabolic reactionmechanisms they have very great prac-

NADH-CoO tical importance for therapeutic medicine.

eductaseThis knowledge was gained from bio-

FADH) (onthaem chemical studies of biological nature in

Fe)

the field of nutrition, which at first seemedtotally unconnected with yeast fermenta-tion or cellular respiration. In the 1890's

-H2) the Dutch physician Eijkman. made theimportant discovery that a severe andwidespread disease, manifesting itselfwith symptoms of nervous dysfunction,

:yt b) called beriberi, by which people in theDutch East Indies were affected, could be

Ctochrome c reproduced in fowls by feeding them areductase diet of polished rice from which the hulls

y(contoininq

It cl ) non-hoem had been removed and that this disease

could be cured by feeding the birds withyt c J unpolished rice of the hulls.

I) ~~Here we have an excellent example of theIt a

Cytochrome validity of one of the remarks which Ioxidase made at the beginning of my lecture,

when I referred to the vital importancei for biochemical research of choosing the

right biological systems. The particular

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iwtritional deficiency with crtacteristLcsymptoms, such, as head retractions, is& reaonly in birds, best ,of all in pigeons (Fig. 5).

qemonpstratea

. 5.--Thiamine-deficient pigeon showing typical head-drop symptom.

In 1912 Frederick Gowland Hopkins, in whose laboratory- Ihad the good fortune to spend two very happy and fruitfulyears, reported his now famous experiments (Fig. 6) that ratssupplied with a calorically adequate diet of carbohydrate, pro-tein, and fat, would not grow unless 2 ml. of milk per day wasadded to their diet. The observation made it clear that thereeXisted in the diet some hitherto unknown factor or factors,present in trace amounts, in the absence of which some import-si f metabolic systems in the intact animal did not properlyfunction, and that this caused characteristic pathological

,deficiency syndromes which could be cured by, the addition ofthe lacking factor to the diet. The similarity between the defi-

- clency diseases and the lack of activity of the fernientation-eiiezyme system in the absence of cozymase was evident.

90o

70

50

s0DAYS 20 40

FIG.. 6.-Graph to illustrate the effect of milk on growthand life. Lower curve, rats on artificial diet alone.Upper curve, six similar animals receiving in addition 2 ml.

of milk each per diem.

The identity of vian d e* factorsw rwhen it was found that one of t vitamins an - hfactor discovered in yeast by P. Gyorgi and termed vitamin B2,was riboflavin. As, has already been mentioned, riboflavinphosphate linked to adenylic acid in pyrophosphate linkage(Fig. 3) is the coenzyme of the dehydrogenase termed NADdehydrogenase, which catalyses the transfer of.,electrons fromreduced cozymase to the cytochrome system and therefore hasa vital role in the respiratory chain. Later the flavine-adeninenucleotides were found to be cofactors of several otherimportant oxidation enzymes.The recognition of identity of vitamins and coenzymes was

one of the most important achievements of biochemical researchand opened up a field with vast perspectives which continues tobe actively explored.

6. Isolation of New Cofactors

In these studies the ingenuity of thebiochemist had to beprimarily directed towards finding. an appropriate biologicaltest system and the right nutritional conditions in whichnutritional deficiencies would show up. Once these conditionswere found, the isolation of the factor responsible for thedeficiency symptoms was a matter of routine, though sometimeslaborious and time-consuming.Once the close similarity of the basic metabolic reactions in

cells of higher animal tissues and microbial cells had beendemonstrated beyond doubt the possibility suggested itself ofusing nutritional deficiencies of microbial- growth, which is morerapid than the growth of animals and 'can be followed undermore readily controllable and reproducible conditions, forrevealing the existence of new growth factors. Fildes was oneof the pioneers of the development of this method, Which wassuccessfully used for the discovery of several importantvitamins. This work has been recently recognized by the awardof the Copley, medal of the Royal Society.

Furthermore, the old method of Harden and Young of usingcell-free enzyme systems for the recognition of new cofactors,either by dialysis or by other detection techniques, has con-tinued to give rewarding results also-in more recent tumes: thediscovery of coenzyme A by Lipman, which had such far-reaching repercussions for our understanding of -the biosynthetic'pathways of fatty acids, steroids, rubber, and many other naturalproducts, is a particularly brilliant example. He found that liverextracts were capable of acetylating sulphanilamide, lost thisproperty on dialysis, and regained;it'when boiled liver extractor a concentrate of dialysate was added. Fig. 7 lists some of theimportant cofactors 'discovered by nutritional or biochemicaltechniques.

Discovery of New Cofactors

ABThrouglh Nutritional and Clinical Studies Through Studies of Ifolated Enzyme Systems

On On' Cofactors Enzyme Systems th.oughAnimals Micro-organismtI which Discovered

Vitamin A

Thiamine ((I)Riboflavin (B2)Nicotinic acidPyridoxinePantothenic acidFolic acidVitamin B1',Vitamin CVitamin KTocopherol

Biotin

p-Aminobenzoic acidLipoic acidFolic acidVitamin BLsStrepogeninMevalonic acid

M Ao- anddinucleotides

NADNADP

PAD

ATPUTPGTP

PolynucleotidesTransfer RNAMessenger RNACoenzyme A

Coenzyme Q

Alcoholic fermentationGlucose-6-phosphatedehydrogenase

Glucosee6-phosphatedehydrogenase

GlycolysisGalactoisomeraseProtein synthesis

Protein synthesisProtein synthesisAcetylation of

sulphanilamideRespiratory chain

FIG. 7

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Biochemical Research-Chain

As can be clearly seen, animal biochemistry and chemicalmicrobiology are inextricably mixed up in this field, for therevelation as well as the preparation of cofactors, and in many

cases the isolation of the cofactors, involved large-scale produc-tion of micro-organisms. Each of these substances has openedup a new important chapter of biochemistry. The study oftheir function forms an essential part of the field of enzymologyto which a large proportion of the biochemical research efforthas been and still is devoted. Most of these cofactors have beenshown to form part of important enzyme systems, and as suchto possess a highly important metabolic function.

7. Elucidation of Biosynthetic Pathways

The elucidation of the pathways of metabolism is one of themost important tasks of functional biochemistry. Very great

progress has been made in this field, and we are now familiarwith the essential steps of most of the important metabolicpathways.

This achievement is a major landmark in biochemistry andhas been brought about by enzymological studies in cell-freesystems, in conjunction with studies designed to follow the fateof metabolites in the intact cell, organ, or organism.Three phases in this research development can be

distinguished: (1) the pre-isotope era, (2) the isotope era, and(3) the era of biochemical genetics.

Pre-isotope Era

In the pre-isotope era the most important information was

obtained from enzymological studies of alcoholic fermentationand glycolysis in cell-free extracts of the Buchner type, whichled to the complete elucidation of the pathway involved(Fig. 8). Closely connected with and actually emanating from

AA

glucose qlucose-6-phosphate-q'lucose-l-phosphate qlycogen+PJ(hexokisase) (phospho-_heso-somerase)

fructose-6-phosphate1j (phospho-hexokinase)

heiose-d s-phosphate(a Idol in)

ATP 2 triose-phosphate 4di-hydroxy-acetone-phosphate)\~~~(qlyceraldehyde-3-phos phate

Csriose-phosphate-isomerase)glycerolaehyde-3-phosphate -2H

CADP) (DPN) (t riose-phosphaite dehydroqenase)l-3-di-phosphoglyceric acid

.TP (phosphoqlyceric acid transphosphorylase)3-phosplsoqlyceric acid

( phoioholyceromutase) (DNP)

2-phosphoglyceric acid

U ( enolose)CADP) (enoD) ptosphopyruvicacid

Cphosphoentl-transphosphoryla se)pyruvic acid +2t

U Clactic dehydrogenase)lacticacid

FIG. 8.-Glycolytic Meyerhof-Embden system.

these studies was the discovery by the Coris of the enzyme

phosphorylase, which brings about the phosphorylysis ofglycogen and starch to glucose-l-phosphate and, by reversalof this reaction, the synthesis of starch and glycogen throughcondensation of glucose-l-phosphate units in the presence ofa small amount of polysaccharide primer with the eliminationof phosphate (Fig. 8); glucose-l-phosphate was first found bythe Coris as a breakdown product of glycogen in muscle extracts.

Phosphorylase, as well as most of the enzymes involved inalcoholic fermentation and glycolysis, has been obtained in the

crystalline state. These investigations led to the elucidationof function of several important new cofactors, with repercus-

sions far beyond the limited field under study. The discoveryof the function of vitamin BI as the coenzyme of carboxylasewhich catalyses the decarboxylation of pyruvate to acetaldehydeand CO. by Lohmann was a further important milestone; itsimportance for pyruvate oxidation in animal tissues had pre-

viously been recognized by Peters. The mechanism by which,

D

BRrrHMEDICAL JOURNAL 215

in the light of the latest experimental evidence, vitamin B bringsabout anaerobic and aerobic decarboxylation of pyruvate isshown in Fig. 9.

CHCCOOCOOH + N"-C -CH2-0- C-CH,

C HcC1 .CHCHOH HCC'NCNHNH,N CS'CH2-C CH2 0- P-0- 0-

N''lC- CH2OH Nt-C-CH3H IN C C 0C ,, ,H _ Iz IC2 CH - -P °

Wz*H HC -CH NN+ t-C -H3 -9H

NH2 CH2-CH2-0-P -o - 0

anaerobic in yeast aerobic in animal tissues 0

ffi ffi S-S-lipoic aci dCHSCHO+CO2 S*COCC 3 SN

+ thiamine - H-CH2-+ 2 thiamine pyrophosphatepyrophosphate (C

COOHNADH I CoASH S S

CoA-S-CO-CH3 +CH2-CHd-CCH+NADoxidation via citric( H2),eacid cycle COON

FIG. 9.-Non-oxidative and oxidative pyruvate decarboxylation.

The discovery of another cofactor of alcoholic fermentationand glycolysis with tremendous repercussions was that ofadenosine triphosphate (ATP) (Fig. 10), which was shown tobe the main chemical mediator for the storage and transferof oxidative energy. The names of Meyerhof, A. V. Hill,Lohmann, Karl Meyer, Lipman, and many others are asso-

ciated with the discovery of the functions of ATP.

NH2C-NH2 0

NC C-N O OH O0 0

>CH I 1 11 11N-C -N < tCH-C- -CM-CN-C,-O-P-O-P-O-P -ON

N ON ON

FIG. 10.-Adenosine triphosphate (ATP).

Its importance became clear only slowly, over the course ofmany years, and to a large part in the second era of the studyof intermediate metabolism, when isotopes became available.It was found capable of undergoing several types of reactions,of which the most important ones are (Fig. 11):

1. The transfer of the terminal phosphate to a wide variety ofsubstances, sugars, phosphorylated sugars, nucleotides, nucleosides,pyruvate, creatine, arginine, cofactors such as pyridoxine andmevalonic acid and mevalonic 5-phosphate. In all cases thesubstances became more susceptible to enzymic attack and thereforemetabolically more reactive through phosphorylation; in some cases

the energy of the terminal ATP phosphate link is retained in thephosphorylated products and enables them to undergo energy

requiring condensation reactions. Thus, for instance, Popjack hasshown that molecules of mevalonic acid 5-pyrophosphate undergocondensation with each other to give squalene and higher terpenoids.

2. It can undergo enzymic pyrophosphorylytic cleavage to giveAMP and a pyrophosphate ester, for instance in the reaction withthiamine, which leads to the formation of thiamine pyrophosphate,the biologically active form of thiamine or with UMP to giveUTP. The latter, after condensation with glucose-l-phosphate,through the action of another pyrophosphorylase, to give UDPG,was found by Leloir to have an important metabolic function incarbohydrate metabolism as the coenzyme of galactose-4-isomerase,converting galactose phosphate into glucose phosphate, and as

coenzyme of glycogen synthesis.3. It can undergo pyrophosphorylytic cleavage to give mixed

anhydrides, for instance, with carboxylic acids and amino-acids,which are thus activated for synthesis of long-chain fatty acids andof proteins respectively, the latter via transfer R.N.A. and" messenger " R.N.A. (see below).

4. It can undergo condensation with other nucleotides throughenzymic pyrophosphorylysis to give the dinucleotide coenzymes ofthe respiratory chain NAD and FAD and mononucleotide

triphosphates.5. ATP can undergo enzymic condensation with other ribo-

nucleotide triphosphates containing purine and pyrimidine bases to

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give high molecular RNA under the action of a polymerase and theelimination of pyrophosphate. This reaction, like the above-mentioned polymerization of glucose-i-phosphate to starch orglycogen, needs a high polymer primer, which in this case is highmolecular RNA or DNA. The primer also acts as template. If,for instance, DNA of different origin is used as primer, the resultingRNA corresponds in its composition and nucleotide sequence to theparticular DNA added. This observation is obviously of greatimportance for the transmission of genetic information.

REACTIONS OF ATPI. Kinases

ReactantGlucoseGlucose-6-phosphatePyruvateCreatinePyridoxineMevalonate5-PhosphomevalonateRibonucleosidesAMP

ProductGlucose-6-phosphate + ADPGlucose- 1,6-diphosphatePhosphoenol pyruvateCreatine phosphatePyridoxine phosphate5-Phosphomevalonate5-PyrophosphomevalonateMonoribonucleotides2 ADP

BRITISHMEDICAL JOURNAL

our studies, which I shall briefly mention later, on the metabol-ism of nervous tissue, the mode of action of insulin, and otherquestions regarding intermediate metabolism.The spectrophotometric studies of Britton Chance are of

particular interest in this connexion ; quantitative spectro-photometric scanning of some of the most important knownredox components of the respiratory chain and simultaneouspolarographic determination of oxygen consumption show thataddition of ADP immediately leads to increased respirationand characteristic changes in the state of oxido-reduction of theredox components until it is fully phosphorylated (Fig. 12).The respiratory chain and the machinery for oxidative phos-

phorylation are carried by a subcellular fraction, obtained bycentrifugal fractionation of broken-up cells: the mitochondria.We owe very largely to the work of Green and his colleagues,

2. PyrophosphokinasesReactant Product

Thiamine Thiamine pyrophosphate--AMPUMP (uridinemonophosphate) UTP (uridinetriphosphate)+AMP

3. Carboxyl activation through pyrophosphorylysisReactant Product

R-CH2-COOH AMP-O-CO-CH2R + PPR-CH-(NH2)COOH AMP-O-CO-CH(NH2)R + PP

4. Dinucleotide formation through pyrophosphorylysisReactant Product

NMP (nicotinamide ribose monophosphate) NAD+PPFMP (flavine monophosphate) FAD + PP

5. Polynucleotide formationProduct

'dApdeoxyribonucleotide fdTp +nPP

polymerase dCp

dGpI n

ribonucleotide

polymerase

ribonucleotide

phosphorylase

FIG. 1 1

t i _-13mm=660o-m- p30Ot-

A0P 0 CYTOCHROME CI OXIDATION

551- S40 Om-O o/l OOOS

fAp

Up + nPPCp

iGpJ n

fAp

Up +nP

CpGp

Kornberg discovered that DNA itself can be synthesizedenzymically from a mixture of deoxyribonucleotide triphos-phates, also with the elimination of pyrophosphate. A primeris required, too, for this reaction.

Finally, mention must be made in this connexion of thephosphorylase discovered by Ochoa and Grunberg-Managowhich condenses nucleotide diphosphates containing bothpurine and pyridine bases with the elimination of phosphate,and does not need a primer. This enzyme condenses singlenucleotide diphosphates of one base composition to give poly-adenylic, polycytilic, etc., acids, or of different base composi-tions, giving rise to high molecular ribonucleic acids withvarying proportions and varying sequence of bases. As willbe discussed later, the artificial synthetic nucleotides haveproved very useful tools in studies of protein biosynthesis.ATP is generated principally by oxidative energy-that is,

the energy generated during electron transfer at some step ofthe respiratory chain ; the mechanism of this process has been,and still is, the object of intense study by many investigators,but despite the immense amount of work expended is still veryimperfectly understood.We ourselves are actively engaged in a study of this problem,

and shall continue for some time to come, in connexion with

=1~~3 mm-

ir AOI

. u2= r!CYTOCOE IN

Jr4B ' ~- OXIOATION

ADP

-t t 13i T I; | \ 13^" | ~~~~~~FL AVOPvq~ti

Ws=M+ oxio.^ 014

s-46 SiO ^ lot [o/I t ooos -

-1 I~~~5I

.1 I- iC7771-7- 1v i i M%.4 ia I I -A 4.-A I a I

I

P EOUCE0PYRIDINENUCLEOTIDEOXIOAT ION

Jr: I = 30,pM_~ ~~~ .N >ADP r7

-:340 3L4 rm)--log to/ - G 4 ~-

FIG. 12.-Influence of substrate and ADP on redox systems of respiratorychain. (Britton, Chance, and Williams, G. R., 7. Biol. Chem., 1955, 21,

409.)

216 23 January 1965 Biochemical Research-Chain

A

Reactant

( ndATP

ndTTP

ndCTP

ndGTP

( nATP

nUTP

nCTP

nGTP

( nADP\

nUDP I

nCDP 5

nGDP

oc F ff f II A I YA i I

-----L-A pl#%A% 0% di 9 i HAWOf

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systematically and consistently continued over many years, therecognition of the very important fact that in these particles allthe numerous enzymes involved in the process of oxidativephosphorylation are present in a well-coordinated structuralunit and that the structural arrangements in themselves areone of the most important factors responsible for the efficiencyof the system.The recognition that heterogenicity is an intrinsic property

of many of the most important enzyme systems, both fordegradative and for synthetic processes, signifies a mostimportant advance in biochemical thinking and should beconstantly borne in mind. We cannot hope to progress veryfar in our understanding of metabolic reactions if we limitourselves to the study of the structure of soluble enzymes inhomogeneous solutions, which is of course a much simplerproposition than the study of water-insoluble heterogeneoussystems composed of proteins, lipids, and carbohydrates inwhich each component fulfils an important role.A better understanding of reactions in the solid state appears

to be an essential prerequisite for the understanding of thefunctioning of metabolic reactions.

Other important advances in our knowledge of intermediatemetabolism in the pre-isotope era was the recognition of thedirect pathway of glucose oxidation by Dickens (Fig. 13), thedegradative pathway of fatty acids by sl-oxidation via the corre-sponding keto acids by Knoop, and the consolidation of theconcept of metabolic cycles, which had the widest repercussionsin the urea (Fig. 14) and tricarboxylic acid (Fig. 15) cyclesproposed by Krebs.The investigations of Krebs are characterized by extensive use

of manometric analytical techniques for the determination ofmetabolites, using the apparatus introduced by Warburg.

H-C-OH C02-C /HO-C- H

H-C-OH H+ H-C-OHNHO-C-H 0 H2O H-C-OH NADPH NADP

H-C-O CH20PO;NADPH+HH- C b-Phospho-D- D-R

NADP, C H20PO; gluconateH, ,OH b PhosphoD-o

C gluconolactoneH-C-OH fHc, OH

HO-C-H H-C-OHH- - O ADP+H 0H-C-ON

N- TP NIHHCo-Glucose-b- C CH2OPO

phosphate H-C-OH D-Ribose-5-phosphate t

HO-C-H 0H-C-OH TPP

H-C'C H20OH

CH20H D-Glucofe H.O

/OH H-C-OHC.IC H20P.03

-iO-C-N 0 o-Glyceroldehyde-3-H-C-ON phosphateH-C D-!

CH20PO \v-Fructose-6-

phosphateATP

ADPH+ \ H-C-OH

CH20PO3 H-C-OHIOH CH20PO

Ho-~i \ D- E rythrose- 4- phcH-C-OHI

CH20PO;, H-C-OHa Fructose-I,6- I

diphosphate CH20P0; TPP

CH2OH o-Clyceraldehyde-3-phosphateC=OC H20P03

Clihydroxyocetone-phosphate

C H20HC=O

H-C-OHH-C-OH

C HN0PO3

Ribulose-5-phosphate

CH20HC=O

HO-C- NH-C-OH

C H20PO3D-Xylulose-5-phosphote

CH20HC=O

HO-C-HH-C-OHH-C-OHH-C-OH

CH20PO3Sedoheptulose-7-

phosphate

CH2 OHC=O

HO-C-H)l H-C-OH

CH2OPO3osphate D-Xylulose-5-

phosphate

-a

Fig. 13.-The pentose phosphate cycle.

BRMISHMEDICAL JOURNAL 217

Isotope Era

The introduction of heavy and radioactive tracers and ofthe various chromatographic techniques, used in combination,led to an almost explosive expansion of the field of the studyof metabolic pathways.Not only were many of the concepts of the pre-isotope era

confirmed-for instance, the existence of the urea and tri-carboxylic acid cycles-but much new knowledge accumulated.

0-

N4, + HCO_ + 2ATP NH2- COO' P O

0o

NH2( CH2)3C H-NH2COOH

urea-.J arqinase

NHC-NH2NH +

(CH2)3C H-NH,COOH

NH-CO-NH2

corbomyl phosphate (cH2)3transcarbomylose CH-NH2

COOHCOOH AT P.

ICH-NH2 arginino succinote synthetaseC H2COOH

NH COOHasportate 11 N

C - NH - CHCOOH NH CH2CH (CH2)3 COOHCH CH-NH2COOH COOH

arqinine fumarate arqininosuccinate

Fig. 14.-The urea cycle.

C6 H,206

C H3CO-COOHI 2H--CO2

active acetateactive acetate + COOH -C H2CO*COOH

oxaloacetic acid

COOH-C H2CtOH)*- CH2-COOHCOOH

citric acid

COOH -*CH2CH*CHOH-COOHCOOH

isocitric acid

I -2HCOOH* CH2CH-CO- COOH

COOHoxalosuccinic acid

+HO-2 COOHNCH=CHNCOOH

2H fumaric acid

I -2H

COOH-CH2-CH2-COOHsuccinic acid

-2H-CO2

CO2 * COOH C*CHiCO-COOHc. ketoqlutaric acid

Fug. 15.-The tricarboxylic acid cycle.

Biosynthesis of the porphyrin ring via O-aminoloevulinic acid

t0O-SCoA NH2-CH- COO:HNH2-CH2-COO)H+CH2-CH2-COOH_--i I . __ pyrdoxcl-

t CO-CH2-CH2-COOH + CoASH p osphate

t tNH2-CH2-CO- CH2-CH2-COOH

6-aminolaevulinic acid

tCOOH tCOOHtCOOH CH, tCOOH CHICH2 CH2 CH2 CH2CH t- tCo 6-aminolaevulinic C Ct

CH acid dehydraseCH2," NH I-, -2H20 tC *CH

CH2 NHNH2

NH2

4 pyrrols - porphyrin+6 "CO2CH2 CH2

CH3 CH CH3 CH

Ct==H CCH CN ~~~NH

CHNH

\ NH N* *tC' 'C=HC-C C

t. 1. oC=CfCH, CH, CH2 CH13

tCOOII) tCOOH

Fig. 16.-Biosynthesis of the porphyrin ring via C -ami'nolaevulinic acid,

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218 23 January 1965 Biochemical Research-Chain

Highlights in this development are the elucidation of thepathways of the synthesis of the porphyrin nucleus fromglycine and succinate via 8-amino-leuculinic acid by a seriesof simple reaction steps (Fig. 16), of the purine nucleus(Fig. 17), of the fatty-acid chain (Fig. 18), of the steroids andterpenes via mevalonic acid (Fig. 19), and the pathway ofphotosynthetic CO2 fixation (Fig. 20).

Simultaneously with the elucidation of the various metabolicpathways, and as an essential part of this work, the functionof the various coenzymes became understood. For instance,pyridoxal phosphate was shown to be involved in transamina-tions, amino-acid decarboxylation, a, ,B cleavage of /3-hydroxy,a-amino-acids (serine to glycine and formaldehyde), condensa-tion reactions (indole + serine-tryptophan), folic acid in transferof one-carbon compounds and for methyl formation, biotin inCO2 activation for malonyl CoA formation in fatty-acidsynthesis, pseudo-vitamin B12 in transmethylation reactions, inthe isomerization of glutamate to /3-methylaspartate and methylmalonate to succinate.The latest and one of the most exciting developments in this

field is the elucidation of the biosynthesis of proteins, and theinfluence of nucleic acid in this process. A number of con-

CH2-NH2

CO

NH OH OH H °

HC-C-C-C-CH2O°-P-OI l ~~~~~~~~~I

H H

O-glycinamide ribotide

H2C NH 0

C-H

NH OH OH H °-

HC-C- C- C- CH20-P-OI

H0

H 0

N-formyl alycinamidine ribotide

0

C

h42N C N

H2N N OH OHH °

HC-C-C-C C H2°-P-O0

0

5 amino-4-carboxamidoimidozol-ribotide

CH2- NH 0

CO C-H

NH OH OH HII

HC-C-C-C-CH2O-P-06

Po~N-formyl glycinamide ribotide

HC-N

-H20 C CH

N HN'\N OH OH H O-HC-C-C-C-CH2 0-P-0O

H H 0

0d<

verging lines of research, at first without any apparent con-nexion between each other, led to the present state of know-ledge: (1) the discovery of the chemical nature of the trans-

Synthesis of fatty acids via malonyl coenzyme A

~bicotinCH3-CO-SCOA+ C°2-ATPCH2-CO-SCoACOOH

CH-3(CH2-CH2)n- CO-SCoA 4- CH2-CO-SCoA-bCOOH

CH3-(CH2-CH2)t CO-CH27CO-SCoA+ CoASH+CO2NADP I H+

CH3-(CH2-CH2)n-CHOH CH2 CO SCoA

C H3-(CH2-C tH2)n- C.H=CH -CO-SCoANAOP IH +

C H3-(C H2-C H2)n- CH2-C H2-CO -SCoA

CH3-(CH2-C H2)nil -COOH+ CoASH

Fig. 18

2CH3-CO-SCoA - CH3-CO-CH2-CO SCoA C3_COSCoAacetyl CoA acetoacetyl CoA condensinq enzyme

CH3 CH3OH-C-CH2-COOH OH-C-CH2-COOH

¢H2 ~~NADP HCH2 H-- CH 2

co CH2 OHSCoA

3-hydroxy, 9 methyl- mevalonic acidqlutaryl-CoA

qlutamine

CO2

NH2(aspartateqlutomate)

OH

Cl unit N C N

ATP HC C CH

NN- OH OH H

HC-C- C-C-CH20-P-O

0inosinic acid

Fig. 17.-Synthesis of inosinic acid

mevalonic acid + ATP-- mevolonic acid-S-phosphatemeva lonic acid-S-phosphate mevalonic acid-5-pyrophosphate

+ ATPCH3

OH-C- CH2-COOH

CH2 O O

CH20- P-O-P O-AP

C02 °6

-H20CH3 CH3C=CH2 somerase C-IJH,

CH2

CH20-P-O- P-O CHO- P-O-P- OO 0 0 0

is ,pentenyl pyrophosphare vy-dimethylallylpyrophosphate(Activce soprene)

CH3 0 0O

C ICN2o P-pOPo isopentenyl pyrophosphate

CH3 CH 0 0

H3 CH3

".C -CH2 /C s

CH3 CH CH2 CHgeranylpyrophosphate

*-H3

CH3 CH

carboxydismutaser system H2C- oTl

H2COEPI HO-CH triose phosphateO dehyd rogenase

C-C-OH CO2 TPNH H2C - OSATP

C02 C-O 2 - HO-CHH20 -H20

HCOH CO2 2 =H

IH20[P1 HC-20HG/ 3-PClyceraldehydeCH2O0I HC-OH e

_Of

H2C-OEffI / </ot ~ aldolase H2C-O

3-P-Glyceric acid

IHO-CH

H2C-O HNC=O H2C-O0[I HC=O HC-OH

C-O HC-OH / CO HC-OH HC-OH

HC-OH C- OH H2C-OH HC-OH H2C-O[J

H C-OH HC-ON / P-Dihydroxyocetone H2C-O[B Fructose-lb-DiP

H2C-Ot0 H2C- o[E Erythrose-4-PRibulose-l-DP' Ribose-S-P /tH20 hsate

tATP E t / transketolase hoptsphosphopentokinase y. H2l-UN

H2C-OH H2C-\O C=OI /1 C=O CO=O HO-CH

H2C ON HCH NiH-CH HC-OH

NCON~~ NC -OH HC-OHC= O HC-OH < 1HC- OH \ HC- OHHC- OH H2C- OH NC-OH HC- OH H2C- O[

phosphataseHC-OH C=O HC-OH HC-ON Fructose-6-P

H2C-Om HO-CH H2C-O[0 H2C- \

Ribulose-S-P HC-OH Sedoheptulose -7-P Sedoheptulose-l, 7-DiP

H2C- O*mphosphoketopentose transketolase

epimeiFase Xylulose P

Fig. 20.-The photosynthetic carbon-reduction cycle.

0- 0- isopentenylpyrophosphote

11 u pyrophosphorolysisO 0

CH3 CH3 01- 0

,CH2 cz"I0H2 oC+N CH2O-P-O-P-0-

CH2 CH CH2 CHfornesyl pyrophosphate

2 iarnesylpyrophosphateCH3 CH3 CH3

C CH2 C CH2 C CH2

(<3 CH CH2 CH CH2 CH

CH3 CH3 CH3C CH2 CH2 C CH2

CH 3 CH CH2 CH CH2 CH

squa lene

CH3N/CH2 CH CH3

CH2 C CH2 C

CH2 CH CH CH3C N3

CH CH CH2 squaleneCH2 CNCNI

CH2 C C CH2

;tc / ,\ HX/HCH CH21 CH3H2CH /CH3

C CN2 CH CH2 CCN3 CN3 3 CH3

laniosterolICH CN2 CH2 CH3

Y\ /\ /

CH CH2 CH

CN3

cholesterol

HO CJCH3CH3

HO

Fig. 19.-Pathway of steroid synthesis via meva-Ionic acid and squalene.

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Biochemical Research-Chain

forming agent as DNA, already referred to ; (2) biochemicaland genetic studies on viruses and bacteria; (3) the enzymaticsynthesis of polynucleotides ; (4) the discovery that proteinsynthesis can be brought about in cell-free enzyme systems, ofmammalian and bacterial origin, when suitably supplemented.It has been recognized (Fig. 21) that this occurs in severalstages, the first of which is the activation of amino-acids alreadyreferred to. This is followed by the attachment of the activatedamino-acid to a ribonucleic acid of low molecular weight,termed transfer nucleic acid (each amino-acid having its owntransfer ribonucleic acid), and finally the amino-acid is trans-ferred from the transfer of nucleic acid to the ribosomes andincorporated into the protein. This is done under the directinginfluence of a ribonucleic acid, of molecular weight of about150,000, which, as has already been mentioned, is formed underthe directing influence of the chromosomal DNA and is itscopy in miniature; it has been termed "messenger nucleicacid " because it conveys the genetic message for specific proteinsynthesis for the chromosomes to the amino-acid-assemblingmachinery of the protein-synthesizing enzyme system. Thisitself is non-specific, like a printing machine which can repro-duce any set of type. The artificial ribonucleotides have provedvery useful tools in the elucidation of the role of messenger

FORMATION OF TRANSFER RNA ACTIVATION OF AMINO ACIDS

sRNA R-CH-COOH-enzymer

2CTP 3FFPNH2

ATP ATP

c1- H enzyme (PP)sRNA-Cy-Cy-O- P-O-HC-C CH-OH R-CH-COO-AMP- enzyme

H I II0 OxC H-OH NH2

transfer RNA activated amino acid

adenine

|-o- TV Enzyme, AMP, PP

FORMATION OF TEMPLATE sRNA- Cy-Cy-O-P-O-CH2-C-CHO-CO-CH-Rchromosome DNA R

messenger RNA I\ _ _adenln~~~~~~~~~~aene

r osos e

transfer RNA

PROTEIN

FIG. 21.-Protein synthesis.

ribonucleic acids for specific protein synthesis. With theirhelp it could be shown that the sequence in which the amino-acids are incorporated into proteins is determined by thesequence of nucleotide units in the messenger ribonucleic acids,each unit probably consisting of three nucleotides (so-callednucleotide triplets). With the help of synthetic nucleotides ofdifferent nucleotide composition and sequence it could beestablished which specific sequence of ribonucleotides in thetriplets was required for the incorporation into proteins of eachindividual amino-acid. As the messenger ribonucleic acids are

formed under the influence of chromosomal DNA and are

their exact copy, nucleotide triplet sequence determines thespecific genetically determined amino-acid sequence in proteins.The sequence of ribonucleotides in the ribonucleotide tripletsfor incorporation of individual amino-acids is known as

"'genetic code" (Fig. 22), which appears to be universal.Fractionation of the protein-synthesizing cell-extracts by

centrifugation in a density gradient has shown that the amino-acid-assembling system is associated with a subcellular particu-late fraction, the ribosomes, which needs a high gravity fieldfor sedimentation.

High-speed ultracentrifugation in density gradients in whichgravity fields of 100,000 g and more are reached and whichhas to be continued in some instances for periods of severaldays has become, despite the complexity and cost of the equip-ment, an indispensable and widely used technique, not onlyfor the preparation of subcellular fractions, but also in the

BRITISHMEDICAL JOURNAL 219

nucleic-acid field, for analytical as well as preparative purposesaimed at the separation of the metabolically active nucleic acidsof different molecular weight. The ultracentrifuge was devel-oped many years ago by Svedberg for the determination ofmolecular weights of proteins and used only for this limitedpurpose by a small number of protein chemists specializing inthis field. Now it has become a standard piece of biochemicalequipment and represents a good example of the ever-increasingcomplexity and costliness of modern biochemical methodology.

"Modified" Suggested Functional TripletsAmino-acid Base

Pairs ==U °-A n=C

Lysine ... A`A AUA AAAAsparagine ... °AA,C°A CUAUAA CAAHistidine ... A`C AUC ACCGlutamine ... °AC,GG° UAC AAC,GGAGlutamic acid A°G AUG AACTyrosine ... A°U AUU ACUThreonine ... OCA,°GC UCA ACA CCA,CGCProline ... COC CUC CAC CCCAlanine ... COG CUG CAG CCGSerine ... C°U,°CC,°CG CUU,UCC ACGAspartic acid GOA GUA GCAMethionine ...°GA UGAArginine ... G°C,G°A GUC GAA GCCGlycine ... G°G GUG GAG GCGTryptophan ... OGG UGGCysteine ... G°U GUUIsoleucine ... °UA,°AU UUA AAU CAUValine ... U°G UUGLeucine ... U°C,°CU,°AU,°GU UUC,UAU,UGU CCUPhenylalanine U°U UUU UCU

FIG. 22.-Suggested assignments of messenger RNA triplets to codingfunctions in the amino-acid code.

Era of Biochemical GeneticsThe introduction of microbial genetic methods for the study

of intermediate metabolism by Tatum and Beadle is anothermilestone in biochemical research. The concept that enzymicreactions are gene-controlled was arrived at on the basis ofthe study of certain metabolic abnormalities in man (inbornerrors of metabolism) and the genetic transmission of antho-cyanine pigments of known constitution in plants; the namesof Garrod, Rose Moncrieff, and Haldane are prominently asso-ciated with this idea. Beadle and Tatum, using the fungusNeurospora crassa, were able to demonstrate that each enzymicstep in a chain of biosynthetic events is controlled by one gene.Here again much of their success depended on choosing thisparticular organism.The general principle of the method which they used for

studying the metabolic pathway of a given metabolite-forinstance, an amino-acid, a purine base, etc.-was the following:a wild strain of Neurospora crassa which was capable of grow-ing in a simple medium was treated with mutagenic agents (xrays, ultra-violet rays, chemicals). Mutants with blocks inthe biosynthetic reaction sequence of the metabolite under studywere thus obtained which were not able any more to grow,or grew only at a retarded rate, unless supplemented with themetabolite under study, or its precursors after the geneticblock. In some cases precursors of the metabolite in the reac-tion chain before the metabolic block accumulated and couldbe isolated and their constitution elucidated. It was also foundpossible in some cases to demonstrate the absence of an enzymebringing about the reaction interrupted at the genetic block ofthe biosynthetic reaction sequence. These metabolic geneticblocks were later shown to be due to changes brought about inone single Mendelian gene by the mutagenic agent.With this method steps of the biosynthetic pathways of many

important metabolites were established-amino-acids, vitamins,purines and pyrimidines, sulphate, nitrate, etc. A particularlyimportant outcome of this work, emanating from its applicationto the biosynthesis of the amino-acids phenylalanine, tyrosine,and tryptophan, was the discovery of the pathway of bio-synthesis of the aromatic ring system via shikimic acid (Fig. 23).The analytical techniques employed in the study of pathwaysof intermediate metabolism have also become very complex.

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220 23 January 1965 Biochemical Research-Chain MRITALJISOHRNChromatographic separation methods of all kinds-paper,

column, and gas chromatography-have made a tremendousimpact in this field, as they have in all other fields of analyticaland preparative chemistry. Used in combination with variousmethods for determining the isotope content of tracers, theydominate the field. For determination of 14C in the form of14CO2 the low background anticoincidence counter has found

COOH COOHCHO C-OP COOH /HCOH CH2 Co HO, COOH COOH

phosphoenol IHHO¢H pyruvic 0. ¢ 2 A\I JOHHCOH CHO >_ HOCH __ __ | HO OH

I ICHOH ~~~~~~~~~~~~OHHCOH CHOH COHHOH OOH smic acidCH20H CHOH CHOH

glucose OH OH COOHCH20F CH2OP 5-dehydroquinic acid 5-dehydroshikimicerythrose-4- 2-keto-3-deoxy- acidiphosphate 7-phospho-glucoheptonic a.

COOH C OOH COOH

phosphoenolphyruvate CH2 CH2 OHB1 1I COOH

TO H O ot OCOH C\fcr,\\I\OH P O" O/'tOOH 0'"COOH/OH OH OH .

Z, phosphate ciorismic acid

COOH COOHCHNH2 CO NH2

CH2 CH2 HOOC CH2CO-C00H COOH

/ / /0j NH2

phenylolonine tyrosine prephenic acid tryptophan

FIG. 23.-Biosynthesis of aromatic ring system.

wide application, but is rivalled by the considerably more costlyscintillation counter which is suitable for /3 as well as for yradiation, and therefore allows the determination of tritium,iodine, etc., in addition to 14C. It has the great additionaladvantage that it allows the determination of radioactivitydirectly in fractions coming from column chromatography,

without first having to dry aliquots or to combust aliquotsto CO2.The chromatographic as well as the isotope-determination

methods have been automated to a large degree. For the studyof protein biosynthesis and similar problems the automaticamino-acid analyser has become an indispensable tool, whichallows not only the neat separation of complex mixtures ofamino-acids and their individual quantitative determinationbut also simultaneously the determination of their specificactivity. Heavy isotopes are also extensively used in studiesof metabolic pathways and the mechanism of action of theenzymes involved, particularly 180 and 15N, but also 2H. Forthe quantitative determination of the former mass spectrometricmethods are used.

Looking back from our present position on the achievementsattained in the field of metabolic pathways in the course ofonly thirty years, the progress is certainly impressive. How-ever, despite all this progress, we are still as far away as everfrom our final goal, to understand the biochemical functioningof the cell.

It is true we have discovered a great number of new bio-chemical reactions and reaction sequences, but it is surprisinghow little we know about their significance in the living cell,their relation to the specific functions in a particular cell kind,and the way in which the various reaction rates are regulatedand interdependent. Yet these are problems of the very greatestimportance, theoretical as well as practical, for all fields ofbiology, and not least for medicine-for metabolic disordersare caused by changes in metabolic reaction rates.

This brings me to the discussion of a field of research inwhich we are actively engaged at present and which will formone of the main topics of research in the newly constitutedMetabolic Reactions Research Unit of the Medical ResearchCouncil, to be housed in our new Biochemistry Department:the mode of action of hormones.

[The conclusion of this Inaugural Lecture will be publishedin next week's issue.]

Primula Dermatitis

ARTHUR ROOK,* M.D., F.R.C.P.; HAROLD T. H. WILSON,t M.D., F.R.C.P.

Brit. med. 7., 1965, 1, 220-222

Most doctors and many gardeners know that primulas maycause dermatitis, but general practitioners are often unfamiliarwith the pattern of the rash produced. It was felt, therefore,that a clinical description would be helpful.

The Primulaceae

The primula family comprises some 22 genera and 600species. The genera include Primula, Androsace, Soldanella,Lysimachia, Glaux, Anagallis, and Cyclamen.The genus Primula contains over 200 species, including the

common primrose and the cowslip. Those most commonlygrown as house-plants are P. obconica (Fig. 1) andP. malacoides. P. sinensis is retailed in smaller quantities,while P. kewensis, P. floribunda, P. denticulata, and P. mollis

also appear in florists' shops from time to time. With veryrare exceptions only P. obconica causes dermatitis.

* Dermatologist, Addenbrooke's Hospital, Cambridge.t Dermatologist, Central Middlesex and Royal Northern Hospitals,

London.

Historical

P. obconica (Fig. 1) was introduced to Britain from Chinain 1880 and rapidly became established as a popular green-house plant throughout Europe and the United States. Thefirst accounts incriminating this plant as a cause of dermatitiswere published in American horticultural journals and werereceived sceptically in England. However, reports of similarexperiences in this country soon followed; one indignantgardener vigorously condemned " this vegetable viper." A yearor two later reports began to appear in the medical journals,first in America and soon after in Britain (Sym, 1890 ; Clarke,1890; Ferguson, 1890). So numerous were the reports thatit was predicted (Cooper, 1899) that such a poisonous plantwould be grown less and less frequently; but this prophecy

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