Technical Information Philosophy of the Tracer Methods

A. A. BENSON Scripps Institution of Oceanography, University of , San Diego, La Jolla, California 92093

ラ ジ オ ア イ ソ トー プ が サ イ ク ロ ト ロ ン で 製 造 さ れ は じめ た1930年 代 か ら40年 代 に か け てErnest Lawrence教 授 を 中 心 にSam Ruben博 士,Martin Kamen博 士 ら は ラ ジ オ ア イ ソ トー プ の ト レ ー サ ー 実 験 を 開 始 し た,本 稿 は サ イ ク ロ ト ロ ン で 製 造 さ れ た 半 減 期20分 の11Cを 使 っ て 寸 秒 を 惜

ん で 行 わ れ た 活 気 に 満 ち た 初 期 の こ ろ の 様 子 。Kamenお よ びRuben両 氏 に よ る14Cの 発 見 と, そ れ に 続 く輝 か し い 多 くの 成 果 が 得 られ た 熱 気 に 満 ち た カ リ ホ ル ニ ア 大 学,ロ ー レ ン ス 研 究 所 の 人 々 の 活 躍 ぶ り,放 射 性 人 間 と い わ れ たKamen博 士 の 奮 斗 ぶ り,壮 絶 なRuben博 士 の 殉 職 の 事

件 な ど,当 時 い っ し ょ に 協 同 研 究 を して お ら れ たAndrewA. Benson教 授 が 直 接 語 ら れ た き わ め て 貴 重 な 資 料 で あ る 。 こ の 講 演 は1976年9月10日 他 団 体 と 日本 ア イ ソ トー プ 協 会 農 業 生 物 部 会 の 共 催 で 行 わ れ た 。

It is a privilege to discuss tracer methodo- radioisotope discovery and a birhtplace for logy with you today. Especially because much of radioisotope methodology. its development involved exciting experi- In many ways, Berkeley was an ideal ences with many Japanese colleagues who site for these developments. Ernest Law- rence**(Fig. 1) had invented the played such important roles in designing and executing dramatic experimental studies in 1931. He also had a strong interest in of nhotometabolism. Those adventures be- supporting biological applications of the gan a delightful and productive series of scientific and cultural ex- changes which have enriched both our lives and our Science. I have been told that tracer ap- plications of radioisotopes began in Manchester where George Hevesy used an artificially produced lead to confirm his suspicions that the landlady of the boarding house was actually re-using the left- over meat from the plates. Pro- gress in depth, however, awaited invention of the cyclotron and its application for production of a va- riety of radioactive useful for biological research. Berkeley, the birthplace of the cyclotron, the- refore became the major center for

† ト レ ー サ ー 法 の 哲 学,A.A.ペ ン ソ ン 教 授(右 図)カ リホ ル ニ ア 大 学,ス ク リ プ ス 海 洋 研 究 所,ラ ホ ヤ,カ リホ ル ニ ア92093 The author acknowledges support of U.S. National Science Foundation grants 5-S-285 and FJ-5053 in mak- ing this exchange possible.

(66) May 1977 A.A. Benson: Philosophy of the tracer method 349

by •gthe complexity of Nature•h.

In this environment Sam Ruben emerged

as the capable chemist and

as the physicist-chemist through whom Ernest

Lawrence would make his contribution to

Biology. By making it possible for Martin

Kamen to devote part of his efforts to col-

laborate with Sam Ruben and many other

chemists and biologists, Lawrence had con-

structed a most important organization for

biological research. Together, Ruben and

Kamen made more progress than any scien-

Fig. 1 Professor Ernest O. Lawrence at the tists I have known. It was not easy. They

controls of the first cyclotron in the often worked around the clock, and usually early 1930's. It was located in the Old Radiation Laboratory (ORL) from 8 to 2, seven days a week. Ruben's where it was later displaced by the family saw all too little of him. The labo-

laboratory of Professor ratory problems were technical as well as Melvin Calvin. biological. Instability of electronic cir-

newly available isotopes. Both chemists cuitry and unreliability of the home-made

and physicists in Berkeley had strong back- Geiger-Muller counter tubes, put together

grounds in thermodynamics and physical with sealing wax and temporary electrical

as a result of the influence of connectors, could ruin their carefully

Gilbert N. Lewis who had built the College planned experiments of a few hours duration. of Chemistry and outlined the training of With a 20-minute half-life, five hours was

its students with the textbook by Lewis and the maximum duration of 11C experiment.

Randall. All students understood the ther- Ruben's laboratory was in a three-story

modynamics of equilibrium, partition, ad- stuccoed wood frame building, fondly known

sorption, and diffusion, each very important as •gThe Rat House•h, the main entrance being

in development of radioisotope methodology. at the second floor. in-

All chemists were familiar with high vacuum habited the ground floor until he left for techniques, mercury diffusion pumps, Mac- the and nuclear Leod gauges, and liquid nitrogen applica- energy research. Ruben •glived•h in the rear tions so necessary for C* and H* syntheses of the ground floor, his laboratories being and analyses. There was little real knowl- located here and there in all three. The edge of biology or but plenty building had no ventilation system, no fume

of self-assurance stemming from the spirit hoods, bare wiring, bad lights, a noisy stair-

of G.N. Lewis and his feeling that Nature way, and little or no sunshine. Yes it was is always simple when we understand it. a great place to work and build. One could

This philosophy differs from that of many drill holes in the walls or connect wires modern scientists who appear overly awed anywhere. The lights were on most of the

** In 1953-55 devoted much of night and there were no inhibitions about

his talents to development of the three-color doing experiments.

electron gun for his color TV system. With Many 11C experiments were done nights the experience and talents of Harry Powell, or Sundays, when the cyclotron was avail- Chief Glassblower, whose shop was adjacent to the •g37 inch•h room, Lawrence went far able to Martin Kamen. At seven or eight towards success of the color TV picture tube P.M., the whole building would be ablaze which was finally achieved by Sony. It is with light and ready for the experiment. interesting that such an invention was actually Every scaler was on, every hotplate and taking place in the glass shop adjacent to the laboratory for photosynthesis studies. combustion furnace was on FULL, every

(67) 350 RADIOISOTOPES Vol.26, No. 5

GM tube was re-checked for plateau and the costs in every activity of our lives.

refilled if necessary, every vacuum system The precautions prescribed today by univer-

was running. Every preparation was com- sities and governmental agencies often do

plete for the experiment. All too often, not allow scientists to decide between risks

the eager group of students and helpers was (potential for progress) and costs. They

dismayed by the phone message from seem far too stringent to allow scientists to

Kamen, •gCyc's Sick,•h which meant that dedicate themselves to research in the way

the cyclotron had broken down and no that Sam Ruben and Martin Kamen did.

radioisotope would be available-a great Nowadays we are allowed to take many

disappointment. Usually, however, Martin risks in every day life and it seems only

Kamen succeeded in exhausting the 11C0 and proper that we expect to take similar risks 11CO2 gas mixture from the B203 target of associated with our dedication to science. I

the cyclotron with his evacuated brass •gas- feel that there is a need at this time for

pirator•h chamber (Fig. 2). Working with scientists to take a firmer stand in determin-

•g hot" cyclotron targets was hazardous, to ing the relative costs of the risks they take

say the least, and the •gCO2 in the aspirator in their lives and work. They should be in

was equally so. At those times, Ruben a far better position to estimate these costs

would never allow Kamen to remain in the and risks than those impersonal government

Rat House laboratory after delivering the regulations which have spread their in- "CO2 . Ruben would tell him to •ggo away, fluence throughout our lives. Of course,

you are too hot". Usually, Kamen and the regulations have produced a safer world,

Ruben worked together feverishly preparing no one will deny that. The costs, however,

samples, after which Kamen would retire should be considered as conscientiously as

to a safe distance to reduce the `background' we have considered the precautions. and wait for Ruben to finish counting. I At the beginning Kamen would bring the suspect that Martin Kamen should be in- hot aspirator chamber containing the (11CO vestigated, biochemically, for his remarka- +11CO2) to the Rat House for combustion, bly effective DNA repair mechanisms, as but later the combustion was done by well as for the basis of his remarkable dis- Kamen at the Rad Lab-in fact most of the coveries of C-14, photometabolic mecha- runs were started this way with Kamen car- nisms, and comparative biochemistry of rying the •hCO2 to the Rat House in a glass cytochromes. U-tube immersed in liquid air in a Dewar

flask. The graduate students, Mary Belle

Allen, Charlie Rice, Pete Yankwich, and I,

as an Instructor fresh out of CalTech, were

all ready for our individual parts in these

experiments in which we had been •gbriefed•h

by Ruben and Kamen in the classroom up-

stairs the day before. There could have

been vociferous arguments about the proce- Fig. 2 Brass Aspirator' chamber used by Martin dures but Ruben and Kamen always came Kamen to recover the •gC oxides produced in the cyclotron target. to agreement. It was absolutely essential

The radiation hazards such as Ruben and that every priceless moment of the C-11's

Kamen and the other cyclotron workers life be used. After the experiment there

exposed themselves to would not be allowed was no radioactive waste or contamination

today. In many ways scientific progress, to worry about. Every sample was •gdead•h.

like any other progress, whether it be in The results could be discussed and new ex-

business, exploration, or even art, involves periments for next day planned. It is no

risks. And we must balance risks against wonder that Sam Ruben, who wanted to be

(68) May 1977 A.A. Benson: Philosophy of the tracer method 351

recognized by an Associate Professorship,

published at least 16 papers in 1942, many of them milestones in biochemical history.

Ruben and Kamen's interest in thermo-

dynamics directed their interests toward the

energetics and mechanisms of photosyn-

thesis. It became a major part of their re-

search effort. Ruben was an avid disciple

of the thoughts of Lipmann and of Kalckar

whose concern for free energy of phospho-

rylation and of 'high energy' phosphate

esters elicited their ideas for involvement

of such energy in photosynthesis. Ruben's

concept of CO2 reduction was simple. He

envisaged CO2 fixation by RH to form

RCOOH, a process expected to require 10 Fig. 3 Professor Sam Ruben, photographed kcal. He and Kamen had deduced from in the 'Rat House' in 1942 holding a the chemical properties of RCOOH that it bottle of the first large sample of was a hydroxycarboxylic acid (whose barium 14 C-labeled barium carbonate produced from neutron irradiation of ammo- salt they had decarboxylated by heating). nium nitrate solutions in tanks sur- Ruben envisaged further condensation of rounding the cyclotron. R-COOH using energy of ATP and C2 pro-

ducts of CO2 fixation. In one discussion, necessary to use the •gscreen-wall•h Geiger Ruben and Kamen also suggested an aromat- counter tube invented by Willard Libby in ic system with considerable resonance ener- the lab down-stairs in The Rat House. By gy, bound to the C-H bond as •hR•h, might using a brass screen instead of a metal

provide the necessary negative free energy cylinder the 14C betas will enter the func- for the carboxylation. tional volumes of the GM tube and be reg- Carbon-14 was •ginvented•h by Kamen and istered by the scaler. The sample had to be Ruben. Kamen had developed enough ' plated' on the interior of a glass cylinder knowledge about radioisotope production to which slid over the screen so that half the

believe that C-14 should exist. Lawrence radiation could enter the counter. For this urged him to search for a way to produce I built a gadget to roll the cylinder while I it. He and Sam Ruben tried valiantly to could apply the sample uniformly with a

product it from C-13. It was difficult. They pipette and dry the solvent continuously finally succeeded (1940) and when I started with a blower (Fig. 4). This was the fore-

with Sam Ruben in 1942 they had accumu- runner of my 'turntable' for application lated the 200 mg of Ba14CO3 he holds in the of samples to planchet disks for counting

photograph (Fig. 3). This he gave to me to by later GM tubes with large end-windows use to identify and isolate •gRCOOH•h. (Fig. 5).

We designed our experiment to maximize With 14C measurement made more reli-

dark CO2 fixation. We used Chlorella in able I could determine the 'partition coeffi- N2, giving them the •gCO2 for 20 minutes to cient' of R19COOH, an hour, then exhausting the gas through

sodium hydroxide to trap all the unused

•h CO2. Thus our stock became more and •¬ between water and ether as a function of more dilute as the experiments progressed. pH. Fortunately, the 'RCOOH' had a rea- Measuring the 'RCOOH' produced was not sonable partition coefficient, 1/7, between

the simple matter as it is today. It was ether and water at pH 2. With data for

(69) 352 RADIOISOTOPES Vo1.26, No. 5

Berkeley, Sam had no one to do this for him and, still, no fume hoods. He had broken an arm in an auto accident driving to an experimental site in northern California a few days before and was trying to transfer liquid phosgene from a glass ampoule with only one hand. Phosgene boils at 8° and the old glass ampoule should Fig. 4 ' Apparatus for preparation of uniformly thin have been cooled carefully in ice but, samples of 14C-labeled products of photosyn- as was his nature, Sam could not wait; thesis. Glass cylinder for Geiger-Mueller he plunged in into a Dewar of liquid counting with the Libby Screen Wall Counter. nitrogen. The result was tragic. Al- though he took all precautions to im- mobilize himself after calling the nearby University Hospital, he had ingested a deadly quantity of the liquid droplets induced in the spattering of the warm liquid phosgene coming into contact with the liquid air. Science lost one of its most promising young Fig. 5 Turntable with hot air blower for uniformly thin application of samples on glass or alumi- talents. His brave wife and growing num planchets to be counted with large end- family lost a father they had known window Geiger-Mueller counters. all too little. His discoveries and his

Kdistribution at a range of pH values the pKa philosophy of tracer science have left the of RCOOH could be estimated. It was a world much enriched. relatively strong organic acid. From the liquid partition studies of

At this point the war had demanded RCOOH with Ruben I found further oppor-

Ruben's time more and more. He was en- tunity with Melvin Calvin to whom Ernest gaged in a meteological project studying Lawrence now gave his support in the Ra- movement of gas clouds. Kamen and Ruben diation Laboratory. With Lawrence's sup- had discussed the possibility that death of port, and the transfer of the original •gan- experimental goats from phosgene could be tique•h •g37 inch•h Cyclotron to UCLA, we

an indirect result of an immune reaction to designed a laboratory and began work in

novel proteins produced in the lung by the adjacent room in ORL, the •gOld Radia- phosgene. Being a `divalent' acid chloride, tion Laboratory•h. It was necessary to cover CI-CO-C1, it can react with two amino the floor with linoleum because it was groups to alter protein conformation or to yellow with uranium compounds. We had tie two proteins together. I synthesized C-11 to excavate a dungeon and cover it with

phosgene to make such studies. To do so three feet of concrete to avoid the neutron

in a few minutes took some ingenuity but clouds floating from the adjacent 60•V cyclo-

we succeeded. tron. Backgrounds were variable indeed.

For our work I had been transferring Later we could occupy the 37" room itself phosgene from the 20-year old Kahlbaum and the group grew rapidly with the newly glass ampoules to steel cylinders for Sam to available space. It was an exciting place use in his work. These old 500 ml ampoules and the stream of visitors from over the

could have crystallized during their long world enriched our viewpoints and our

storage in sawdust-filled boxes in the chem- potential for progress.

istry department storeroom. When I left Using more 14C02 I could produce more

(70) 353 May 1917 A.A. Benson: Philosophy of the tracer method

R-14COOH from dark anaerobic Chlorella. synthetic 14C-labeled product with authentic Finally the ether extract crystallized! It Kahlbaum (Berlin) •gglycerinsaure•h. The was recrystallized and melted at 140° with result was convincing. The RCOOH was an equivalent weight of 59. It was succinic actually 3-phosphoglyceric acid. R-H must acid -not an exciting revelation of photo- be a C2 compound! synthesis but it was good experience. Dur- The history of Science is never written ing this time I had help from many distin- by the scientists involved in making dis- guished scientists, Ernest Lawrence, Edward coveries. That would be too painful, too McMillan, Emilio Segre, George Hevesy, embarrassing, to reveal the mistakes and Mme. Irene Curie-Joliot, Glenn Seaborg, disappointments along the way. Each dis- Otto Meyerhof, and Carl Neuberg, all of covery yields such a simple answer or con- whom added their touch to solution of the cept that it should have been obvious, problems at hand. Succinic acid became an simple, and straightforward to prove. The intermediate in the first C4-photosynthesis story of 14C in photosynthesis is now being cycle. completed by Dr. Arthur Norberg of the Calvin had designed novel chelating Bancroft Library, U.C. Berkeley. agents for transuranic elements and the Ber- keley chemists knew well the power of liq- Search for the C2 Acceptor uid partition for separating and character- After the preliminary •gbrute-force•h search izing radionuclides in pauciatomic quanti- for the acceptor by trying every C2 sub- ties. The method was free of the problems stance on the shelf we had to proceed more of absortion. From liquid-liquid partition methodically. Acetate, acetaldehyde, gly- the plant biochemists developed the fixed colic acid, glycine, glycol, and ethanol, all liquid phase silica gel column chromatogra- labeled synthetically with 14C, had all failed. phy for fatty acid separation. The next Glycolaldehyde was a candidate but less step, to delete the glass column, was the easily prepared. Stepwise degradation of paper chromatographic method of Martin hexose and PGA gave little clue to the and Synge. It's application in our labora- identity of the acceptor, Aronoff had shown tory stemmed from the arrival of William that hexose was 3,4-labeled. Alan Bassham

Stepka, a student of F.C. Steward where began his graduate work in chemistry with

C.E. Dent from the Martin and Synge labo- degradation of succinic and glyceric acids, ratory had begun paper chromatographic finding equal 2,3-labeling in PGA, a further studies at Rochester. Stepka's experience clue to the nature of acceptor. accelerated our progress tremendously.

Sometimes it seemed we could separate and The Chemical Approach recognize a new intermediate every day. Following the approaches established in

Melvin Calvin's contacts with Dow the early work of Ruben and Kamen I

Chemical Co. introduced ion exchange to saturated the carbon system with 14C by 2

our laboratory. The 14C-labeled products min Ps (two minutes steady state photosyn-

of shortest time photosynthesis clung most thesis at maximal light intensity) and then tenaciously to the anion exchange resins. removed the 14C02 by a stream of N2, He,

The earliest product being much more diffi- or C02-free air. These experiments resulted

culty eluted from the resin than later pro- in two discoveries. ducts, Calvin deduced that it should be a Two new labeled intermediates appeared dibasic substance possibly phosphoglyceric in 14C radiograms of photosynthesis pro- acid. After preparation of 14C-glyceric acid ducts of Rhodospirillum rubrum. They ap- from the radioactive material and conver- peared to be sugars but not any of the sion to a derivative by classical organic classical hexoses. They were produced by techniques I could cocrystallize the photo- action of phosphatase upon the more water-

(71) 354 RADIOISOTOPES Vol. 26, No.5 soluble phosphorylated components from major carboxylation product. He had dem- the chromatogram. By periodate degrada- onstrated an important part of C4 photo- tion Al Bassham estimated the -CH2OH con- synthesis ! Kawaguchi eluted and 'phospha- tent of one to be 14%. •gIt must have been tased' each of the sugar phosphates and a result of incomplete labeling of a Ce chromatographed each to give the kinetics sugar•h; so we continued to work on the of their formation. substance. Finally it became clear, after Students today do dot realize the impor- conversion to a polyalcohol and periodate tance of our (Warburg's) choice of Chlo- degradation, Bassham had been correct in rella and Scenedemus (Gaffron) for these the first instance. A whole year had been studies. Had we tried to use barley or rice spent. The sugar was sedoheptulose whose or soybean leaves we would have made no unique anhydride derivative and the authen- progress by the kinetic approach. The algae tic sedoheptulose, isolated from Sedum acre have at least ten times higher levels of by Arnold Nordal, gave final proof of its phosphorylated intermediates than do the identity. A C7 sugar in photosynthesis ! higher plants. Wheat leaves produce sucrose Now, the nature of the acceptor became in 4 seconds, Chlorella takes two minutes ! unlimited. The much greater pool sizes allowed us to The other unknown, lying beyond ala- find and to identify the intermediates of nine and far beyond fructose in the chro- sucrose synthesis in algae. matogram, was a strange one. Its phos- When the algal pools of phosphorylated phate esters had a tendency to yield phos- intermediates were saturated with 14C (two phoglyceric, phosphoglycolic, and phospho- minutes PS was sufficient) we could flush erythronic acids upon alkali treatment in out the 14C02 and look for the acceptor air. Paralleling the work of the Horecker which should accumulate. With N2 or He and Racker laboratories we recognized the flushing one compound increased three-fold unknown as ribulose diphosphate, different in concentration-ribulose diphosphate. It from the ribulose-5-phosphate identified by was clear that here was the C2 acceptor in Horecker and Smyrniotis. Fortunately ri- disguise: C1+C5=2C3, a logical but yet un- bulose could be easily made from arabinose; predicted carboxylation process. By this both it and sedoheptulose gave unique color process, the free energy change for carbox- reactions on the paper chromatograms which ylation was -13 kcal instead of the usual added confirmation of their identities. +10 kcal anticipated by Ruben and Kamen. Nature had shown herself far more clever The Kinetic Approach than the chemists. Ribulose-5-phosphate is Theoretically, a kinetic examination unique among sugars. It is a real ketone; of photosynthetic intermediate formation it cannot form the hemiketal structures of should reveal the sequence of their forma- other sugar phosphates. This accounted for tion. In fact, however, the multiple label- the dramatic free energy change and for the ing and cyclic nature of the process pre- unique reactivity of ribulose mono- and cluded profound conclusions. The initial diphosphate. carboxylation reactions were delineated by In CO2-free air, ribulose diphosphate did the . elegant work of Dr. Shinichi Kawagu- not accumulate. In fact it disappeared rap- chi, now Professor of , idly. This was the beginning of our under- Osaka City University, Kawaguchi's careful standing of photorespiration. In 1950 Pro- work and superb skill produced several fessor Hiroshi Tamiya came to Berkeley. series of radiograms of 5 sec to 10 min PS It was the beginning of the development of Scenedesmus which gave clear-cut resutls. our deep respect for Japanese biology and

PGA was not the only •gfirst product of enhancement of our respect for the breadth photosynthesis•h. Malic acid, also, was a sand depth of Japanese culture. By his

(72) 355 May 1977 A.A. Benson: Philosophy of the tracer method charm and by his knowledge and experience ture, Calvin presented the evidence and in German, French, and Japanese labora- underlying concepts before a large audience tories he enriched our lives and our science. at the Federation meetings in San Francisco From his wartime studies on inhi- (1954). It was a thrilling paper for all of bition of photosyntheses he knew there us. When he finished, Professor C.B. van must be a photo-respiratory system. To- Niel, long respected as the leader in micro- gether we tried to test such ideas by experi- biology and photosynthetic metabolism, ex- ment but could produce no clear-cut con- tended his hand in congratulation. With clusions. It was still too early to recognize deep emotion he expressed his opinion that the role of oxygen in its reaction with the the struggle to understand photosynthesis yet unknown ribulose diphosphate. Once was now over. It was a touching moment, we saw the products of photosynthesis indeed. channeled into phosphoglycolate and free R. Clinton Fuller was engaged as a mi- crobiologist familiar with sensitive bioassay glycolic acid in sugar beet leaves, however it was clear to me that some intermediate from thioctic acid. His repeated efforts was being attacked by oxygen. I had shown yielded negative results. Finally I labeled that ribulose diphosphate yielded phospho- Chlorella with 35S by culture overnight in 35SO4 nutrient . The radiogram revealed a glycolate under alkaline conditions in vitro. It was too early to discern that this was the huge radioactive lipid-like spot in the area oxygenase function of ribulose diphosphate expected for thioctic acid. It was really carboxylase (Tolbert and Anderson; Osmond exciting for all of us, especially Melvin and Akazawa). Calvin. It was truly a Nobel idea. But, alas, Fuller could find no thioctic in the I believe that the most brilliant idea on radioactive product (it was later shown to quantum conversion of the time was Melvin be the chloroplast sulfolipid, Fig. 6). One Calvin's concept of thioctic acid participa- by one, each of the pillars of circumstantial tion in photosynthesis. Studies by Gunsalus, evidence for thioctic acid involvement in Reed, and the Lederle group revealed the the quantum conversion crumbled; Nature role of thioctic (lipoic) acid in pyruvate had not understood the great idea. decarboxylase and the instability inherent in the 5-ring disulfide system. Calvin con- ceived a scheme for its participation as quantum acceptor from a. The yellow lipoic acid absorbs at 330 nm, hardly able to accept a quantum from chlorophyll. With a number of brilliant preliminary ex- periments he and Baritrop and others ac- cumulated evidence that thioctic acid could act as a radical initiator for methyl metha- crylate polymerization, so that it could act Fig. 6 The plant sulfolipid. The major 35S- as a quantum acceptor for chlorophyll, that labeled product in green plants grown in 35504- nutrients. it might react with water in the excited state to yield one-SH and one-SOH (sulfinic acid). Now, thirty-five years later, CO2 fixation The former could contribute to pyridine is nearly understood. But we do not under- nucleotide reduction; the latter could yield stand photosynthesis, even with the efforts a hydroperoxide and revert to the closed ring of the scientific progeny of Keita Shibata disulfide. This concept appeared to tie to- and Otto Warburg. There is still a Green gether all the requirements of photosynthe- Secret! sis and the search for thioctic acid in real In conclusion I would like to express my photosynthetic tissues began. At this junc- personal appreciation to my many Japanese

(73) 356 RADIOISOTOPES Vol.26, No.5

colleagues, many of whom are here today. tosynthesis experiments with 11C02. It is a As a token of our respect for your generous simple but meaningful token of our mutual assistance and brilliant contributions to the respect which has been reinforced by many development of radiotracer methodology, exciting and rewarding experiences with our Martin Kamen and I thought it appropriate fellow scientists in Japan. that Professor Miyachi and the Radioisotope Centre should preserve this historic Geiger I am grateful to Professor Martin Kamen counter tube made by Sam Ruben in 1940- for his assistance in reading the manuscript 41 and used in his laboratory in many pho- and providing it with accurate information.

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