Photosynthesis Research 27: 73-82, 1991. (~) 1991 Kluwer Academic Publishers. Printed in the Netherlands.
Personal perspective
Experiments* William A. Arnold
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA (correspondence: 102 Balsam Road, Oak Ridge, TN 37830, USA) Received 24 October 1990; accepted 24 October 1990
Key words: autobiography, photosynthetic unit, efficiency of photosynthesis, excitation energy transfer, delayed light emission, thermoluminescence, electroluminescence Abstract
In this article, I have provided a brief history of my life. After tracing my family background and my early interest in physical sciences, I discuss how I entered biology under the influence of Robert Emerson. I have always enjoyed doing experiments and this led to new measurements and analyses of 'chlorophyll unit', efficiency of photosynthesis, excitation energy transfer, delayed light emission, thermoluminescence and electroluminescence in photosynthetic organisms. It is my view that discoveries are made because we follow our scientific curiosities.
On looking back over a long life, 1904 until now, I realize I have been an extremely lucky man and it must be the luck of the Irish because on my mother's side both parents were Irish, and on my father's side his mother was Irish. We lived at first, in Douglas, Wyoming, where my father had a lumber yard and a contracting business. My brother Mike was also born in Douglas. Then my father and a friend bought two hundred acres in Pleasant Hill, Oregon, built themselves houses, put in a fruit orchard and a creamery and sent their children to the small two-room, two-teacher school. In our barn was a fine tool shop. The only rule Pop made was that we were to report to him at once if we broke a tool. My brother Joe was born in this period. It was a great place to raise children if you discounted the fact that we did not have electricity or indoor plumbing. The son of one of our neighbors gave me his old high school physics book. By the time I was in the eighth grade I had done all the experiments which could be set up in the shop. My * This article was written at the invitation of Govindjee.
favorite subject was arithmetic and I was excited to see how it was used in all the experiments. Along came World War I. Pop volunteered and was sent to the Presidio in San Francisco to the Officer's Training School for Field Artillery. Near the end of the three month course, the army found from the records his experience in the lumber business, and transferred him to the Air Corps and moved him to Portland, Oregon, to help get out spruce to make airplanes. The family moved back from San Francisco to the ranch in Oregon and waited for Pop to return. In 1920, due to my father's ill health, we moved to California and bought a small ranch and raised chickens. I went to the nearest high school which was in Van Nuys, and started my sophomore year. The high school was astonishing. There were more students in my class than in the whole school in Pleasant Hill. I made friends with some boys who were making radio receivers and with their help I too soon had a 'radio shack'. My sister, Dorothy (Dody) was born in 1921. Our algebra teacher had worked at Mt. Wilson Observatory; she took a number of us on an
74 overnight visit to the observatory. We looked through the telescope, and the next morning were shown the solar tower. Every month or so my chemistry teacher (he also taught physics) took us to Cal Tech for the Friday night lecture. Both teachers emphasized that mathematics was the basic tool of science. I decided I wanted to become an astronomer or physicist. After graduation in 1923, I entered Cal Tech. My father helped me through the first year and then I worked at all sorts of jobs (waiting tables, telephone co., Edison Co.). In 1926, I was given a job as research assistant to Dr S.J. Barnett who was Head of the Physics Department at UCLA and was doing research at Cal Tech on what was then called Gyromagnetic Anonomaly. The work was supported by the Department of Terrestrial Magnetism at the Carnegie Institution. The experiments had to be done between ll:30p.m, and 6:00a.m. when the street cars were not running. Each experiment consisted of (at about 10 p.m.) measuring the three components of the earth's magnetic field. This information was used to adjust the DC current in three Helmholtz coils in order to reduce the field to zero at the sample. After the last car had gone by, the data for the experiment could be taken for four or five hours; then the three components of the earth's field were measured once more to see if the data could be used. As Barnett's assistant, I had a key to the Physics building, and could use the library and special shop that was for the faculty and graduate students. This experiment had originally been done by Einstein and de Haas. On one of his visits to Cal Tech, Einstein spent an afternoon with us discussing the experiment. When Jean and I were married in 1929, I was still working for Dr Barnett. In 1930, I returned to being a full time student. There were a number of courses required for graduation which I had not taken. It proved impossible to fit them into any schedule. Elementary Biology came at the same time as another course that was required. My advisor in the Physics Department sent me to see Dr T.H. Morgan who was head of the Biology Department. He suggested that I take the course in Plant Physiology, which was being taught by Robert Emerson, a brand new
professor in Biology. Dr Morgan said if I passed the course it would be accepted in place of Elementary Biology.
With Robert Emerson: Photosynthetic unit I liked Emerson and I liked the course which was largely about photosynthesis. Emerson had worked in Otto Warburg's Laboratory (in Berlin) and much of the discussion was about Warburg's work. He was very interested in an experiment first done by Brown and Escombe (1905) and then by Warburg (1919-1920). These experiments showed that for photosynthesis, reducing the intensity of a beam of light with a rotating sector was very different than using neutral filters or inverse square. He felt that the phenomenon was important, and he was considering adding a light source with a rotating sector to his Warburg apparatus. I had a friend in the Physics Department who was helping out one of the local suppliers of neon signs. He had told me how they were made and how they were used. I suggested to Emerson that he could use the neon signs simply by putting them in the water under the Warburg vessel. Neon light has a number of lines which fall under the absorption band of chlorophyll. No change would have to be made in the Warburg apparatus itself. The power in Pasadena was 50 cycles; so we could change from 100 flashes per second to 50 by a simple rectifier tube. A few days later Emerson said he had received permission to use part of the research fund for the work, and if I would set up the neon light system he would give me credit for the lab work that was part of his course. I was delighted. The experiment worked fine. When I graduated in the Spring of 1931, Emerson asked me to stay on as his assistant to make a more detailed study of the effects of flashing light. Since I had been unable to find a place to do graduate work in astronomy I agreed to continue as his assistant a while longer. The short stay grew to fifteen months. It was interesting and fun. The laboratory had as visitors: Hans Gaffron, Cornelius Van Niel, Kaj Linderstrom-Lang. During this period, Marston
75 Sargent arrived from Harvard to take his PhD with Emerson. Marston and I became good friends and he later married Jean's sister. The Biology Department was setting up a Marine Station at Corona del Mar, California. We moved our work down there for part of the summer. The next summer we spent with Dr Van Niel at the Hopkins Marine Station in Pacific Grove. The work went well and resulted in two papers. The concept of 'Photosynthetic unit' was born. Emerson put my name on these papers as co-author (see Emerson and Arnold 1932a,b). I was only an undergraduate student. Later, I was able to test the assumptions in the original work and show the generality of the 'chlorophyll unit' in various phyla of plants (Arnold and Kohn 1934).
Graduate work at Harvard and postdoctoral work at Berkeley: Kinetics and efficiency of photosynthesis Emerson urged me to give up astronomy and do graduate work in biology. Finaly, I agreed to his writing a letter to Dr W.J. Crozier at Harvard asking if he would take me as a graduate student. To my great surprise I was admitted to graduate school and made a research assistant in Physiology. In the fall of 1932, Jean and I went to Wellesley, MA, to share a house with True and Adele Robinson. True had been a classmate of mine at Cal Tech and was also a graduate student in Physiology. Dr Crozier, the head of the department, was my thesis advisor. He proved to be an ideal advisor. He seemed to know all of the biological literature. Any question I asked he answered with the author, journal and year of the paper which answered my question. My thesis subject was photosynthesis. He was specific about courses. I was to take all undergraduate courses starting with freshman Biology. At that time Crozier was trying to have the term General Physiology encompass Bacteria, Animal and Plant Physiology, Biochemistry, Biophysics, etc. While he did not succeed, it made his department an exciting place to work, since the graduate students were each using a
different organism. True was working on oat plants. Stacy French was working on yeast. Charlie Winsor was working on snails. Henry Kohn was working on blow flies. I was working on Chlorella and Carol Haskins, Nick Werthessen, J.N. Stannard were working on other organisms. From this group I quickly learned the great value of making a short experiment with another graduate student: 1. The experiment may work, even lead to a paper. 2. Even if the experiment fails you both learn something new. 3. You do not have to write a grant proposal. My thesis work at Harvard involved the first study on the kinetics (Arnold 1933a, 1935) and the effect of ultraviolet light on photosynthesis (Arnold 1933b), on the efficiency of photosynthesis that I found to be 35% (published only much later: Arnold 1949) and on the photosynthesis unit that we called 'chlorophyll unit' (Arnold and Kohn 1934). Other papers during this period dealt with UV effects on bacteria and yeast (see e.g., Blank and Arnold 1935a,b and Oster and Arnold 1935b,c). Inhibitory effects of UV (2537A) on photosynthesis of green algae and chloroplasts were pursued even much later by Stanley Holt (Holt et al. 1951). We also constructed a photoelectric densitometer (Stier et al. 1934) and examined the significance of Talbot's law in photobiology (Arnold and Winsor 1934). It was a period of activity and joy. When I graduated from Harvard, in 1935, I was given a Sheldon Fellowship. I decided to go to Berkeley to audit Robert Oppenheimer's couse in Quantum Mechanics, and continue work on the Callendar Radio Balance. While a graduate student, I had audited a course in Atomic Physics and had learned about the balance, which had been used to measure heat production of radioactive substances. It would measure very small amounts of heat. In the book, Photosynthesis, page 328, H.A. Spoehr had written in 1926 ' . . . o f using the difference.., in the heating effect between a leaf which is photosynthetically active and one which is not, as a means of measuring photosynthesis'. I had made a radio balance and used it to
76 measure the efficiency of photosynthesis, as part of my thesis. The value I found 35%, as noted above, was far too low. In Emerson's course I had learned that one carbon dioxide used four quanta; so something much be wrong with the balance. The research at Berkeley was done with Dr A.R. Davis in the Plant Nutrition Department. I made a new and better radio balance, tested every part of it, and after a year's work I was still finding the same low value. I published it only much later (Arnold 1949). This, of course, is the accepted value now.
With Cornelius Van Niel In the fall of 1936, we moved to Pacific Grove, California, to work with Van Niel, first on a General Education Board Fellowship and then as his assistant. I started experiments on the purple bacteria. A quantitative method for measuring bacteriochlorophyll from photosynthetic bacteria was established (Van Niel and Arnold 1938). In May 1937, our first child, Elizabeth, was born. The Hopkins Marine Station was an exciting place to work. Among those there were S.F. Carson and E.H. Anderson who later were members of the Biology Division in Oak Ridge. Dr Francis Lloyd had moved to Carmel and was writing his book on Carnivorous Plants. He came to our seminars, and he and I did an experiment on Drosera. Dr Max Delbrfick came to take Van Niel's famous Microbiology course. With him, I made an experiment on bacteriophage.
Fellowship at Copenhagen Dr Warren Weaver of the Rockefeller Foundation came to visit Van Niel and while he was there we had a long talk. He asked me if I would like to go to some place in Europe for a year's study. At that time, Dr G. Hevesy, in Neil Bohr's Lab, was far ahead in the use of radioactive tracers in Biology. So I said I would like to go to work with him in Copenhagen. (I did not know until later on reading a paper by Dr
Weaver that the Rockefeller Foundation, whose main interest was in medicine, had decided that Biology had to make more use of mathematics and physics. They decided they would support Biology students who had a foundation in mathematics or physics.) In 1938, Jean, Elizabeth and I left San Francisco on a Norwegian Freighter bound for Rotterdam. We took the train from Rotterdam to Copenhagen and when we were settled in an hotel, Mrs Hevesy found us an international pension in which to live. I told Hevesy that I was most interested in learning how they measured the activity of the samples, and he suggested that I work with Dr Hilde Levi who was in charge of the counter lab. She started me making the small Geiger-Miiller counters. Most of the work in Hevesy's Laboratory was with 32p. They used counters with mica windows 4-6 milligrams per square centimeter. Dr Hevesy wanted to extend his experiments to 35S which is a soft beta emitter. They needed counters with much thinner windows; so this was an ideal problem for me to start on. Dr Otto Frisch suggested the window would be stronger if mounted on a cylindrical surface rather than a plain. With this idea we made windows of less than 1 milligram per square centimeter (Arnold et al. 1939).
Deuterium experiments Besides the work on the counters, I carried out two research problems which were not published. Bohr's Laboratory had some very pure D20 from Norway. I wondered if the hydrogen bacteria would burn D E as well as H E. Would large amounts of D be found in the bacteria? I isolated some hydrogen bacteria and grew them on the D 2 plus O z mixture made by electrolyzing the D 2 0 . They would u s e D E just as well as H E. The bacteria were dried and burned with oxygen. The water formed was taken to the Carlsberg Laboratory where Dr Lindstrom-Lang (whom I had known at Cal Tech in 1932) determined the D content with his density method. The D content was the same as the water in which the bacteria grew. The bacteria had been grown in ordinary water since the DzO was too precious to use in the culture media.
77
Attempts to make
14C
While I was in Copenhagen my friend Stan Carson was spending part of his time in Berkeley with Dr Sam Ruben using 1~C to trace reactions in bacteria. He wrote the total time involved in the experiment had to be less than 3 or 4 h due to the 20 min half life of 11C. This limitation on the time meant many of the experiments we wanted to do were impossible. Dr Frisch pointed out that the irradiation of nitrogen with neutrons almost certainly made 14C. The calculations of the masses involved indicated that X4C might be radioactive and have a long half life. I became very interested in this possibility. Using the laboratory neutron source, I irradiated 5 kilos of ammonium nitrate for about a month. I extracted the possible ~4C and counted the sample. The count was not statistically above background. As you know, Ruben and Martin Kamen about that time did show that ~4C was radioactive. Their experiment with the long half life of 5700 years made my thirty-day exposure look silly. I was in the laboratory the day that Dr O. Frisch made the experiment which showed that Uranium atoms split into two parts and released a large amount of energy when hit by neutrons. He said to me, 'you work in a microbiological lab. What do you call the process in which one bacteria divides into two?' I answered, 'binary fission'. He wanted to know if you could use the word fission alone and I said you could. Later that day when he sent the famous telegram to Bohr, who was in the United States, he used the word 'fission' in quotation marks. Dr Bohr when he made the announcement removed the quotation marks and fission became the accepted term. Dr Lise Meitner was Dr Frisch's aunt, and like Drs Hevesy, H. Levi, and Frisch, had left Germany on account of the Nazis. She came over from Sweden and spent a couple of weeks in the laboratory. Among the other well known scientists I was privileged to meet at the institute, were Drs Werner Heisenberg, Enrico Fermi and Otto Hahn.
Return to USA
The Rockefeller office in Paris sent us a letter advising us to leave when we felt it necessary.
We returned in August 1939, on the Norwegian ship Stavangerfjord to New York. Back in Pacific Grove I learned that J.L. Magee, T.W. Dewitt, E.C. Smith and F. Daniels, in 1939, had published a paper using the heating effect as suggested by Spoehr to measure the efficiency of photosynthesis. They had not used the radio balance, but they had obtained the same low efficiency as I had. So I did no more on the problem. Excitation energy transfer from phycocyanin to chlorphyll a
Emerson and Dr C.M. Lewis were working on the quantum yield problem at the Department of Plant Biology of the Carnegie Institution, that is located on the Stanford Campus. We saw them several times each month. On one visit, Emerson told me that he and Lewis had found, on making the action spectrum for the blue-breen alga Chroococcus, that light absorbed by phycocyanin was used in photosynthesis. He asked me to see if the energy absorbed by phycocyanin was being transferred to chlorophyll or was the phycocyanin doing photosynthesis. A few simple experiments showed the energy was being transferred to chlorophyll a. I went up to Berkeley, and told Dr Oppenheimer about the problem. He pointed out this transfer was analogous to 'internal conversion' of gamma rays, if both the separation of emitter from absorber and wave length of the radiation were multiplied by a factor of 10 4. We agreed that I was to write a paper on the subject. However, it was published only much later (Arnold and Oppenheimer
1950). War time research
In July 1940, our second daugher, Helen, was born in Pacific Grove. 1941 proved to be the high point of my university career. I was made Assistant Professor of Biophysics in June. I had visions of spending winters on the campus of Stanford, and summers at the Hopkins Marine Station. However, I received a letter from Princeton University asking me to take part in an investigation of anti-aircraft fire. This was for the Office of Scientific Research and Development. I
78 took the letter to the President of Stanford. He said that after such a request they would have to let me go, and the smart thing to do was volunteer. In December, we had an early Christmas with Jean's folks in Pasadena, and then took a train to Buckroe Beach, Virginia, a small town within walking distance of Fortress Monroe, where the experiments were to be done. At that time, anti-aircraft fire was the responsibility of the Coast Defense. The group from Princeton was working on the M1 height finder. In order to know the path the target plane had flown, they photographed it with two theodolites several miles apart. At each instrument a photograph was taken several times per second. Each picture was to show the location of the plane with respect to the cross hairs, and the time, azimuth, and elevation. They used the light developed by Dr H.E. Edgerton to stop the moving dials. My knowledge of how these lights worked was the reason why I was there. The same set-up was used to test radar instruments sent down from the radiation laboratory at MIT. Later in the summer of 1942, the armed forces were reorganized. The anti-aircraft fire was given to the Air Force. All of the field tests were moved to a new Air Force Base at Camp David just north of Wilmington, North Carolina. The optical tests on the range finder were moved to the Eastman Kodak Company in Rochester, New York. I was told over the phone to report to Kodak. We lived in Rochester for two years. By the summer of 1944, it was clear that radar was much better than the height finder. Tennessee Eastman (a branch of Kodak) was operating the so-called Y-121 facility in Oak Ridge, TN. They were using the large mass spectrometers which had been designed at the big cyclotron in Berkeley to separate the isotopes of uranium. They needed a number of physicists; so I was loaned to Tennessee Eastman with the understanding I could be called back if needed. We moved to Oak Ridge in September 1944. I spent the next two years on insulator problems.
The Oak Ridge appointment At the end of the war there was much talk in Oak Ridge about setting up a real Biology Divi-
sion as a part of the X-101 facility. This new department was to study the effect of radiation on living systems and make use of radioactive tracers. I applied to Dr Paul Henshaw, the acting director, for a job, was hired, and we have been in Oak Ridge ever since. After Dr Alexander Hollaender was appointed Director of the Biology Division we were given the use of five or six buildings on the edge of Y-12 facility. They had been built during the war to do Uranium chemistry, but had not been used. As the assistant director, I was in charge of getting these buildings into shape. (I recorded two contributions of Physics to Agriculture (Arnold 1947).) In 1948-49, the Society of Plant Physiologists was planning a meeting in Chicago. Dr Farrington Daniels was organizing a symposium on photosynthesis as a part of the meeting. The quarrel between Warburg and Emerson as to the number of quanta per oxygen worried all of us. Dr Gaffron asked me to give a paper on the radio balance to add a little to the argument about the quantum yield. This was the paper I didn't write in 1936 because I was then convinced that four quanta was right. The paper, as noted earlier, was published only in 1949 (Arnold 1949). On one of his trips to Oak Ridge, I saw Dr Oppenheimer and he reminded me that we were writing a paper together on energy transfer in photosynthesis. The paper was, as also noted earlier, published only in 1950 (Arnold and Oppenheimer 1950). By that time a number of people had written on energy transfer, and L.N.M. Duysens had started work on his marvelous thesis. The concept of excitation energy transfer through depolarization of chlorophyll a fluorescence was pursued soon thereafter (Arnold and Meek 1956).
Delayed light emission By 1950 I was entrenched in the Biology Division of Oak Ridge National Laboratory working on the problem of photosynthesis in green plants; the experiments involved the green alga Chlorella and isolated chloroplasts from higher plants. In June, Dr Bernard Strehler came to the
79 Laboratory. Strehler had a brand-new Ph.D., a tremendous amount of energy, and lots of ideas about almost everything. One day he appeared at the laboratory door and said, 'Arnold, how would you like to make one of the fundamental discoveries in plant physiology?' My answer was 'OK, if it won't take too long.' Strehler then explained that he had been thinking about photosynthesis and that it seemed to him that ATP (adenosine triphosphate) must be required and that it would be silly for the plants to use respiration as the source. So the chloroplasts must make ATP. Strehler had done his thesis on bioluminescence under the direction of Dr William McElroy at the John Hopkins University. This work had led to the invention of a simple method of measuring ATP, and the device consisted of a small light-tight box with two compartments. A test tube could be put into the first compartment through a small light-tight door; then a shutter could be opened so that the photomultiplier in the second compartment could 'see' the test tube. The test tube contained a 'concoction' made from dried firefly tails that would emit light upon addition of ATP. We mixed a suspension of chloroplasts with the firefly suspension, illuminated the mixture for a minute for so, and put it into the box. The photomultiplier gave a signal for some seconds. We thought at first that we had made Strehler's 'fundamental discovery in plant physiology'. But when we ran the controls, firefly suspension alone, we found that the light was coming from chloroplasts. Simple experiments with coloredglass filters showed that the light emitted by the chloroplasts was red and was probably coming from chlorophyll. Light emission from green plants seconds after illumination was totally unexpected. It was weak. We knew from the absorption spectrum of chlorophyll that the natural lifetime of prompt fluorescence was very short (
Personal perspective
Experiments* William A. Arnold
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA (correspondence: 102 Balsam Road, Oak Ridge, TN 37830, USA) Received 24 October 1990; accepted 24 October 1990
Key words: autobiography, photosynthetic unit, efficiency of photosynthesis, excitation energy transfer, delayed light emission, thermoluminescence, electroluminescence Abstract
In this article, I have provided a brief history of my life. After tracing my family background and my early interest in physical sciences, I discuss how I entered biology under the influence of Robert Emerson. I have always enjoyed doing experiments and this led to new measurements and analyses of 'chlorophyll unit', efficiency of photosynthesis, excitation energy transfer, delayed light emission, thermoluminescence and electroluminescence in photosynthetic organisms. It is my view that discoveries are made because we follow our scientific curiosities.
On looking back over a long life, 1904 until now, I realize I have been an extremely lucky man and it must be the luck of the Irish because on my mother's side both parents were Irish, and on my father's side his mother was Irish. We lived at first, in Douglas, Wyoming, where my father had a lumber yard and a contracting business. My brother Mike was also born in Douglas. Then my father and a friend bought two hundred acres in Pleasant Hill, Oregon, built themselves houses, put in a fruit orchard and a creamery and sent their children to the small two-room, two-teacher school. In our barn was a fine tool shop. The only rule Pop made was that we were to report to him at once if we broke a tool. My brother Joe was born in this period. It was a great place to raise children if you discounted the fact that we did not have electricity or indoor plumbing. The son of one of our neighbors gave me his old high school physics book. By the time I was in the eighth grade I had done all the experiments which could be set up in the shop. My * This article was written at the invitation of Govindjee.
favorite subject was arithmetic and I was excited to see how it was used in all the experiments. Along came World War I. Pop volunteered and was sent to the Presidio in San Francisco to the Officer's Training School for Field Artillery. Near the end of the three month course, the army found from the records his experience in the lumber business, and transferred him to the Air Corps and moved him to Portland, Oregon, to help get out spruce to make airplanes. The family moved back from San Francisco to the ranch in Oregon and waited for Pop to return. In 1920, due to my father's ill health, we moved to California and bought a small ranch and raised chickens. I went to the nearest high school which was in Van Nuys, and started my sophomore year. The high school was astonishing. There were more students in my class than in the whole school in Pleasant Hill. I made friends with some boys who were making radio receivers and with their help I too soon had a 'radio shack'. My sister, Dorothy (Dody) was born in 1921. Our algebra teacher had worked at Mt. Wilson Observatory; she took a number of us on an
74 overnight visit to the observatory. We looked through the telescope, and the next morning were shown the solar tower. Every month or so my chemistry teacher (he also taught physics) took us to Cal Tech for the Friday night lecture. Both teachers emphasized that mathematics was the basic tool of science. I decided I wanted to become an astronomer or physicist. After graduation in 1923, I entered Cal Tech. My father helped me through the first year and then I worked at all sorts of jobs (waiting tables, telephone co., Edison Co.). In 1926, I was given a job as research assistant to Dr S.J. Barnett who was Head of the Physics Department at UCLA and was doing research at Cal Tech on what was then called Gyromagnetic Anonomaly. The work was supported by the Department of Terrestrial Magnetism at the Carnegie Institution. The experiments had to be done between ll:30p.m, and 6:00a.m. when the street cars were not running. Each experiment consisted of (at about 10 p.m.) measuring the three components of the earth's magnetic field. This information was used to adjust the DC current in three Helmholtz coils in order to reduce the field to zero at the sample. After the last car had gone by, the data for the experiment could be taken for four or five hours; then the three components of the earth's field were measured once more to see if the data could be used. As Barnett's assistant, I had a key to the Physics building, and could use the library and special shop that was for the faculty and graduate students. This experiment had originally been done by Einstein and de Haas. On one of his visits to Cal Tech, Einstein spent an afternoon with us discussing the experiment. When Jean and I were married in 1929, I was still working for Dr Barnett. In 1930, I returned to being a full time student. There were a number of courses required for graduation which I had not taken. It proved impossible to fit them into any schedule. Elementary Biology came at the same time as another course that was required. My advisor in the Physics Department sent me to see Dr T.H. Morgan who was head of the Biology Department. He suggested that I take the course in Plant Physiology, which was being taught by Robert Emerson, a brand new
professor in Biology. Dr Morgan said if I passed the course it would be accepted in place of Elementary Biology.
With Robert Emerson: Photosynthetic unit I liked Emerson and I liked the course which was largely about photosynthesis. Emerson had worked in Otto Warburg's Laboratory (in Berlin) and much of the discussion was about Warburg's work. He was very interested in an experiment first done by Brown and Escombe (1905) and then by Warburg (1919-1920). These experiments showed that for photosynthesis, reducing the intensity of a beam of light with a rotating sector was very different than using neutral filters or inverse square. He felt that the phenomenon was important, and he was considering adding a light source with a rotating sector to his Warburg apparatus. I had a friend in the Physics Department who was helping out one of the local suppliers of neon signs. He had told me how they were made and how they were used. I suggested to Emerson that he could use the neon signs simply by putting them in the water under the Warburg vessel. Neon light has a number of lines which fall under the absorption band of chlorophyll. No change would have to be made in the Warburg apparatus itself. The power in Pasadena was 50 cycles; so we could change from 100 flashes per second to 50 by a simple rectifier tube. A few days later Emerson said he had received permission to use part of the research fund for the work, and if I would set up the neon light system he would give me credit for the lab work that was part of his course. I was delighted. The experiment worked fine. When I graduated in the Spring of 1931, Emerson asked me to stay on as his assistant to make a more detailed study of the effects of flashing light. Since I had been unable to find a place to do graduate work in astronomy I agreed to continue as his assistant a while longer. The short stay grew to fifteen months. It was interesting and fun. The laboratory had as visitors: Hans Gaffron, Cornelius Van Niel, Kaj Linderstrom-Lang. During this period, Marston
75 Sargent arrived from Harvard to take his PhD with Emerson. Marston and I became good friends and he later married Jean's sister. The Biology Department was setting up a Marine Station at Corona del Mar, California. We moved our work down there for part of the summer. The next summer we spent with Dr Van Niel at the Hopkins Marine Station in Pacific Grove. The work went well and resulted in two papers. The concept of 'Photosynthetic unit' was born. Emerson put my name on these papers as co-author (see Emerson and Arnold 1932a,b). I was only an undergraduate student. Later, I was able to test the assumptions in the original work and show the generality of the 'chlorophyll unit' in various phyla of plants (Arnold and Kohn 1934).
Graduate work at Harvard and postdoctoral work at Berkeley: Kinetics and efficiency of photosynthesis Emerson urged me to give up astronomy and do graduate work in biology. Finaly, I agreed to his writing a letter to Dr W.J. Crozier at Harvard asking if he would take me as a graduate student. To my great surprise I was admitted to graduate school and made a research assistant in Physiology. In the fall of 1932, Jean and I went to Wellesley, MA, to share a house with True and Adele Robinson. True had been a classmate of mine at Cal Tech and was also a graduate student in Physiology. Dr Crozier, the head of the department, was my thesis advisor. He proved to be an ideal advisor. He seemed to know all of the biological literature. Any question I asked he answered with the author, journal and year of the paper which answered my question. My thesis subject was photosynthesis. He was specific about courses. I was to take all undergraduate courses starting with freshman Biology. At that time Crozier was trying to have the term General Physiology encompass Bacteria, Animal and Plant Physiology, Biochemistry, Biophysics, etc. While he did not succeed, it made his department an exciting place to work, since the graduate students were each using a
different organism. True was working on oat plants. Stacy French was working on yeast. Charlie Winsor was working on snails. Henry Kohn was working on blow flies. I was working on Chlorella and Carol Haskins, Nick Werthessen, J.N. Stannard were working on other organisms. From this group I quickly learned the great value of making a short experiment with another graduate student: 1. The experiment may work, even lead to a paper. 2. Even if the experiment fails you both learn something new. 3. You do not have to write a grant proposal. My thesis work at Harvard involved the first study on the kinetics (Arnold 1933a, 1935) and the effect of ultraviolet light on photosynthesis (Arnold 1933b), on the efficiency of photosynthesis that I found to be 35% (published only much later: Arnold 1949) and on the photosynthesis unit that we called 'chlorophyll unit' (Arnold and Kohn 1934). Other papers during this period dealt with UV effects on bacteria and yeast (see e.g., Blank and Arnold 1935a,b and Oster and Arnold 1935b,c). Inhibitory effects of UV (2537A) on photosynthesis of green algae and chloroplasts were pursued even much later by Stanley Holt (Holt et al. 1951). We also constructed a photoelectric densitometer (Stier et al. 1934) and examined the significance of Talbot's law in photobiology (Arnold and Winsor 1934). It was a period of activity and joy. When I graduated from Harvard, in 1935, I was given a Sheldon Fellowship. I decided to go to Berkeley to audit Robert Oppenheimer's couse in Quantum Mechanics, and continue work on the Callendar Radio Balance. While a graduate student, I had audited a course in Atomic Physics and had learned about the balance, which had been used to measure heat production of radioactive substances. It would measure very small amounts of heat. In the book, Photosynthesis, page 328, H.A. Spoehr had written in 1926 ' . . . o f using the difference.., in the heating effect between a leaf which is photosynthetically active and one which is not, as a means of measuring photosynthesis'. I had made a radio balance and used it to
76 measure the efficiency of photosynthesis, as part of my thesis. The value I found 35%, as noted above, was far too low. In Emerson's course I had learned that one carbon dioxide used four quanta; so something much be wrong with the balance. The research at Berkeley was done with Dr A.R. Davis in the Plant Nutrition Department. I made a new and better radio balance, tested every part of it, and after a year's work I was still finding the same low value. I published it only much later (Arnold 1949). This, of course, is the accepted value now.
With Cornelius Van Niel In the fall of 1936, we moved to Pacific Grove, California, to work with Van Niel, first on a General Education Board Fellowship and then as his assistant. I started experiments on the purple bacteria. A quantitative method for measuring bacteriochlorophyll from photosynthetic bacteria was established (Van Niel and Arnold 1938). In May 1937, our first child, Elizabeth, was born. The Hopkins Marine Station was an exciting place to work. Among those there were S.F. Carson and E.H. Anderson who later were members of the Biology Division in Oak Ridge. Dr Francis Lloyd had moved to Carmel and was writing his book on Carnivorous Plants. He came to our seminars, and he and I did an experiment on Drosera. Dr Max Delbrfick came to take Van Niel's famous Microbiology course. With him, I made an experiment on bacteriophage.
Fellowship at Copenhagen Dr Warren Weaver of the Rockefeller Foundation came to visit Van Niel and while he was there we had a long talk. He asked me if I would like to go to some place in Europe for a year's study. At that time, Dr G. Hevesy, in Neil Bohr's Lab, was far ahead in the use of radioactive tracers in Biology. So I said I would like to go to work with him in Copenhagen. (I did not know until later on reading a paper by Dr
Weaver that the Rockefeller Foundation, whose main interest was in medicine, had decided that Biology had to make more use of mathematics and physics. They decided they would support Biology students who had a foundation in mathematics or physics.) In 1938, Jean, Elizabeth and I left San Francisco on a Norwegian Freighter bound for Rotterdam. We took the train from Rotterdam to Copenhagen and when we were settled in an hotel, Mrs Hevesy found us an international pension in which to live. I told Hevesy that I was most interested in learning how they measured the activity of the samples, and he suggested that I work with Dr Hilde Levi who was in charge of the counter lab. She started me making the small Geiger-Miiller counters. Most of the work in Hevesy's Laboratory was with 32p. They used counters with mica windows 4-6 milligrams per square centimeter. Dr Hevesy wanted to extend his experiments to 35S which is a soft beta emitter. They needed counters with much thinner windows; so this was an ideal problem for me to start on. Dr Otto Frisch suggested the window would be stronger if mounted on a cylindrical surface rather than a plain. With this idea we made windows of less than 1 milligram per square centimeter (Arnold et al. 1939).
Deuterium experiments Besides the work on the counters, I carried out two research problems which were not published. Bohr's Laboratory had some very pure D20 from Norway. I wondered if the hydrogen bacteria would burn D E as well as H E. Would large amounts of D be found in the bacteria? I isolated some hydrogen bacteria and grew them on the D 2 plus O z mixture made by electrolyzing the D 2 0 . They would u s e D E just as well as H E. The bacteria were dried and burned with oxygen. The water formed was taken to the Carlsberg Laboratory where Dr Lindstrom-Lang (whom I had known at Cal Tech in 1932) determined the D content with his density method. The D content was the same as the water in which the bacteria grew. The bacteria had been grown in ordinary water since the DzO was too precious to use in the culture media.
77
Attempts to make
14C
While I was in Copenhagen my friend Stan Carson was spending part of his time in Berkeley with Dr Sam Ruben using 1~C to trace reactions in bacteria. He wrote the total time involved in the experiment had to be less than 3 or 4 h due to the 20 min half life of 11C. This limitation on the time meant many of the experiments we wanted to do were impossible. Dr Frisch pointed out that the irradiation of nitrogen with neutrons almost certainly made 14C. The calculations of the masses involved indicated that X4C might be radioactive and have a long half life. I became very interested in this possibility. Using the laboratory neutron source, I irradiated 5 kilos of ammonium nitrate for about a month. I extracted the possible ~4C and counted the sample. The count was not statistically above background. As you know, Ruben and Martin Kamen about that time did show that ~4C was radioactive. Their experiment with the long half life of 5700 years made my thirty-day exposure look silly. I was in the laboratory the day that Dr O. Frisch made the experiment which showed that Uranium atoms split into two parts and released a large amount of energy when hit by neutrons. He said to me, 'you work in a microbiological lab. What do you call the process in which one bacteria divides into two?' I answered, 'binary fission'. He wanted to know if you could use the word fission alone and I said you could. Later that day when he sent the famous telegram to Bohr, who was in the United States, he used the word 'fission' in quotation marks. Dr Bohr when he made the announcement removed the quotation marks and fission became the accepted term. Dr Lise Meitner was Dr Frisch's aunt, and like Drs Hevesy, H. Levi, and Frisch, had left Germany on account of the Nazis. She came over from Sweden and spent a couple of weeks in the laboratory. Among the other well known scientists I was privileged to meet at the institute, were Drs Werner Heisenberg, Enrico Fermi and Otto Hahn.
Return to USA
The Rockefeller office in Paris sent us a letter advising us to leave when we felt it necessary.
We returned in August 1939, on the Norwegian ship Stavangerfjord to New York. Back in Pacific Grove I learned that J.L. Magee, T.W. Dewitt, E.C. Smith and F. Daniels, in 1939, had published a paper using the heating effect as suggested by Spoehr to measure the efficiency of photosynthesis. They had not used the radio balance, but they had obtained the same low efficiency as I had. So I did no more on the problem. Excitation energy transfer from phycocyanin to chlorphyll a
Emerson and Dr C.M. Lewis were working on the quantum yield problem at the Department of Plant Biology of the Carnegie Institution, that is located on the Stanford Campus. We saw them several times each month. On one visit, Emerson told me that he and Lewis had found, on making the action spectrum for the blue-breen alga Chroococcus, that light absorbed by phycocyanin was used in photosynthesis. He asked me to see if the energy absorbed by phycocyanin was being transferred to chlorophyll or was the phycocyanin doing photosynthesis. A few simple experiments showed the energy was being transferred to chlorophyll a. I went up to Berkeley, and told Dr Oppenheimer about the problem. He pointed out this transfer was analogous to 'internal conversion' of gamma rays, if both the separation of emitter from absorber and wave length of the radiation were multiplied by a factor of 10 4. We agreed that I was to write a paper on the subject. However, it was published only much later (Arnold and Oppenheimer
1950). War time research
In July 1940, our second daugher, Helen, was born in Pacific Grove. 1941 proved to be the high point of my university career. I was made Assistant Professor of Biophysics in June. I had visions of spending winters on the campus of Stanford, and summers at the Hopkins Marine Station. However, I received a letter from Princeton University asking me to take part in an investigation of anti-aircraft fire. This was for the Office of Scientific Research and Development. I
78 took the letter to the President of Stanford. He said that after such a request they would have to let me go, and the smart thing to do was volunteer. In December, we had an early Christmas with Jean's folks in Pasadena, and then took a train to Buckroe Beach, Virginia, a small town within walking distance of Fortress Monroe, where the experiments were to be done. At that time, anti-aircraft fire was the responsibility of the Coast Defense. The group from Princeton was working on the M1 height finder. In order to know the path the target plane had flown, they photographed it with two theodolites several miles apart. At each instrument a photograph was taken several times per second. Each picture was to show the location of the plane with respect to the cross hairs, and the time, azimuth, and elevation. They used the light developed by Dr H.E. Edgerton to stop the moving dials. My knowledge of how these lights worked was the reason why I was there. The same set-up was used to test radar instruments sent down from the radiation laboratory at MIT. Later in the summer of 1942, the armed forces were reorganized. The anti-aircraft fire was given to the Air Force. All of the field tests were moved to a new Air Force Base at Camp David just north of Wilmington, North Carolina. The optical tests on the range finder were moved to the Eastman Kodak Company in Rochester, New York. I was told over the phone to report to Kodak. We lived in Rochester for two years. By the summer of 1944, it was clear that radar was much better than the height finder. Tennessee Eastman (a branch of Kodak) was operating the so-called Y-121 facility in Oak Ridge, TN. They were using the large mass spectrometers which had been designed at the big cyclotron in Berkeley to separate the isotopes of uranium. They needed a number of physicists; so I was loaned to Tennessee Eastman with the understanding I could be called back if needed. We moved to Oak Ridge in September 1944. I spent the next two years on insulator problems.
The Oak Ridge appointment At the end of the war there was much talk in Oak Ridge about setting up a real Biology Divi-
sion as a part of the X-101 facility. This new department was to study the effect of radiation on living systems and make use of radioactive tracers. I applied to Dr Paul Henshaw, the acting director, for a job, was hired, and we have been in Oak Ridge ever since. After Dr Alexander Hollaender was appointed Director of the Biology Division we were given the use of five or six buildings on the edge of Y-12 facility. They had been built during the war to do Uranium chemistry, but had not been used. As the assistant director, I was in charge of getting these buildings into shape. (I recorded two contributions of Physics to Agriculture (Arnold 1947).) In 1948-49, the Society of Plant Physiologists was planning a meeting in Chicago. Dr Farrington Daniels was organizing a symposium on photosynthesis as a part of the meeting. The quarrel between Warburg and Emerson as to the number of quanta per oxygen worried all of us. Dr Gaffron asked me to give a paper on the radio balance to add a little to the argument about the quantum yield. This was the paper I didn't write in 1936 because I was then convinced that four quanta was right. The paper, as noted earlier, was published only in 1949 (Arnold 1949). On one of his trips to Oak Ridge, I saw Dr Oppenheimer and he reminded me that we were writing a paper together on energy transfer in photosynthesis. The paper was, as also noted earlier, published only in 1950 (Arnold and Oppenheimer 1950). By that time a number of people had written on energy transfer, and L.N.M. Duysens had started work on his marvelous thesis. The concept of excitation energy transfer through depolarization of chlorophyll a fluorescence was pursued soon thereafter (Arnold and Meek 1956).
Delayed light emission By 1950 I was entrenched in the Biology Division of Oak Ridge National Laboratory working on the problem of photosynthesis in green plants; the experiments involved the green alga Chlorella and isolated chloroplasts from higher plants. In June, Dr Bernard Strehler came to the
79 Laboratory. Strehler had a brand-new Ph.D., a tremendous amount of energy, and lots of ideas about almost everything. One day he appeared at the laboratory door and said, 'Arnold, how would you like to make one of the fundamental discoveries in plant physiology?' My answer was 'OK, if it won't take too long.' Strehler then explained that he had been thinking about photosynthesis and that it seemed to him that ATP (adenosine triphosphate) must be required and that it would be silly for the plants to use respiration as the source. So the chloroplasts must make ATP. Strehler had done his thesis on bioluminescence under the direction of Dr William McElroy at the John Hopkins University. This work had led to the invention of a simple method of measuring ATP, and the device consisted of a small light-tight box with two compartments. A test tube could be put into the first compartment through a small light-tight door; then a shutter could be opened so that the photomultiplier in the second compartment could 'see' the test tube. The test tube contained a 'concoction' made from dried firefly tails that would emit light upon addition of ATP. We mixed a suspension of chloroplasts with the firefly suspension, illuminated the mixture for a minute for so, and put it into the box. The photomultiplier gave a signal for some seconds. We thought at first that we had made Strehler's 'fundamental discovery in plant physiology'. But when we ran the controls, firefly suspension alone, we found that the light was coming from chloroplasts. Simple experiments with coloredglass filters showed that the light emitted by the chloroplasts was red and was probably coming from chlorophyll. Light emission from green plants seconds after illumination was totally unexpected. It was weak. We knew from the absorption spectrum of chlorophyll that the natural lifetime of prompt fluorescence was very short (
