The Path of Carbon In Photosynthesis MELVIN CALVIN, Radiation University
Fig. 1. Algae farm used by Dr. Calvin and his colleagues for photosynthesis studies
•pvrEHYO»NE is familiar with what the *•* term photosynthesis means. It is used commonLy to describe that process by which grreen plants convert the energy of sunlight (electromagnetic e n e r g y ) into the potential energy of reduced carbon compourads, simultaneously evolving moleculajr oxygen. In 1946, the long-lived radioisotope o f carbon, carbon-14, became available and w e set out to pursue the work ont the path of carbon itself from carbon cEioxide to the reduced carbon com pounds, using labeled carbon as the means of tracing that path. T h e r-process o f photosynthesis can be divided, both theoretically and physically, into t w o rather distinct parts and Fig. 2 shows d-iagrammatically that sort of divi sion. Tine three elements that w e are pri marily concerned with—carbon, hydrogen, and oxygen—come into the plant as carbon dioxide and water; through the agency of light, oxygen is evolved and reduced car bon is generated. The action of the light can be separated fairly distinctly, both physicaUy arid theoretically, from the re duction of C 0 2 . I n F5g. 2 , the right side, in a sense, is t h e iphoto part of photosynthesis and the left is the synthetic part of photosynthesEs. N o w , with the radioactive car bon wet were able to trace the synthetic part frown CO a through a sequence of intermedLates into the reduced carbon that the plant generated. T h e photo part, 1622
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In the first of two articles describing his studies of photosynthesis, Dr. Calvin explains the path of carbon in w h a t he calls the "synthetic" part of the process
however, does not seem to involve carbon, or C 0 2 , or the direct reduction of C 0 2 , and so for a number of years w e confirmed the theoretical separation. Toward the end of the last five years w e more or less com forted ourselves with the notion that all w e had learned from our studies with radiocarbon was something that we should call phytosynthesis rather than photosynthesis. These studies on t h e part of carbon, which for a long time w e thought was completely divorced from the photo part of the reaction, have l e d us to make a suggestion, at least, of what the primary quantum conversion act of photo synthesis is. First w e should review the path of car bon in photosynthesis—how w e have studied it and what kind of information has resulted from this study. The mc hod
of studying a system such as this ii straight forward. O n e simply arranges ι green plant of s o m e sort in a steady state of photosynthesis ( F i g . 3 ) . T h e plant ab sorbs C0 2 > light, and water, and t h e CO is converted through a series of inter mediates into reduced compounds ( carbo hydrates) o£ some sort or other, evolving oxygen in a steady state condition. At ί t i m e t = O, labeled CO2 is injected int< the entering stream of C 0 2 and i t s patl traced inside t h e leaf or green plant. It i quite clear that if o n e waits long enough labeled carbon will find its w a y into carbo hydrates and other reduced materials c t h e plant. T h e purpose of the experimen is to shorten the t i m e during which t b carbon has been following this path unti t h e earnest compounds into w h i c h tha carbon i s incorporated can b e found. Thus
Mel vin Calvin, professor of chemistry at t h e Univer sity of California, Berkeley, is also director o f t h e bioorganic group of the Radiation Laboratory there. H i s several well known contributions in t h e field of theoretical organic chemistry include investigations o n t h e relationship of· electronic structure to color, work in the chemistry of ^chelate compounds, and pioneering work o n the mechanism of photosynthesis using iostopic tracers. During the war h e undertook a n investigation for the National D e f e n s e Research Committee on the problem of oxygen storage a n d regeneration for u s e in submarines.
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not in our lifetime) and some other method of analysis, much more rapid and general, had to be found. This was .ac 2 tually presented by the method of paper chromatography, invented by Consden, Martin, and Synge in England, which was suitable for our purposes. The method of paper chromatography hardly needs any general description. It depends upon a difference in the rela tive solubilities of organic substances in two or more solvents. We have done most /Ν of our work with two-dimensional chromatograms. With these, one can use the co ordinates of the compound with respect to the origin and the series of solvents used as a means of identification of that compound, provided the coordinates can be determined. In order to determine the coordinates on the paper with respect to the origin, one must be able to find the compound on the paper. The amount of compound we use is so small that even if it were colored it wouldn't be visible. The method was originally developed by Consden, Martin, and Synge for use with amino acids; it em ploys a spray test that will develop a color on the paper wherever there is an amino acid. However, we did not know what JFig. 2., Diagrammatic conception of photosynthesis. "Photo", part of the photo-/ we were looking for, chemically; all we "synthetic process is* ' represented in - right-hand, side of diagram; "syn thetic"- part of photosynthesis is represented by section on, left-hand side knew was that we were looking for a com pound containing radioactive carbon. This general fact provided the universal detec tion method we required—the radioactivity itself. One could work slowly over the the path along which it travels and the active compound containing very much of paper, using a Geiger counter, and find branches and cycles it might go through the total radioactivity that had been fixed. out where the radioactivity was. can be traced. W e are using this method today when It was clear that by this method we would In order to do this steady state experi not achieve our goal very soon (perhaps we have a great deal of time or when we ment, it is necessary to have more or less reproducible organisms. W e use green algae for this continuous culture system (although blue-green algae and purple Fig. 3. XHagrammatic representation of a steady state of photosynthesis. In study bacteria are sometimes used); these are ing photosynthesis in laboratory* green plants are arranged in this steady state. harvested every day and a new culture Plant, is -placed in a situation corresponding to situation in box at right medium drawn in. In the exposure ap paratus (Fig. 4 ) , the algae suspension is placed in a flat, circular vessel which we call the lollipop, and light from sources located on either side passes through infra red filters; COa enters in continuous flow through a bubbler tube. At a time t = 0, labeled CO a is allowed to enter vith the main stream of C0 2 - At suitable intervals of time, the stopcock is opened and the algae suspension is drawn into alcohol. Thus, the biological reactions that are go ing on are stopped in as mild a manner as possible so as not to destroy or convert the compounds into which the radiocarbon has been incorporated. Then, analyses of the extract from the algae are made in order to determine which compounds in the extract contain radiocarbon and to de termine the order of appearance of radio activity in those compounds. The method that was first attempted for this analytical purpose was the usual method of isolation; that is, we wanted to try to extract from the solution the various organic materials that were present and determine which one of them contained radioactive carbon. This was a very labori ous, slow procedure, and after about a year of it we had identified only a single radio
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to allow us to draw any conclusions as to what occured first. At least w e eliminated a large number and w e r e practically limited to the phosphates as early products of one-minute photosynthesis. Sucrose, al anine, serine, and others in t h e 10-minute picture had not yet appeaFecL Practically everything showing any intensity was phos phate of one sort or another. However, one minute was still too long, and as is shown in Fig. 8 w e went down to fractions of a minute and found prac tically nothing but phosphoglyceric acid. At still shorter times, there w a s still less of the other compounds. O n e could quantitate the whole thing by simply making a series of such papers at different times (one, two, four, eight seconds, and so forth), detennining the amount of radio activity in each one of t h e s e spots, and making graphs such as those shown in
Fig. 4. The exposure apparatus used in studying photosynthesis in a steady state ex periment. T h e algae suspension is placed in the flat, circular vessel called the lollipop. Light from the sources on either side passes through infrared filters are in a very great hurry. T h e reason for this is that when w e are in a hurry w e usually know approximately where t o iook on the paper and for what w e are looking. W e can put the counter down on the spot where we think the compound should be and see if it i s there. When w e don't know what we a r e looking for and don't know where it might b e located, w e would have to use a very small Geiger counter and explore the entire paper with the counter. This isn*t practical yet, s o w e had to turn to another method. It was provided for us by the fact that the beta particles from carbon-14 are relatively soft, do not go very far, and do expose photographic film in t h e w a y that light does. Thus, w e had the ideal method: The x-ray film is placed i n contact with the paper and wherever there is a radio active spot on the paper, that is, a com pound containing radioactive carbon, t h e x-ray film is exposed. When the x-ray film is developed, a black spot appears.
its darkness being relatively proportional to the amount of radioactivity in the com pound. W e can thus locate radioactive compounds readily and easily. Fig. 6 shows such a chromatogram made from 10-minute photosynthesis in C u 0 2 by Scenedesmus. This is a photograph of the x-ray film after it has been developed. However, the specific compounds are not identified by this method. The proper identification of these compounds consti tuted the major activity of our laboratory for about five years. There are still spots which are not identified. W e hope that we will be able to identify them one of these days. It can b e seen from Fig. 6 that 10 minutes was too long for determining the early compounds of photosynthesis. Su crose, as well as a variety of other com pounds, is known to occur late in the photosynthetic scheme. So, w e shortened the time to one minute (Fig. 7 ) , but there were still far too many compounds labeled
Figs. 6, 7 , and 8 below s h o w x-ray films exposed through contact with paper chromatograms o n which products of photosynthesis have been absorbed. Each compound contains radioactive carbon in proportions indicated by t h e darkness of the spot. Fig. 6 shows the results of 10minute photosynthesis, Fig. 7 shows results of 1 minute photosynthesis, and Fig. 8 shows results of 1 0 second photosynthesis
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