by Isaac Asimov
After the removal of nitrogen, by way of arginine, the remaining carbon skeletons of the amino acids can, by various routes, be broken down to carbon dioxide and water, producing energy. (For the overall metabolism of carbohydrates, fats, and proteins, see figure 12.5.)
Figure 12.5. The overall scheme of metabolism of carbohydrates, fats, and proteins.
Tracers
The investigations of metabolism by all these devices still left biochemists in the position of being on the outside looking in, so to speak. They could work out general cycles, but to find out what was really going on in the living animal they needed some means of tracing, in fine detail, the course of events through the stages of metabolism—to follow the fate of particular molecules, as it were. Actually, techniques for doing this had been discovered early in the century, but the chemists were rather slow in making full use of them.
The first to pioneer along these lines was a German biochemist named Franz Knoop. In 1904, he conceived the idea of feeding labeled fat molecules to dogs to see what happened to the molecules. He labeled them by attaching a benzene ring at one end of the chain; he used the benzene ring because mammals possess no enzymes that can break it down. Knoop expected that what the benzene ring carried with it when it showed up in the urine might tell something about how the fat molecule broke down in the body—and he was right. The benzene ring invariably turned up with a two-carbon side chain attached. From this, he deduced that the body must split off the fat molecule’s carbon atoms two at a time. (As we have seen, more than forty years later the work with coenzyme A confirmed his deduction.)
The carbon chains in ordinary fats all contain an even number of carbon atoms. What if you use a fat whose chain has an odd number of carbon atoms?
In that case, if the atoms are chopped off two at a time, you should end up with just one carbon atom attached to the benzene ring. Knoop fed this kind of fat molecule to dogs and did indeed end up with that result.
Knoop had employed the first tracer in biochemistry. In 1913, the Hungarian chemist Georg von Hevesy and his co-worker, the German chemist Friedrich Adolf Paneth, hit upon another way to tag molecules: radioactive isotopes. They began with radioactive lead, and their first biochemical experiment was to measure how much lead, in the form of a lead-salt solution, a plant would take up. The amount was certainly too small to be measured by any available chemical method, but if radiolead was used, it could easily be measured by its radioactivity. Hevesy and Paneth fed the radioactively tagged lead-salt solution to plants; and at periodic intervals, they would burn a plant and measure the radioactivity of its ash. In this way, they were able to determine the rate of absorption of lead by plant cells.
But the benzene ring and lead were very “unphysiological” substances to use as tags. They might easily upset the normal chemistry of living cells. It would be much better to use as tags atoms that actually take part in the body’s ordinary metabolism—such atoms as oxygen, nitrogen, carbon, hydrogen, phosphorus.
Once the Joliot-Curies had demonstrated artificial radioactivity in 1934, Hevesy took this direction at once and began using phosphates’ containing radioactive phosphorus. With these he measured phosphate uptake in plants. Unfortunately, the radioisotopes of some of the key elements in living tissue—notably, nitrogen and oxygen—are not usable, because they are very shortlived, having a half-life of only a few minutes at most. But the most important elements do have stable isotopes that can be used as tags. These isotopes are carbon 13, nitrogen 15, oxygen 18, and hydrogen 2. Ordinarily, they occur in very small amounts (about 1 percent or less); consequently, by “enriching” natural hydrogen, say, in hydrogen 2, one can make it to serve as a distinguishing tag in a hydrogen-containing molecule fed to the body, The presence of the heavy hydrogen in any compound can be detected by means of the mass spectograph, which separates it by virtue of its extra weight. Thus, the fate of the tagged hydrogen can be traced through the body.
Hydrogen, in fact, served as the first physiological tracer. It became available for this purpose when Harold Urey isolated hydrogen 2 (deuterium) in 1931. One of the first things brought to light by the use of deuterium as a tracer was that hydrogen atoms in the body are much less fixed to their compounds than had been thought. It turned out that they shuttle back and forth from one compound to another, exchanging places on the oxygen atoms of sugar molecules, water molecules, and so on. Since one ordinary hydrogen atom cannot be told from another, this shuttling had not been detected before the deuterium atoms disclosed it. What the discovery implied was that hydrogen atoms hop about throughout the body, and that if deuterium atoms were attached to oxygen, they would spread through the body regardless of whether the compounds involved underwent overall chemical change. Consequently, the investigator must make sure that a deuterium atom found in a compound got there by some definite enzyme-catalyzed reaction and not just by the shuttling, or exchange, process. Fortunately, hydrogen atoms attached to carbon do not exchange, so deuterium found along carbon chains has metabolic significance.
The roving habits of atoms were further emphasized in 1937 when the German-born American biochemist Rudolf Schoenheimer and his associates began to use nitrogen 15. They fed rats on amino acids tagged with nitrogen 15, killed the rats after a set period, and analyzed the tissues to see which compounds carried nitrogen 15. Here again, exchange was found to be important. After one tagged amino acid had entered the body, almost all the amino acids were shortly found to carry nitrogen 15. In 1942, Schoenheimer published a book entitled The Dynamic State of Body Constituents. That title describes the new look in biochemistry that the isotopic tracers brought about. A restless traffic in atoms goes on ceaselessly, quite aside from actual chemical changes.
Little by little the use of tracers filled in the details of the metabolic routes. It corroborated the general pattern of such things as sugar breakdown, the citric-acid cycle, and the urea cycle. It resulted in the addition of new intermediates, in the establishment of alternate routes of reaction, and so on.
Thanks to the nuclear reactor, over a hundred different radioactive isotopes became available in quantity after the Second World War, and tracer work went into high gear. Ordinary compounds could be bombarded by neutrons in a reactor and come out loaded with radioactive isotopes. Almost every biochemical laboratory in the United States (I might almost say in the world, for the United States soon made isotopes available to other countries for scientific use) started research programs involving radioactive tracers.
The stable tracers were now joined by radioactive hydrogen (tritium), radiophosphorus (phosphorus 32), radiosulfur (sulfur 35), radiopotassium (potassium 42), radiosodium, radioiodine, radioiron, radiocopper, and most important of all, radiocarbon (carbon 14). Carbon 14 was discovered in 1940 by the American chemists Martin David Kamen and Samuel Ruben and, to their surprise, turned out to have a half-life of more than 5,000 years—unexpectedly long for a radioisotope among the light elements.
CHOLESTEROL
Carbon 14 solved problems that had defied chemists for years and against which they had seemed to be able to make no headway at all. One of the riddles to which it gave the beginning of an answer was the production of the substance known as cholesterol. Cholesterol’s formula, worked out by many years of painstaking investigation by men such as Wieland (who received the 1927 Nobel Prize in chemistry for his work on compounds related to cholesterol), had been found to be:
The function of cholesterol in the body is not yet completely understood, but the substance is clearly of central importance. Cholesterol is found in a large quantity in the fatty sheaths around nerves, in the adrenal glands, and in combination with certain proteins. An excess of it can cause gallstones and atherosclerosis. Most significant of all, cholesterol is the prototype of the whole family of steroids, the steroid nucleus being the four-ring combination you see in the formula. The steroids are a group of solid, fatlike substances, which include the sex hormones and the adrenocortical hormones. All of them
undoubtedly are formed from cholesterol. But how is cholesterol itself synthesized in the body?
Until tracers came to their help, biochemists had not the foggiest notion.
The first to tackle the question with a tracer were Rudolf Schoenheimer and his co-worker David Rittenberg. They gave rats heavy water to drink and found that its deuterium turned up in the cholesterol molecules. This effect in itself was not significant, because the deuterium could have got there merely by exchanges. But, in 1942 (after Schoenheimer had tragically committed suicide), Rittenberg and another co-worker, the German-American biochemist, Konrad Emil Bloch, discovered a more definite clue. They fed rats acetate ion (a simple two-carbon group, CH3COO–) with the deuterium tracer attached to the carbon atom in the CH3 group. The deuterium again showed up in cholesterol molecules, and this time it could not have arrived there by exchange: it must have been incorporated in the molecule as part of the CH3 group.
Two-carbon groups (of which the acetate ion is one version) seem to represent a general crossroads of metabolism. Such groups, then, might very well serve as the pool of material for building cholesterol. But just how do they form the molecule?
In 1950, when carbon 14 had become available, Bloch repeated the experiment, this time labeling the two carbons of the acetate ion, each with a different tag. He marked the carbon of the CH3 group with the stable tracer carbon 13, and he labeled the carbon of the COO– group with radioactive carbon 14. Then, after feeding the compound to a rat, he analyzed its cholesterol to see where the two tagged carbons would appear in the molecule. The analysis was a task that called for delicate chemical artistry, and Bloch and a number of other experimenters worked at it for years, identifying the source of one after another of the cholesterol carbon atoms. The pattern that developed eventually suggested that the acetate groups probably first formed a substance called squalene, a rather scarce thirty-carbon compound in the body to which no one kad ever dreamed of paying serious attention before. Now it appeared to be a way station on the road to cholesterol, and biochemists have begun to study it with intense interest. For this work, Bloch shared the 1964 Nobel Prize in physiology and medicine with Lynen,
THE PORPHYRIN RING OF HEME
In much the same way as they tackled the synthesis of cholesterol, biochemists have gone after the construction of the porphyrin ring of heme, a key structure in hemoglobin and in many enzymes. David Shernin of Columbia University fed ducks the amino acid glycine, labeled in various ways. Glycine (NH2CH2COOH) has two carbon atoms. When he tagged the CH2 carbon with carbon 14, that carbon showed up in the porphyrin extracted from the ducks’ blood. When he labeled the COOH carbon, the radioactive tracer did not appear in the porphyrin. In short, the CH2 group entered into the synthesis of porphyrin but the COOH group did not.
Shemin, working with Rittenberg, found that the incorporation of glycine’s atoms into porphyrin can take place just as well in red blood cells in the ~est tube as it can in living animals. This finding simplified matters, gave more clear-cut results, and avoided sacrificing or inconveniencing the animals.
He then labeled glycine’s nitrogen with nitrogen 15 and its CH2 carbon with carbon 14, then mixed the glycine with duck blood. Later, he carefully took apart the porphyrin produced and found that all four nitrogen atoms in the porphyrin molecule came from the glycine. So did an adjacent carbon atom in each of the four small pyrrole rings (see the formula in chapter 10), and also the four carbon atoms that serve as bridges between the pyrrole rings. This left twelve other carbon atoms in the porphyrin ring itself and fourteen in the various side chains. These were shown to arise from acetate ion, some from the CH3 carbon and some from the COO– carbon.
From the distribution of the tracer atoms, it was possible to deduce the manner in which the acetate and the glycine enter into the porphyrin. First, they form a one-pyrrole ring; then two such rings combine, and finally two two-ring combinations join to form the four-ring porphyrin structure.
In 1952, a compound called porphobilinogen was isolated in pure form, as a result of an independent line of research by the English chemist R. G. Westall. This compound occurs in the urine of persons with defects in porphyrin metabolism, so it was suspected of having something to do with porphyrins. Its structure turned out to be just about identical with the one-pyrrole-ring structure that Shemin and his co-workers had postulated as one of the early steps in porphyrin synthesis. Porphobilinogen was a key way station.
It was next shown that delta-aminolevulinic acid, a substance with a structure like that of a porphobilinogen molecule split in half, could supply all the atoms necessary for incorporation into the porphyrin ring by the blood cells. The most plausible conclusion is that the cells first form delta-aminolevulinic acid from glycine and acetate (eliminating the COOH group of glycine as carbon dioxide in the process), that two molecules of delta-aminolevulinic acid then combine to form porphobilinogen (a one-pyrrole ring), and that the latter in turn combines first into a two-pyrrole ring and finally into the four-pyrrole ring of porphyrin.
Photosynthesis
Of all the triumphs of tracer research, perhaps the greatest has been the tracing of the complex series of steps that builds green plants—on which all life on this planet depends.
The animal kingdom could not exist if animals could feed only on one another, any more than a community of people can grow rich solely by taking in one another’s washing or a man can lift himself by yanking upward on his belt buckle. A lion that eats a zebra or a man who eats a steak is consuming precious substance that has been obtained at great pains and with considerable attrition from the plant world. The second law of thermodynamics tells us that, at each stage of the cycle, something is lost. No animal stores all of the carbohydrate, fat, and protein contained in the food it eats, nor can it make use of all the energy available in the food. Inevitably a large part—indeed, most—of the energy is wasted in unusable heat. At each level of eating, then, some chemical energy is frittered away. Thus, if all animals were strictly carnivorous, the whole animal kingdom would die off in a very few generations. In fact, it would never have come into being in the first place.
The fortunate fact is that most animals are herbivorous. They feed on the grass of the field, on the leaves of trees, on seeds, nuts, and fruit, or on the seaweed and microscopic green plant cells that fill the upper layers of the oceans. Only a minority of animals can be supported in the luxury of being carnivorous.
As for the plants themselves, they would be in no better plight were they not supplied with an external source of energy. They build carbohydrates, fats, and proteins from simple molecules, such as carbon dioxide and water. This synthesis calls for an input of energy, and the plants get it from the most copious possible source: sunlight. Green plants convert the energy of sunlight into the chemical energy of complex compounds, and that chemical energy supports all life forms (except for certain bacteria). This process was first clearly pointed out in 1845 by the German physicist Julius Robert von Mayer, who was one of those who pioneered the law of conservation of energy, and was therefore particularly aware of the problem of energy balance. The process by which green plants make use of sunlight is called photosynthesis, from Greek words meaning “put together by light.”
THE PROCESS
The first attempt at a scientific investigation of plant growth was made early in the seventeenth century by the Flemish chemist Jan Baptista Van Helmont. He grew a small willow tree in a tub containing a weighed amount of soil, and found, to everyone’s surprise, that although the tree grew large, the soil weighed just as much as before. It had been taken for granted that plants derive their substance from the soil. (Actually plants do take some minerals and ions from the soil, but not in any easily weighable amount.) If they did not get it there, where did they get it from? Van Helmont decided that plants must manufacture their substance from water, with which he had supplied the soil liberally. He was only partly right.
A century later, the English physiologist S
tephen Hales showed that plants build their substance in great part from a material more ethereal than water—namely, air. Half a century later, the Dutch physician Jan Ingen-Housz identified the nourishing ingredient in air as carbon dioxide. He also demonstrated that a plant does not absorb carbon dioxide in the dark; it needs light (the photo of photosynthesis). Meanwhile Priestley, the discoverer of oxygen, had learned that green plants give off oxygen. And, in 1804, the Swiss chemist Nicholas Theodore de Saussure proved that water is incorporated in plant tissue, as Van Helmont had suggested.
The next important contribution came in the 1850s, when the French mining engineer Jean Baptiste Boussingault grew plants in soil completely free of organic matter. He showed, in this way, that plants can obtain their carbon from atmospheric carbon dioxide only. On the other hand, plants would not grow in soil free of nitrogen compounds: hence, they derive their nitrogen from the soil, and atmospheric nitrogen is not utilized (except, as it turned out, by certain bacteria). From Boussingault’s time, it became apparent that the service of soil as direct nourishment for the plant was confined to certain inorganic salts, such as nitrates and phosphates. It is these ingredients that organic fertilizers (such as manure) add to soil. Chemists began to advocate the addition of chemical fertilizers, which served the purpose excellently and eliminated noisome odors as well as decreasing the dangers of infection and disease, much of which could be traced to the farm’s manure pile.
