Next, Kossel began to explore something that had failed to excite Miescher: how nuclein was put together. Miescher deduced that nuclein was C29H49N9O22P3 but this formula gave no insight into its structure – which would be like trying to guess what a painting shows, knowing only how many tubes of each pigment the artist had used. Miescher ran out of steam without making any attempt to tackle the all-important question of how those 112 atoms were arranged in space.
This was the start of Kossel’s lifelong obsession with the ‘Bausteine’ or building-blocks of biochemistry – the small, simple substances which the cell assembles into the complex molecules that underpin the processes of life. For example, the Bausteine of proteins were the amino acids, of which fifteen were then known to exist. Proteins were believed to consist of large numbers of amino acids joined together, although a huge leap of faith was still needed to accept that a string of such lowly compounds could construct a towering edifice like haemoglobin.
Kossel set out to break down nuclein into its building-blocks, just as others had torn apart proteins to create a rich soup of amino acids. Boiling yeast nuclein, or heating it for two weeks in a sealed glass tube with barium salts, eventually yielded two simple organic compounds (xanthine and hypoxanthine) which both belong to the chemical family known as ‘bases’. These experiments enabled Kossel to write an authoritative book, Studies of Nuclein and its Breakdown Products (1881), which was good enough to win him the prestigious Directorship of Chemistry at the Institute of Physiology in Berlin.
There, he built up a lively research group and, with the help of the local slaughterhouse, continued his assault on nuclein. Certain animal tissues turned out to be rich sources of nuclein, although this was slow work. In 1885, Kossel boiled nuclein derived from the pancreas in acid and isolated two different bases. One was already known: guanine, named after the bird-droppings from which it was first extracted. The second was new to science, and he christened it ‘adenine’ (from the Greek for ‘gland’), after the pancreas.
It took another eight years for the slaughterhouse to offer up its next treasures, from a lump of offal that lies deep in the neck of the calf. Butchers know it as ‘gullet sweetbreads’; to scientists, it is the ‘thymus’. Chefs can do surprisingly delicious things with it, but the thymus looks better under the microscope than on the dinner plate: packed full of lymphocytes with massive nuclei. Over the next two years, the thymus lived up to the promise of those nuclei. From thymus nuclein, Kossel’s student Albert Neumann extracted two more bases, both previously unknown. He and Kossel called them ‘thymine’ and ‘cytosine’ (from the Greek for ‘cell’).
These were not casual discoveries. The isolation of adenine, for example, took several months and 100 kilograms of pancreas, and began with thirty calves and 200 litres of sulphuric acid. And that was just the start. Analysing these compounds and working out their molecular structures – the task that Miescher had never even started – was even more daunting.
The year of Friedrich Miescher’s death, 1895, was also a time of transition for Albrecht Kossel (Figure 6.1). He had been married for nine of his forty-two years, had two children and had endured twelve years in Berlin, whose inhabitants he had never liked and now loathed. In April 1895, he moved to Marburg as Professor of Physiological Chemistry, just a few weeks before Felix Hoppe-Seyler died mid-experiment on Lake Constance. Kossel wrote Hoppe-Seyler’s obituary and began his thirty-five-year stint as editor of the journal which his former chief had founded, relaunched as Hoppe-Seylers Zeitschrift für Physiologische Chemie.
While in Marburg, Kossel began looking into other substances in the nucleus. He discovered histidine, a new amino acid which lurked in protamine, the basic protein that Miescher had crystallised from salmon sperm. Next, he found completely novel proteins in the nucleus that were tightly associated with nucleic acid, which he called ‘histones’. Unlike protamine, histones were found in all tissues, not just fish sperm. He had an intuition that they would turn out to be important – maybe even more important than the substance whose building-blocks had kept him busy for the last several years.
After five fruitful years in Marburg, it was time to move on – to Heidelberg, where the people were nicer and where he remained for the rest of his life. During his twenty-four-year term there as Professor of Chemistry, Kossel brought fame and glory to the university; he also served time on the dark side of academia, as Spectabilis (Dean) and Magnificus (Pro-Rector). Throughout, the Kossels lived in No. 7 Akademiestrasse on the university campus. In Basel, Miescher had been obliged to walk to the end of his riverside garden to reach the nearest source of fresh salmon sperm; Kossel had only to go downstairs to his lab, which occupied the ground floor of the family home.
Figure 6.1 Albrecht Kossel.
To celebrate the start of the new century, Kossel returned to yeast nuclein, and showed that it contained the same adenine, guanine and cytosine as the thymus. But when he instructed his Italian student Alfredo Ascoli to isolate the fourth base, thymine, they drew a mystifying blank. Instead, Ascoli found another base in yeast nuclein. This turned out to be uracil, which had been made in the laboratory from uric acid (hence the name) but had not previously been found in nature. To compound the puzzle, they failed in their attempts to pull uracil out of nuclein extracted from either thymus or pancreas.
The bases were the most characteristic and exciting of these building-blocks. They were the distinctive chemical fingerprint of nuclein, as they were not found in proteins, carbohydrates or fats. Analysis of the bases also revealed that ‘nuclein’ was not a single chemical substance. Nuclein extracted from the pancreas or thymus – or other animal sources such as salmon testes and the red blood cells of birds – always contained adenine, guanine, cytosine and thymine. By contrast, yeast nuclein lacked thymine and instead contained Ascoli’s uracil, in addition to the familiar adenine, guanine and cytosine. And then someone isolated ‘yeast nuclein’ from wheat. This was taken as evidence that there were two distinct types of nuclein – one unique to animal tissues and the other to plants, hallmarked respectively with thymine and uracil.
Kossel dug other building-blocks out of the debris obtained by boiling nuclein in water or acid. One constant feature of both ‘animal’ and ‘plant’ nucleins was their high phosphorus content – the chemical oddity that had originally made Miescher realise that this was not a protein. This suggested that phosphate groups were embedded somewhere in the nuclein molecule.
One further hint emerged from Kossel’s experiments, tantalising and inconclusive. Extracts of yeast nuclein left behind a peculiar, almost rubbery residue when the bases and phosphate had been removed. It defied accurate analysis but could reduce certain iron salts. This was a diagnostic reaction of the ‘pentose’ sugars which contain a five-cornered ring structure (‘hexose’ sugars, such as glucose, have a six-sided ring). The same conclusion was reached by Olof Hammarsten in Uppsala, who further found that the sugar did not match any known pentose. It turned out that animal nucleins contained a sugar, which was different from the elusive pentose of yeast nuclein and even harder to pin down; first impressions suggested that it might be a hexose.
As yet, none of this provided any clue about how the phosphates, sugars and bases might all be tacked together to make nuclein – or whether its structure might hold any biological significance.
Meanwhile, a new era in the chemistry of the nucleus had been quietly ushered in. Richard Altmann, Professor of Anatomy in Leipzig, had re-baptised nuclein to emphasise both its source and a distinctive chemical property. He gave it the name ‘Nucleinsäure’, which translates into English as ‘nucleic acid’.
Before long, ‘thymus nucleic acid’ and ‘yeast nucleic acid’ had entered the biochemical vernacular. ‘Thymonucleic acid’ soon followed; this rolled off the tongue so smoothly that it remained in common use until the early 1950s, long after the mystery of those elusive sugars had been cracked and a snappier, chemically accurate name had been introduced.
> The shape of things to come
The bases fall into two families, the pyrimidines and the purines. The pyrimidines are simpler, being built around a hexagonal skeleton – the ‘pyrimidine ring’ – made of carbon (C) and nitrogen (N) atoms. Cytosine, thymine and uracil are all pyrimidines. Purines are more complex, consisting of the pyrimidine ring with a pentagon of N and C bolted to its side. Adenine and guanine are purines.
The bases are shown in Figure 6.2, which pays an insultingly poor tribute to the hard work that Kossel’s team and others put into deducing their structures. Finding a new substance – which might be marked out by a novel combination of properties such as melting-point or solubility – was only half the battle. Back then, the shapes of molecules were conjured up in the minds of chemists, starting with the chemical formula and respecting the number of links that each atom can form with other atoms: just one for hydrogen (H), two for oxygen (O), three for nitrogen (N) and four for carbon (C). A plausible structure ties up all the available links and leaves none dangling in mid-air – as a careful look at thymine will confirm. Hundreds or thousands of iterations might be needed before the right shape crystallises out of the jumble of letters (five Cs, six Hs, and a pair of Ns and Os, in the case of thymine).
Figure 6.2 The bases found in DNA and RNA. DNA contains two purines, adenine and guanine (top), and two pyrimidines, cytosine and thymine (bottom). RNA also contains adenine, guanine and cytosine, but with uracil instead of thymine.
Kossel’s theoretical structures were later confirmed by the next generation of pioneering chemists, from X-ray photographs of crystals of the bases. This technique pinpointed each atom in the molecule, and their positions would have registered nearly perfectly if they had been drawn on a glass sheet and laid over Kossel’s drawings. And seventy years after Kossel discovered adenine, Jim Watson sketched out the flat, geometric structures of guanine, adenine, cytosine and thymine, stipulating precise lengths for the sides of the hexagonal pyrimidine ring and the pentagonal side-piece of adenine and guanine. He took his drawings down to the machine-room in the basement of the Cavendish Laboratory in Cambridge, where they cut the shapes out of a sheet of tin. These models of the bases were accurate enough to be inserted into the spidery sculpture of the double helix which was taking shape in the lab upstairs.
Relax – you will not be tested on the molecular structures or chemical formulae of the bases shown in Figure 6.2. However, you might like to fix the basic shapes of the purines and pyrimidines in your mind, as they will play a crucial role later in this story. The shapes of the purines and pyrimidines are the key to the attractive force that pulls together the two helices of DNA. They also dictate how the molecule replicates itself. As such, they are the common template of heredity and life in every living organism. So give them the respect they deserve, because they have made you what you are.
By-products
The bases which Kossel teased out of nucleic acids slotted nicely into the cutting edge of early twentieth-century biochemistry. This was a fast-moving and profitable field, especially as the bases were proving to be a treasure chest of interesting new compounds which brought a mixture of fame, fortune and misery.
Guano, the source of guanine, was scooped by the shipload off Peruvian islands and transported to Europe and America to be turned into a commodity to dye for: a stunning pigment that quickly undercut the market for the fabled Tyrian Purple, once reserved for the robes of Byzantine emperors. Even more successful was Adolf Baeyer’s synthetic dye which soon ruined the Indian farmers who grew indigo and later put the ‘blue’ in ‘blue jeans’. Another of Baeyer’s triumphs was the compound which he named after Barbara, who might have been the saint who protects against lightning strikes, or a barmaid in Munich. Barbituric acid yielded the first barbiturate, which Baeyer called ‘Veronal’ after Verona, the quietest place he knew. Its descendants went on to corner the market for treating insomnia, anxiety and epilepsy, and those sentenced to die by lethal injection.
Two of the first five Nobel Prizes in Chemistry were awarded for research in this field. Emil Fischer was recognised in 1902 (the second year of the prize) for his ‘extraordinary services’ in working out how sugars and purines were synthesised. In 1905, Adolf Baeyer won the prize for ‘his advancement of organic chemistry and the chemical industry’ through barbiturates and his ‘gorgeous pigment’ which brought indigo to the masses.
Five years after that, the 1910 Prize for Physiology or Medicine recognised Albrecht Kossel for his ‘contributions to our knowledge of cell chemistry made through his work on proteins, including the nucleic substances’. His Nobel lecture, given on 10 December 1910, was entitled ‘The chemical composition of the cell nucleus’. Those wishing to hear about the nucleic acids and their Bausteine would not have been disappointed, but he devoted the second half of the talk to the protamines and histones which he believed dominated this ‘morphologically so important structure’. What did all these players do in the nucleus? If Kossel had any ideas, he did not reveal them.
From Rostock to Stockholm
Albrecht Kossel comes across as an odd, mixed bag of a man. His photographs do not make him look like a bundle of fun – bald, drooping silver moustache and an imperious, humourless gaze – and they fit well with his reputation for being able to cut himself off from the ‘everyday worries and troubles’ of the world outside the laboratory.
A pen portrait of Kossel in his natural habitat was provided by Ernest Kennaway, later Professor Sir Ernest Kennaway FRS, who came to Heidelberg as a visiting fellow from Oxford in 1911. Once or twice a week, Kossel’s juniors lined up to be tossed a few crumbs of advice. Kossel had a stock question – ‘Have you found a very interesting salt yet?’ – which harked back to the old-fashioned chemist’s excitement on crystallising a new compound. Impeccably dressed, Kossel prefaced each interrogation with a military-style click of his heels, and then gave the strong impression of being lost in thought about something else.
That formal facade hid two other versions of Kossel. One was pathologically shy; even lecturing to undergraduates could cripple him with performer’s nerves, but he prepared his talks perfectly and his students – who often filled the lecture hall beyond capacity – adored him. The other Kossel emerged at the many dinner parties thrown by his wife Luise, an accomplished hostess. This one had an impish sense of humour and was fond of telling convoluted jokes, with ‘his blue eyes sparkling with merriment’ as he closed in on the punchline. He also knew how to put on a good show. In August 1907, Kossel hosted the Seventh International Physiological Congress and made it a meeting to remember, culminating in an evening boat trip on the Rhine with the sky above Heidelberg Castle ablaze with fireworks.
Despite his shyness and stiffness, Kossel inspired deep loyalty and affection from those who worked with him. When he returned to Heidelberg after collecting his Nobel Prize in December 1910, he was greeted with an extraordinary celebration, organised by the Guild of Students. University teachers and students assembled on the campus and formed a torchlit procession which wound through the streets of the old city until it reached the house in Akademiestrasse where the Kossels lived over the shop. There, the front door was flung open and the new Nobel laureate welcomed them all inside, leading them upstairs past his lab to an impromptu party which went on far into the night.
Politics always left Kossel cold. He was a principled man, whose moral backbone did not flex as freely as those belonging to many of his peers. His wife Luise, the charming socialite, was quite smitten with the new nationalism that was sweeping the country. Kossel was not, because he did not see the need for Germany to bludgeon its way to ‘a place in the sun’. He refused to bend in the winds of change – a stance that would become ever harder to maintain as his nation began its inexorable slide into the First World War.
In late August 1911, just eight months after listening to her husband’s praises being sung in Stockholm for his ‘extraordinary services’ to chemistry, Luise Kossel found herse
lf cruising into New York on board the SS Prinz Friedrich Wilhelm. She and her daughter Gertrude were there to accompany him on a holiday-cum-lecture tour. And Albrecht Kossel, the brilliant but modest man from across the Atlantic, had seized the imagination of the New World.
‘One of the greatest living scientists,’ proclaimed the New York Times, under the headline, ‘seeks life secret in study of cells – may solve cancer problem’. That headline was not entirely accurate, as ‘the foremost authority in the world on cellular life’ took pains to explain (in good English) that he was ‘not seeking the secret of life, but the secrets of cellular composition’; the task of discovering how those secrets fitted into the bigger picture of life was for others. As he put it, ‘The processes of life are like a drama, and I am studying the actors, not the plot. There are many actors, and it is their characters which make the drama. I seek to understand their habits, their peculiarities.’
Kossel’s tour culminated in the prestigious Harvey Lecture which he gave in New York in mid-October. There, he explained his mission to find the Bausteine that were assembled ‘according to a determined plan’ into the ‘larger aggregates’ which kept cells alive. Of course he talked about nucleic acids and bases, but he concluded that the most exciting and versatile Bausteine were the amino acids which made proteins like the histones and protamines, so crucially important in the nucleus.
Kossel bade farewell to the United States with a successful tour and an honorarium of $1,000 under his belt. Back in Heidelberg, he settled into the last twelve years of his career; a grand old man, crowned with the greatest glories that the scientific community can bestow on one of its brethren.
Unravelling the Double Helix Page 10