Biochemistry

History of Biochemistry

by Noel G Coley

Introduction

Biochemistry aims to explain biological phenomena in chemical terms. The problems are highly complex and until the twentieth century progress was slow and unreliable. During the eighteenth century chemistry was dominated by the phlogiston theory and the traditional method of organic analysis by destructive distillation provided no information about elementary composition. Both in theory and techniques therefore, chemistry was wholly inadequate to unravel the mysteries of the vital functions. Yet the importance of fermentation was recognized, photosynthesis was discovered and studies of animal respiration and digestion made useful contributions to early biochemistry. The complex chemical transformations occurring at moderate temperatures during the vital functions led to the belief that in living matter the ordinary laws of chemistry were modified by unknown vital forces. These controlled the special conditions pertaining to living matter, but when life was extinguished ordinary chemical forces once more came into play, resulting in decomposition and decay. Despite growing experimental evidence to the contrary, vitalism persisted among physiologists and chemists into the nineteenth century.
 
The displacement of phlogiston by oxygen from the late eighteenth century greatly facilitated progress. In addition, Antoine Lavoisier's experimental definition of the chemical element was accompanied by a more consistent use of the balance and a rational chemical nomenclature. Combustion analysis, first introduced by Joseph Louis Gay-Lussac in 1811, allowed the empirical formulae of organic compounds to be determined, although more accurate atomic weights were required for reliable results. Chemists analysed plant and animal substances from the beginning of the nineteenth century, while physiologists and some physicians applied themselves to studies of the vital functions in health and disease. However, the development of biochemistry would also require later nineteenth-century advances in physical and organic chemistry. These included structural organic chemistry, electrochemistry, osmosis, colloid chemistry, stereochemistry and molecular energy studies together with physical techniques such as X-ray diffraction, electrophoresis, chromatography and labelling with radioisotopes.
 
Animal Chemistry
 
In the 1780s Lavoisier proposed a mechanism for photosynthesis by which plants take in carbon dioxide and release oxygen; he also began to investigate animal respiration. With Pierre Laplace he measured the heat evolved by small mammals, and similarities between combustion and respiration led him to locate the production of animal heat entirely in the lungs. William Prout, better known for his atomic hypothesis, also studied the vital processes. He thought that digestion and assimilation proceeded in stages involving the action of saliva, gastric juice, pancreatic juice and bile, followed by oxidation in the lungs, where the products of digestion were finally converted into blood. In 1824 Prout discovered hydrochloric acid in gastric juice and he later classified foods as saccharinous, albuminous and oleaginous (cf. carbohydrates, proteins and fats).
 
Michel Eugène Chevreul investigated the nature of animal fats from about 1811. He showed that the fatty acids were analogous to inorganic acids and were capable of yielding salts with bases. Separating them using solvents, he identified each fatty acid by its melting point. The use of a physical constant as a criterion of purity and means of identification was an important step in the development of organic analysis. His results, published in 1823, showed that animal fats, although products of the vital functions, were normal chemical compounds.
 
Jöns Jacob Berzelius and Justus Liebig were among the leading nineteenth-century chemists who advanced biochemistry. Berzelius analysed animal solids and fluids; Liebig endeavoured to extend Berzelius's work by creating a comprehensive metabolic theory. Beginning with the oxygen theory and his own organic analyses, Liebig described the assimilation of foods and the oxidation of muscle tissues releasing energy and excretory products like urea and uric acid. For three decades after its publication in 1842, Liebig's Animal Chemistry, or organic chemistry in its application to physiology and pathology stimulated chemical and physiological research. Unfortunately, Liebig was no physiologist and most of those who tried to find supporting evidence for his theories failed. By about 1870 Liebig's theories had been almost entirely superseded, but the research they engendered helped to advance biochemistry and some nineteenth-century chemists even applied Liebig's ideas successfully. For example, Henry Bence Jones discovered proteins now known to be part of the autoimmune system and Ludwig Thudichum identified some important biochemical substances, including haematoporphyrins in the blood and the main chemical constituents of the brain.
 
Cell Theory
 
In 1838 Theodor Schwann, originator of the cell theory in biology, suggested that fermentation occurred only in living yeast cells. Liebig refused to accept this and proposed an alternative chemical theory of fermentation but Louis Pasteur, opposing Liebig's chemical theory, showed that fermentation depends on the vital functions of living yeast cells and bacteria. The controversy was resolved in 1872 when Pasteur's work received general recognition.
 
Close links between plant and animal life have been identified. Animal metabolism is ultimately dependent on the assimilation of plant material and consequently photosynthesis studies have made important contributions to the elucidation of animal metabolism. In 1817 J. Pelletier and J. B. Caventou suggested the name ‘chlorophyll’ for the green substance common to all plants. The connection between chlorophyll and starch in growing plants was recognized in1862 when Julius von Sachs, a German plant physiologist, suggested that starch in green plants is produced from carbon dioxide and water. In the early years of the twentieth century chromatography revealed two forms of chlorophyll and in 1914, Richard Willstätter and Arthur Stoll showed that both are esters of dibasic acids with methyl alcohol and phytol, a hitherto unknown unsaturated, aliphatic alcohol (C20H39OH). Further investigation of photosynthesis showed that it involved a chemical and a photochemical step. The chemical step is associated with the formation of a chlorophyll–carbon dioxide compound and the photochemical step involves the formation of a peroxide which is then decomposed by an enzyme to yield oxygen, formaldehyde and reconstituted chlorophyll.
 
About 1860 Moritz Traube suggested that to understand the chemistry of life required a correct theory of fermentation. His ‘ferments’ were oxygen-rich agents derived from proteins. In 1878 Willy Kühne coined the term ‘enzyme’ for Traube's ferments. Enzyme action had first been observed in 1833 when Anselme Payen and Jean François Persoz isolated a compound (diastase) which converted starch into sugar. Three years later pepsin was extracted from the stomach wall by Schwann. These discoveries preceded Berzelius's notion of catalysis (1837) which, he predicted, would prove important in plants and animals where complex reactions took place at very moderate temperatures. Further investigation of fermentation resulted in a distinction between ‘organized’ ferments such as yeast and ‘unorganized’ ferments including the enzymes, the biological catalysts, but in 1877 Felix Hoppe-Seyler suggested that there was no fundamental difference between them. Twenty years later Eduard Buchner fermented a sugar solution with a cell-free extract of yeast. This discovery showed that the contents of the cells, rather than their life processes, caused fermentation. This led to a theory of fermentation based on enzyme action and provided the key to the study of cell chemistry.
 
Liebig's idea that animals are incapable of biosynthesis was challenged in the 1850s by Claude Bernard's discovery of the glycogenic function of the liver. In 1867 Carl Voit tried to revive Liebig's theory of the direct assimilation of proteins, but Eduard Pflüger, Professor of Physiology at Bonn, argued that there were constitutional differences between food proteins and tissue proteins. In the 1870s he demonstrated the importance of intracellular respiration, a discovery embraced by Bernard, and suggested a theory of indirect nutrition, whereby animal cells synthesize complex substances from simpler nutrient molecules derived from food. Bernard postulated an internal environment within the cells, where chemical degradation and synthesis takes place. According to Bernard all the principles necessary for the maintenance of animal life are released into the blood. Respiration introduces oxygen, digestion introduces the necessary nutrients together with the secretions of the various organs. The blood is the carrier of all these substances and the cells absorb from it only what they require to maintain the vital functions occurring inside them. The circulation and secretions together ensure, besides the renewal of the internal environment, the removal of waste products, and all these changes are regulated and harmonized by the nervous system, maintaining a steady state (homeostasis). See also Claude Bernard, and History of Blood Chemistry
 
Bernard's theory of the internal environment marks a watershed in the history of biochemistry. It put an end to theories of direct assimilation, replacing them with the breakdown of complex food molecules into smaller nutrient constituents from which new compounds specific to the needs of each cell were synthesized. This theory prefigured one of the most important discoveries of modern molecular biology – the coded programme of protein synthesis – but Bernard's ideas of indirect nutrition and the internal environment were too advanced for many of his contemporaries. Much detailed research was needed to establish them and it was due to British and American scientists including W. M. Bayliss, E. H. Starling, C. S. Sherrington, J. S. Haldane, J. Barcroft, W. B. Cannon and L. J. Henderson that the concept of the internal environment was elaborated from the beginning of the twentieth century.
 
Biological Oxidation
 
It has been recognized from the beginning of the nineteenth century that energy is released during biological oxidation, though the mechanisms by which this was brought about remained in doubt. When C. F. Schönbein discovered ozone in 1840 he suggested that the first step in biological oxidation was the conversion of oxygen to ozone. This idea led to the popular nineteenth-century ‘ozone craze’. Later, in 1903, A. N. Bach and R. Chodat discovered the enzyme peroxidase in plant cells and it was thought that peroxides caused biological oxidation in a two-stage process. In the first stage the enzyme oxygenase formed peroxides; in the second stage peroxidase used these peroxides to oxidize other organic compounds. This theory dominated ideas on biological oxidation until about 1920. Although based on plants, it was widely thought that the oxygenase–peroxidase system also accounted for respiration in animal tissues. It was later found that oxygenase is itself an enzyme (catechol oxidase) capable of oxidizing catechol and similar compounds to quinones and that peroxidase cannot use these as oxidizing agents.
 
From 1920 another theory of biological oxidation was proposed, based on the respiratory systems in yeast, bacteria and animal tissues. According to this theory, oxygen atoms were first activated by combination with iron atoms in a haemoprotein enzyme. Otto Warburg called this the ‘respiratory ferment’; it would later be identified as cytochrome oxidase. A rival school considered that the organic molecules were activated rather than oxygen and that most biological oxidations were dehydrogenations brought about by a new group of enzymes, the dehydrogenases. These have turned out to be the most important factors in biological oxidations; there are now well over 150 known dehydrogenases. By studying these two opposing systems using spectroscopic techniques David Keilin followed the course of respiratory changes in the mitochondria of cells. He discovered the oxidative cytochromes in 1925 and identified many intermediate steps in cell respiration.
 
In 1906 Arthur Harden and W. J. Young observed that the addition of a soluble phosphate to a fermenting sugar solution caused the rate of fermentation to increase. The additional carbon dioxide and alcohol formed was proportional to the quantity of phosphate added and the phosphate was converted into a hexosephosphoric acid. These observations were important for metabolic studies in the mid-twentieth century, which have shown that most vital functions depend on enzyme action. The hydrolytic processes catalysed by enzymes break down starch, fats and proteins into simpler molecules (monosaccharides, fatty acids and amino acids) which then enter complex metabolic pathways also controlled by enzymes. During these processes energy is released, providing animal heat or to be stored for later use in muscular exertion. One of the principal aims of twentieth-century biochemistry has been to discover the details of these metabolic pathways. Considerable progress has been made, though many details still require further elucidation.
 
In 1907 Walter Fletcher and Frederick Gowland Hopkins published the first reliable quantitative data on the proportions of lactic acid in muscle tissue. Lactic acid was known to take part in the biochemical transformations of carbohydrates, linking them with proteins and fats, but these two workers now recognized that its role in the metabolism of animal tissues is pivotal. However, they did not discover that lactic acid holds an intermediate position between sugar and alcohol in the metabolism of muscle tissue.
 
In the early years of the twentieth century Gustav Embden had isolated several intermediate metabolic products from muscle tissues, including adenyl phosphoric acid, also found in the liver. Embden was the first to discover and link together all the steps in the conversion of glycogen to lactic acid. Between 1919 and 1921 Otto Meyerhof, recognizing that muscle is the only tissue in which it is possible to compare the chemical changes occurring with the work done or heat energy evolved, investigated lactic acid formation in muscle tissue as a measure of work done. This led to the glycogen–lactic acid cycle, and made a fundamental contribution to the understanding of muscular action. In all but the briefest, most intense muscular contractions additional adenosine triphosphate (ATP), an important energy-carrying coenzyme, is supplied by the chemical reactions of the glycolytic, or Embden–Meyerhof–Parnas, pathway, particularly applicable to rapid muscular action. In this, the change from glucose to lactate is coupled with the formation of ATP from adenosine diphosphate (ADP). If not immediately required, the energy is stored in the muscle. ATP is created in greater abundance than the numbers of carbohydrate, fat or protein molecules metabolized, creating more of this energy-rich compound than is required for immediate needs. The excess energy is stored in muscle tissue as phosphocreatine, a labile compound that readily absorbs and releases phosphate groups.
 
The lactate, a waste product, diffuses out of the muscle to be transported by the blood to the liver where most of it is converted into glycogen. The rate of oxidation of lactic acid is regulated by the rate of respiration. In prolonged strenuous exercise lactic acid is formed faster than it can diffuse out of the muscle and this results in muscle fatigue. Glycogen from the liver is reversibly converted into glucose and finds its way into the bloodstream, thus completing the cycle.
 
In the 1930s Warburg investigated the relation between the chemical and photochemical steps in photosynthesis using the effects of intermittent illumination on the green unicellular alga Chlorella. His work led to a study of the cytochromes. Warburg and Keilin independently examined the respiratory function of the cytochromes and showed that the respiratory chain in the cells was located in the mitochondria. Alternate oxidation and reduction could be traced through chains of cytochromes, each reducing the next in line until the last, identified as the respiratory ferment, reacted with oxygen. At each stage the small quantities of energy released are stored in high-energy bonds such as the phosphate link in ATP. The whole complex system was summarized in the Krebs cycle, the metabolic pathways of which have been subjected to minute and intensive research ever since. It also became clear that other substances besides the cytochromes were involved and in 1932 Axel Theorell working with Warburg, isolated the first so-called ‘yellow enzyme’ composed of a protein and the nonprotein yellow coenzyme riboflavin (vitamin B2). Keilin isolated the oxidative enzyme cytochrome c and proposed a new explanation of the action of the cytochromes involving activated hydrogen as well as oxygen. These investigations were carried out on heart-muscle preparations, but similar reactions have been detected in the cells of plants, microorganisms and fungi. Thus, they represent a common feature shared by all forms of life. They were essential early stages in the elaboration of the citric (or tricarboxylic) acid cycle proposed by Sir Hans Krebs in 1937. In animal cells the enzymes specific to each step are located in the mitochondria, in plant cells they are found in the chloroplasts, and in microorganisms they are found in the cell walls.
 
A fuller elaboration of the citric acid cycle came after 1940 with the discovery that the three principal food constituents, carbohydrates, fats and proteins, all yield a common product, coenzyme A. This forms citric acid which is then broken down into carbon dioxide and water with the liberation of coenzyme A once more. At each intermediate step in this metabolic chain, hydrogen atoms are transferred from one carrier to another and small quantities of energy are released. Thus, the citric acid cycle is the source of energy refurbishment supporting all the vital processes.
 
Enzyme reaction rates vary widely and depend on activators, or coenzymes, ranging from simple metal ions such as Ca2+ or Mg2+ to complex organic molecules such as vitamins. In 1960 it was found that the coenzyme in energy-producing processes, ATP, is itself fabricated in the cells by a protein enzyme, ATP synthase. The molecular structure of ATP synthase was partially determined in 1994 after 12 years of research. In addition to providing fresh knowledge about how living things produce energy, it is thought that this research may throw new light on the processes of ageing and the causes of degenerative diseases. The large molecules of carbohydrates, fats and proteins are broken down into smaller molecules such as pyruvates, free fatty acids and amino acids, all of which take part in the mechanisms of the citric acid cycle and thus link the metabolic transformations of carbohydrates, proteins and fats. Enzymes are now known to control most biochemical processes, from the breakdown of complex molecules like carbohydrates and proteins during animal metabolism to the synthesis of macromolecules within the cells. Most enzymes have a protein structure and most work with one or more coenzymes, but certain other complex molecules not regarded as enzymes, such as messenger RNA, also show catalytic properties.
 
Structural Organic Chemistry
 
Before the details of such cellular mechanisms could be fully explored, far more information about the chemical structures of large molecules was needed. Thus, progress in biochemistry had to await the development of structural organic chemistry.
 
In 1815 J. B. Biot found that certain natural oils rotated the plane of polarized light. The same substance could exist in two forms both having the same empirical formula, yet capable of rotating the plane of polarized light in opposite directions. The problem facing organic chemists was to correlate optical activity with molecular configuration. The fundamental discovery required to solve this problem was Kekulé's recognition in 1858 that the carbon atom is tetravalent. Using this, J. H. Van't Hoff suggested in 1874 that structural formulae must be three-dimensional. A compound in which a central carbon atom is attached to four different substituents in a tetrahedral structure would have two non-superposable forms one of which is the mirror image of the other. Applying this concept Pasteur investigated molecular asymmetry in the tartrates by separating potassium tartrate crystals mechanically. Optical activity was later used by Emil Fischer to identify the many stereoisomers of the sugars.
 
In 1885 Fischer observed that phenylhydrazine (C6H5.NH.NH2) forms well-defined crystalline compounds (osazones) with the sugars and this allowed him to identify the molecular structures of 16 stereoisomers of glucose. Before 1891 it had been necessary to assume a configuration for each sugar, but from that year onwards Fischer applied himself to the problem of assigning specific configurations to each isomeric sugar molecule and by 1896 he had done so for all the monosaccharides. He retained the straight-chain formulae devised by H. Kiliani, though later studies by W. N. Haworth and others showed that they contain lactone rings. Fischer also examined the degradation of sugars by enzymes and from his study of the saccharases he recognized the specificity of enzymes and concluded that the enzyme and its substrate were related as a key to its lock. Later work has shown this concept to be true to a degree Fischer himself could hardly have suspected.
 
Fischer's other major contribution to structural organic chemistry concerned his elucidation of the molecular structures of the purines, amino acids and proteins. Even before he began work on the sugars Fischer had been engaged in a study of three purine derivatives, caffeine, theobromine and xanthine, all structurally related to uric acid. Ludwig Medicus had given the correct formula for uric acid in 1875; Fischer confirmed it by synthesis. Between 1882 and 1900 he isolated about 130 purine derivatives, thus extending Liebig and Friedrich Wöhler's 1838 studies of the oxidation products of uric acid. Fischer attempted to correlate the molecular structures of these compounds with their physiological properties. Later, in 1914, he returned to this work and succeeded in preparing the first synthetic nucleotide, theophylline d-glucose phosphoric acid, thus linking his work on sugars with his studies of the purines. This work was a first step leading to the study of the nucleo-proteins. Fischer also investigated polypeptides and simple proteins, showing how amino acids are combined in protein molecules. By 1907 he had synthesized a polypeptide containing 18 amino acids. Its molecular weight was 1213 and he calculated that there would be 816 possible optical isomers.
 
In 1877 Traube suggested that enzymes are related to proteins, but the isolation of pure enzymes only began in the 1920s. Jackbean urease was the first enzyme to be crystallized in a pure state, by J. B. Sumner in 1926. J. H. Northrop, who isolated several proteolytic enzymes, crystallized pepsin in 1930. Both were found to be complex proteins and little could be done to determine their structures until special methods were developed. The sequence of amino acids in proteins was later found using a method devised by Frederick Sanger for determining the order of the 51 amino acids in the insulin molecule in about 1950. Another group of biological compounds, the hormones, has also caused considerable difficulty for structural organic chemists in the twentieth century. Those hormones, which like glucagon are polypeptides, have posed problems in the elucidation of their amino acid sequences, but others have been found to be based on the characteristic ring structure of the sterols containing 17 carbon atoms and 28 hydrogen atoms arranged in four rings.
 
Hormones and Endocrinology
 
In the nineteenth century it was recognized that glands such as the spleen, thymus, thyroid and adrenals secrete specific substances which are carried by the blood to other organs in the body. Towards the end of the nineteenth century a number of discoveries revealed the essential nature of these secretions for animal health and it became common to connect the lack or excess of specific endocrine secretions with certain diseases. Adrenaline (C9H13O3N) was discovered in 1894 in experiments on the blood pressure of a dog. A little later vasopressin, a peptide hormone released from the posterior lobe of the pituitary gland, was discovered. This is also capable of increasing the blood pressure. Adrenaline was isolated independently in 1901 by T. B. Aldrich and J. J. Abel in America and shortly before by J. Takamine in Japan. In 1902 William Bayliss and Ernest Starling identified the substance secretin, produced by epithelial cells in the stomach during digestion. This is released into the bloodstream and in the pancreas it causes the secretion of pancreatic juice. Thus, secretin was recognized as a chemical messenger, produced in one organ and carried by the bloodstream to the organ it is intended to stimulate. Starling introduced the term ‘hormone’ to describe such chemical messengers in 1905. However, as secretin is a polypeptide of moderate molecular weight, Bayliss and Starling were unable to isolate it, much less determine its molecular structure. The existence of a number of other mammalian hormones was surmised, but they all proved difficult to isolate and synthesize and only a few had been characterized. It is now known that hormones fall into three main classes: steroids, peptides and proteins, and amino acid derivatives.
 
Hormone secretion is controlled by the pituitary gland situated at the base of the brain. There are three main divisions of this gland, although the whole complex organ weighs only about one gram in the human adult. During the 1920s work on the pituitary gland led to the isolation of six different protein hormones, four of which were found to stimulate other endocrine glands. Since then other hormones have been isolated, several of which control the sexual functions; others govern the rate of growth and the chemical or physiological balance in the animal body. The chemical structures of all have been determined and their chemical syntheses achieved.
 
Adrenaline, a derivative of catechol, was the first hormone to be synthesized in 1904; thyroxin, the active principle of the thyroid gland, first isolated by E. C. Kendall in1914, was synthesized by C. R. Harington and G. Barger in 1926–1927. Thyroxine, stored in the thyroid gland as a protein, is hydrolysed by thyrotrophin, another hormone produced by the pituitary body. Thus one hormone calls another into action. Thyroxine is mainly concerned with the consumption of oxygen and thus the metabolism of all the cells and tissues in the body; it appears to increase the production of a number of enzymes. In 1922 Frederick Banting and Charles Best identified and prepared the pancreatic hormone, insulin, for therapeutic use in diabetes. J. J. Abel obtained crystals of insulin in 1926, but Dorothy Hodgkin using X-ray diffraction elucidated the molecular structure of insulin only in the 1960s. This work complemented Sanger's earlier determination of the complete amino acid sequence for bovine insulin. Sanger's methods opened the way to the determination of the structures of many other complex proteins. He later applied radioisotope labelling using 32P and other techniques to determine the sequencing in ribonuclease synthesized in the early 1960s. The pancreas also produces another hormone, glucagon, a polypeptide containing 29 amino acids in a known sequence.
 
The chemical structures of the steroid hormones produced by the adrenal glands and the sex organs are related to cholesterol. They are all very similar and even small structural changes in their molecules produce profound physiological effects. The steroid hormones secreted by the adrenal cortex control carbohydrate and mineral metabolism; some are concerned with the formation of glucose from proteins and enable the body to withstand stresses such as intense heat or cold, injury and infection. The hormones of the adrenal cortex control carbohydrate and mineral metabolism in the body. Similar compounds, though with more complex structures, are produced by the thyroid gland. Work on the sex hormones was carried out from about 1926. In 1929–1930 Edward Doisy in America, Guy Marrian in Britain and Adolf Butenandt in Germany isolated from the urine of pregnant women two ovarian hormones related to sterol. Butenandt also isolated androsterone from male urine and in 1934 Leopold Ruzicka in Switzerland obtained this hormone from cholesterol. These biologically active substances are metabolites of the ovaries and testes, respectively. The differences in their molecular structures and physiological actions are relatively slight; both are fat soluble.
 
The anterior pituitary gland influences the rate of secretion of the hormones of the adrenal cortex, the gonads and thyroid gland. Its action is influenced by nerve centres in the hypothalamus, the part of the brain immediately above the pituitary gland. The secretion of hormones and the functions they control are therefore coordinated by the nervous system. In some cases there is a ‘feedback’ mechanism whereby the secretions of one endocrine gland stimulates or damps down secretions from another. Thus a circle of action and reaction exists between some of the endocrine organs as the balance of hormone secretion is maintained.
 
The effects of injecting adrenaline are similar to those induced by stimulation of the sympathetic nervous system and this led to the suggestion that the liberation of adrenaline at sympathetic nerve endings might transmit the excitatory or inhibitory impulse to the effector cells of muscles or glands. Thus it seemed that certain hormones might control the functions of the central nervous system. It is now thought that a large number of chemicals can act in this way, but only a few have been so far identified. The first of these, noradrenaline, was isolated by Hans van Euler in 1946. Others include acetylcholine, dopamine and secretonin. Acetylcholine was first isolated in 1914; its function in slowing the heartbeat was identified in 1921 by Otto Loewi, a German physiologist.
 
In the 1930s it was observed that one hormone can act in opposition to another. Frédéric Houssay in Argentina found that extracts of the anterior pituitary gland combat the action of insulin. Frank Young found that administration of anterior pituitary extract causes a persistent diabetic condition. Cyril Long and Francis Lukens discovered an antagonism between insulin and the secretions of the adrenal cortex. These and other observations that hormones can act against each other have proved useful in elucidating the endocrine control of the vital functions and in medical and surgical treatments of certain conditions. However, it is now known that the substance present in an endocrine gland does not necessarily take the same form as that of the hormone in the blood. Furthermore, hormones may themselves undergo metabolic changes, either in the blood or in the tissues, before they are able to cause reactions in the cells or in the enzymes they influence. Thus, increased knowledge in endocrinology has created a situation in which the precise chemical definition of a hormone has become a matter of some difficulty.
 
Vitamins
 
Although dietary deficiencies have long been recognized as the cause of certain diseases, the search for vitamins (a term introduced by Casimir Funk) began about 1912. Gowland Hopkins observed that animals fed on a sufficient quantity of purified foods ceased to grow unless a small amount of milk was added. This drew attention to the ‘vitamin’ question, though for several years the real existence of these elusive dietary factors was disputed.
 
In 1915 Elmer McCollum and M. Davis identified fat-soluble A and water-soluble B as essential accessory dietary factors in rats. These were later named vitamins A and B respectively. The latter was found to prevent beri-beri, while lack of vitamin A retarded growth and caused increased liability to infection of the respiratory system. The antiscorbutic vitamin C was later identified and in 1922 lack of fat-soluble vitamin D was recognized as the cause of rickets. About the same time vitamin E was identified. In 1926 it was discovered that pellagra is a vitamin deficiency disease. The vitamin in this case seemed to accompany the anti beri-beri factor, but was different from it, in that it was much more stable to heat. In 1927 the anti beri-beri factor was labelled vitamin B1 and the heat-stable factor became vitamin B2. This was at first thought to be a single compound, but was later found to be a complex including riboflavin, a yellow pigment with growth-promoting properties found in milk. In 1934 pyridoxine, another component of the B2 complex was identified. Nicotinic acid and nicotinamide (niacin and niacin amide), the anti-pellagra factor, were identified in 1937. For several decades additional vitamins were discovered. Three are of particular importance for human blood: vitamin K promotes the formation of prothrombin in the liver, folic acid prevents anaemia and vitamin B12 (cyanocobalamin) is the anti-pernicious anaemia factor.
 
Each of the four fat-soluble vitamin groups, A, D, E and K, includes several related compounds with biological activity. All contain one or more units of an isoprene structure in their molecules (–C=CH–C.(CH3)=CH–). These vitamins are transported by lymph from the intestines to the blood. Bile salts are required for their efficient absorption. They may be taken up as esters of palmitic acid or combined with a protein. Vitamins A, D and K are stored chiefly in the liver; vitamin E is found in body fat. The action of the fat-soluble vitamins is connected with certain enzymes. Some carotenes also show vitamin A activity. Only alpha and beta carotenes and tryptoxanthin are important in human metabolism and beta carotene is the most active. Water-soluble vitamins include vitamin B1 (thiamin), B2 (riboflavin), B3, B6 (pyridoxine), niacin, vitamin B12, folic acid, pantothenic acid and biotin. In metabolic processes they act as coenzymes. Thus, vitamins B1, B2 and B6 become phosphates, biotin undergoes a change in structure and nicotinic, pantothenic and folic acids form esters. Water-soluble vitamins are not stored in the body to the same extent as fat-soluble ones and any excess is excreted in the urine. In addition to the true vitamins there are other substances with vitamin activity such as choline and p-amino benzoic acid, but these are fabricated in the body, as well as occurring in foods, and are not considered true vitamins.
 
Blood Chemistry
 
The difference in colour between venous and arterial blood was known in ancient times, but that the same blood changed colour as it circulated from veins to arteries through the lungs was discovered by William Harvey in the 1620s. The cellular structure of the blood was also first observed in the seventeenth century. The red blood cells attracted most attention and in the nineteenth century it was realized that they were the oxygen carriers. In 1827 Hans Fischer and his co-workers synthesized a large number of porphyrins, the chemical components of the red cells, and showed how they transport oxygen in the blood. As the only fluid circulating through all the organs, the blood was thought to transport nutrients and remove waste products, maintaining homeostasis in the body, fundamental to Bernard's theory of the internal environment. In 1862 Hoppe-Seyler observed the characteristic absorption spectrum of oxyhaemoglobin and in the same year William Stokes demonstrated the oxidation–reduction of the pigment present in the red cells. The nature of the active part of haemoglobin and the structure of the porphyrin ring was investigated by Ernst Küster in 1913. Max Perutz and John Kendrew determined the complete structure of the haemoglobin molecule using X-ray spectrographic techniques between 1937 and 1959.
 
The white blood cells, or leucocytes, include a proportion of phagocytes, cells that engulf and digest bacteria, protecting the body from disease. In addition, 20–25% of the white cells, the lymphocytes, combine with antigens and remove them from the body, so controlling infections. There are two types of lymphocytes, called B cells and T cells. The B cells produce chemical antibodies on activation by antigens and release them into the bloodstream. In the 1970s the Japanese immunologist Susumu Tonegawa showed that about 1000 pieces of genetic material in the antibody-producing part of B lymphocytes can be shuffled and recombined in different sequences, enabling up to 1 billion different types of antibodies to be formed each specific to a different antigen.
 
Serum albumin accounts for 55% of the total protein in blood plasma. Its main function is to help maintain the osmotic pressure between blood vessels and tissues. Circulating blood tends to force blood out of the blood vessels and into the tissues, but the colloidal nature of albumin and to a lesser extent of other blood proteins, the globulins, keeps the blood within the blood vessels. Albumin also contains two materials necessary for the control of clotting: antithrombin keeps the clotting enzyme thrombin from working unless needed and heparin cofactor is necessary for the anticlotting action of heparin. The fate of cholesterol, another chemical substance found in the bloodstream, was investigated by Henri V. Goldstein in 1972. He found that low-density lipoproteins, the primary cholesterol-carrying particles, are withdrawn from the bloodstream into the body's cells by receptors on the cells' surface. Although dealing with complex chemical substances and processes, studies on the blood tend to be physiological and immunological, rather than biochemical. They form part of medical research and it is in such areas that the overlap between biochemistry, medical chemistry and physiology highlights the difficulties of demarcation and the precise definition of biochemistry as an independent discipline.
 
Molecular Biology and the Nucleic Acids
 
Since the 1950s progress in biochemistry has been led by ‘molecular biology’, a subject concerned with the ultimate physiological organization of living matter at the molecular level. It is virtually impossible to distinguish between biochemistry and molecular biology, since both are concerned with intermolecular transformations within living cells. Since its inception in the late nineteenth century biochemistry has sought to develop a molecular biology in stark contrast to the macrobiology of organs and tissues. Yet modern molecular biology seeks recognition as a separate discipline concerned with the molecular basis of inheritance (genetics) and with protein synthesis in the cells. It has developed from and is related to biochemistry, yet distinct from it. In 1950 W. T. Astbury identified molecular biology with a study of the forms of biological molecules, their development through higher levels of organization and their relationship with genesis and function. The first part of this definition is conformational, the second is informational and for some years these two aspects of molecular biology were studied independently.
 
By the end of the nineteenth century biochemists had come to recognize that complex biological molecules held the key to understanding vital processes in living cells. But, as the methods of classical chemistry were inadequate to determine the structures of large molecules such as proteins, polysaccharides or nucleic acids, biochemists concentrated on the transformations of smaller molecules forming the components of these complex molecules. Molecular biologists, on the other hand, adopted physico-chemical techniques such as X-ray diffraction, developed in the 1920s at the Royal Institution in London by W. H. Bragg who built up a school of crystallography including Astbury, J. D. Bernal and Kathleen Lonsdale. Using this technique it was discovered that the structures of complex molecules could be determined, but even this was simpler than the problems of explaining their functions in terms of their structures. Astbury applied X-ray crystallographic techniques to fibres and discovered that when a natural fibre such as hair is stretched, its diffraction pattern changes due to molecular rearrangements in its structure. Bernal turned to other living materials and with Dorothy Hodgkin and Perutz obtained diffraction patterns for large crystalline protein molecules. Perutz analysed the structure of the haemoglobin molecule and Kendrew made the first three-dimensional analysis of the molecular structure of myoglobin. ATP synthase, an enzyme responsible for synthesizing ATP, the universal energy carrier in living cells, was also examined. In the 1930s, however, further progress was impossible due to the inadequacy of contemporary methods of analysis.
 
In America it was recognized that the helix was a common structural form for large molecules. As X-ray diffraction analysis was unable to cope with such a structure Linus Pauling suggested that model building would offer the only hope of solving these difficult problems. For success, however, precise inter-atomic distances and angles were required. Pauling therefore applied X-ray diffraction techniques to the analysis of amino acids and small peptides in order to determine their dimensions with minute accuracy. By the late 1940s he was able to use his results to construct models of large protein and polypeptide molecules by combining these smaller units. From this work a general theory of the diffraction of X-rays by helical structures was evolved. This would be very important for determining the structures of the nucleic acids. During the same period another group of workers settled on the study of bacteria, believing that the bacteriophage was an ideal subject for the study of heredity because the transfer of hereditary material was not confused by other biological functions such as metabolism. The so-called ‘phage group’ was indifferent, even hostile, to chemistry and perhaps due to this attitude it was only in 1944 that it was realized that the materials under study were actually nucleic acids.
 
The British and American researchers were proceeding independently of each other until James D. Watson, a post-doctoral student from the phage group, came to work in Cambridge. Watson was working on DNA (deoxy ribonucleic acid), a compound first isolated from pus cells in 1869. Its significance as genetic material was recognized in 1944 when it was observed that bacterial DNA changed the genetic material of other cells. In 1953 Francis Crick and Watson proposed the double helix structure for DNA, providing the conceptual framework for understanding DNA replication and protein synthesis. It appeared that the DNA molecule was composed of two helical phosphate-sugar chains running in opposite directions and crosslinked at regular intervals by four organic bases always appearing in pairs (thymine–adenine and cytosine–guanine). The links between the chains are formed by relatively weak hydrogen bonds and the separation of the chains leaves each one as a template for duplication using the small molecules brought to the cells by the blood.
 
In 1955 S. Ochoa in Spain discovered an enzyme, polynucleotide phosphorylase, capable of synthesizing ribonucleic acid (RNA). It was later found that this enzyme degrades RNA in the cells, but under test-tube conditions it runs its natural reaction in reverse. The enzyme has enabled understanding of processes whereby hereditary information in genes is translated through RNA intermediaries into enzymes that determine the functions and character of each cell. With discoveries such as these the ‘informational’ molecular biologists could proceed without further help from the chemical conformationists who were free to move on to other structural problems.
 
Although the way in which molecular biology apparently developed seems to fit this simple scenario, it is incomplete. The development of molecular biology includes many more aspects of biochemical research like plant viruses such as Tobacco mosaic virus. These can be readily extracted from plants and crystallized; they all contain RNA. The mechanisms of nuclear division have also been important, as has the role of RNA in protein synthesis. The contributions of these and other lines of biochemical research cannot be ignored in considering the origins of molecular biology. In 1978 the New York Academy of Arts and Science held a meeting to promote a broader view focusing attention on the history of protein research. Since then developments in molecular biology have involved such important advances as the first complete synthesis of a protein, the detailed mapping of the arrangement of atoms in certain enzymes, the elucidation of intricate mechanisms of metabolic regulation and the molecular action of hormones. These lines of investigation are closely related to medical research and prominent recent researchers in molecular biology are largely drawn from this field. They include immunologists Caesar Millstein and Georges Kohler, geneticists Christiane Nusslein-Volhard and Eric Wieshaus, and Hugh Esmor Huxley, who proposed the sliding filament theory of muscle contraction.
 

Glossary

Phlogiston
A hypothetical substance or ‘principle’ supposed to exist in all combustible bodies, was disengaged as heat, and sometimes light, during combustion, the calcination of metals and animal respiration. Substances such as oils, waxes, charcoal, inflammable air (hydrogen) and sulfur, which burn leaving very little residue, were considered rich in phlogiston. Air saturated with phlogiston would not support combustion or respiration. The theory, which dominated eighteenth-century chemistry, was superseded in about 1789 by Lavoisier's oxygen theory and by 1800 phlogiston had disappeared from most new chemical texts.
Phlogiston theory
Introduced about 1702 by Georg Ernst Stahl (1660–1734), phlogiston (from the Greek ‘phlox’ = flame) was a development of the terra pinguis, inflammable earth, of Johan Joachim Becher (1635–1682).
 

Further Reading

  • Coley NG (1973) From Animal Chemistry to Biochemistry. Amersham, UK: Hulton.
  • Florkin M and Stotz EH (1972–1979) Comprehensive Biochemistry, sect. VI, vols 30–33. Amsterdam: Elsevier.
  • Fruton JS (1990) Contrasts in Scientific Style: Research Groups in the Chemical and Biochemical Sciences. Philadelphia: American Philosophical Society.
  • Holmes FL (1974) Claude Bernard and Animal Chemistry. Cambridge MA: Harvard University Press.
  • Needham DM (1971) Machina Carnis; The Biochemistry of Muscular Contraction in its Historical Development. Cambridge: Cambridge University Press.
  • NeedhamJ (ed.) (1970) The Chemistry of Life. Cambridge: Cambridge University Press.
  • NeubergerA and DeenenLMvan (eds) (1983–1997) Comprehensive Biochemistry, vols 35–38, 40. Amsterdam: Elsevier.
  • Olby R (1994) The Path to the Double Helix: the Discovery of DNA (Reprint of 1974 edn with additions). New York: Dover.
  • Teich M and Needham DM (1992) A Documentary History of Biochemistry 1770–1940. Leicester, UK: Leicester University Press.
  • Weatherall M and Kamminga H (1992) Dynamic Science: Biochemistry in Cambridge, 1898–1949. Cambridge: Wellcome Unit for the History of Medicine.
 
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