Frederick Sanger is surely one of the most outstanding biochemists of modern times. His methods for determining the exact sequence of amino acids in proteins and of nucleotides in deoxyribonucleic acid (DNA ) have won him numerous awards, including two Nobel Prizes in chemistry.
from which he received a B.A. in biochemistry in 1939. He remained at Cambridge as a conscientious objector during World War II and earned his Ph.D. in biochemistry for work on amino acid metabolism and the nitrogen of potatoes with Albert Neuberger in 1943. Later that year he joined Antony C. Chibnall's group at Cambridge and began research on proteins and, in particular, insulin.
The basic principles of protein chemistry were firmly established when Sanger started his work on insulin. It was known that proteins were composed of amino acids linked through amide bonds to form long polypeptide chains. Although the relative number of each of the twenty amino acids could be obtained for a given protein, the particular order, or sequence, of those amino acids in the protein had never been determined. Sanger saw sequence as the key to understanding living matter and set out to determine the exact sequence of amino acids in insulin.
Sanger first needed to characterize the free amino groups in insulin. For this he developed a reagent , dinitrofluorobenzene (FDNB), that reacted with amino groups present in proteins to form an acid-stable dinitrophrenyl (DNP) derivative. The DNP protein was treated with acid to break the polypeptide backbone, and the free DNP amino acid derivatives were isolated and compared to standards prepared from known amino acids. In this way, Sanger determined that insulin was made up of two peptide chains: one (chain A) with an amino-terminal glycine residue and another (chain B) with an amino-terminal phenylalanine. Subsequent work revealed that chain A was composed of twenty amino acids and chain B thirty-one.
The individual chains were then broken down into smaller components: Acid was used to cleave the polypeptide backbone, performic acid was used to break the cysteine disulfide bonds , and proteolytic enzymes were used to hydrolyze the polypeptide at specific sites on the chain. The reaction products were separated from each other and their sequence determined.
Sanger was able to deduce the complete sequence of insulin after twelve years of painstaking research and molecular puzzle solving. The Nobel committee was quick to recognize Sanger's accomplishment and awarded him the 1958 Nobel Prize in chemistry for "his work on the structure of proteins, especially that of insulin."
In 1962 Sanger moved to the Medical Research Council Laboratory of Molecular Biology in Cambridge, where he became interested in nucleic acid sequencing. He and his colleagues developed a cleave-and-sequence method for small ribonucleic acid (RNA) molecules, but they soon realized that a different method was needed to sequence the much larger DNA molecules. For DNA sequencing, he chose to investigate copying procedures.
Sanger eventually settled on a procedure that uses DNA polymerase to copy short fragments (200 nucleotides) of single-stranded DNA obtained from enzyme-catalyzed cleavage of the parent DNA. In addition to the usual radiolabeled deoxyribonucleotide triphosphates, the DNA synthesis cocktail contains a 2′, 3′-dideoxy analog that is incorporated into the growing DNA strand and blocks further DNA synthesis. After repeating the reaction using each of the remaining dideoxy nucleotides, the various chain-terminated fragments are separated by gel electrophoresis and the DNA sequence read directly from the gel. Sanger used this method to sequence a DNA containing more than 5,000 nucleotides and shared the 1980 Nobel Prize in chemistry for "contributions concerning the determination of base sequences in nucleic acids."
Sanger retired in 1983 after forty years of service at the Medical Research Council and after helping to usher in a new era in biology and medicine.
see also DNA Replication; Insulin; Proteins.
Thomas M. Zydowsky
James, Laylin K., ed. (1993). Nobel Laureates in Chemistry 1901–1992. Washington, DC: American Chemical Society; Chemical Heritage Foundation.
Sanger, Frederick (1958). "The Chemistry of Insulin." Nobel e-Museum. Available from <http://www.nobel.se/chemistry/laureates>.
Sanger, Frederick (1980). "Determination of Nucleotide Sequences in DNA." Nobel e-Museum. Available from <http://www.nobel.se/chemistry/laureates>.
The English biochemist Frederick Sanger (born 1918) was awarded the Nobel Prize in Chemistry for his discovery of the chemical structure of insulin.
Frederick Sanger, son of Frederick Sanger, a medical practitioner, was born at Rendcombe, Gloucester-shire, on Aug. 13, 1918. Entering St. John's College, Cambridge, in 1936, he graduated with the degree of bachelor of arts (in natural sciences) in 1939. In 1943 he received his doctorate of philosophy (in chemistry) with a thesis on lysine. He held a Beit Memorial Fellowship from 1944 to 1951 and then joined the staff of the Medical Research Council. He later became director of the Division of Protein Chemistry in the Council's Laboratory for Molecular Biology at Cambridge.
Sanger worked entirely on the chemical structure of the proteins, especially insulin. About 1900 Emil Fisher had succeeded in breaking down proteins into polypeptides, consisting of their ultimate constituents, amino acids. About 25 different amino acids occur in nature, and of these 20 are found in most mammalian proteins.
By 1943 it was known that proteins consisted of long chains of amino acid residues bound together by peptide linkages. A. C. Chibnall and others knew the 51 amino acid residues that composed insulin; they also knew that phenylalanine was at the end of one of the chains. The insulin molecule appeared to consist of a large number of polypeptide chains, and it was held that what was important biologically was the sequence in which the amino acids followed each other in the chains. This sequence was unknown for any protein.
Sanger introduced the reagent fluorodinitrobenzene (FDNB), which reacted with the free amino acid at the end of a chain to form a dinitrophenyl derivative (DNP) combined with that amino acid. The DNP acids were bright yellow. If the chains were then split by hydrolysis, the colored terminal acid of each link could be identified by chromatographic and electrophoretic methods. Sanger at first thought that the insulin molecule contained four long chains; but he later concluded that it consisted of only two chains containing 21 and 30 amino acids respectively. He then split the bridges joining the chains by oxidation with performic acid and dealt with each chain individually. The chain was separated into successively shorter links, and in each link the terminal amino acid was identified. He was able to determine the exact sequence of amino acids in each chain.
Sanger then determined that the two chains were linked by two disulfide bridges of cystine residues, with a third bridge linking two parts of the short chain. The determination of the exact positions of these bridges enabled Sanger, after over 12 years of research, to give a diagram for the structure of insulin. For this work he was awarded the Nobel Prize in Chemistry in 1958.
In 1951 Sanger was awarded the Corday-Morgan Medal of the Chemical Society. In 1954 he was elected a Fellow of the Royal Society and a Fellow of King's College, Cambridge; and in 1958 he was elected a Foreign Honorary Member of the American Academy of Arts and Sciences.
In 1980 Sanger shared the Nobel Prize for Chemistry with two other scientists for work determining the sequences of nucleic acids in DNA molecules. Their combined work has been lauded for its application to the research of congenital defects and hereditary diseases. It also proved vitally important in producing the artificial genes that go into the manufacture of insulin and interferon, two substances which are used to treat a variety of diseases. Sanger retired from research in 1983.
There was a biography of Sanger in Nobel Lectures, Chemistry, 1942-1962 (1964). This work also included his Nobel Lecture, which gave an admirable summary of his work. For the chemical background see P. Karrer, Organic Chemistry (4th ed. 1950). See also the article "Sequences, Sequences, and Sequences" in Annual Review of Biochemistry (1988, pages 1-28), and Nobel Prize Winners (H. W. Wilson, ed. 1987, pages 921-924). □