Moore, Stanford

Stanford Moore was born in Chicago , Illinois when his father, John Howard Moore, was a law student at the University of Chicago (JD 1917). Mom (nee Ruth Fowler) graduated from Stanford University . His parents met in Standord and got married in 1907. They say that it was in memory of the meeting place that the parents gave their son such a name. Stanford began his studies at the age of 4 at a comprehensive school in Winnetka , Illinois . Soon the family moved to Nashville, Tennessee , where his father was offered the position of professor of law at Vanderbilt University, where he worked until his retirement in 1949. John Howard Moore died in 1966 at the age of 85. In Nashville, Moore became a student at the Peabody School of George Peabody College of Education. He was an excellent student during all 7 years of schooling. From the very beginning of Stanford's studies, English and science became interested in him, but later he was lucky to meet a teacher, R.O. Beauchamp, who aroused his interest in chemistry. Entering Vanderbilt University in 1931, Stanford Moore hesitated between aircraft manufacturing and chemistry. But Arthur William Ingersoll, who developed his interest in organic chemistry and the molecular structure of substances, had a great influence on Moore's future. As a result, Stanford chose chemistry as his main subject, graduated with honors from Vanderbilt University in 1935 with a bachelor of arts and received the Founder Medal as the most outstanding student. In the fall of 1935, Moore was awarded a scholarship from the Wisconsin Alumney Research Foundation, which made it possible to continue his studies at the University of Wisconsin. Stanford conducted research under the supervision of Professor Karl Paul Link, who had recently worked in Europe with Fritz Pregl on microanalytical methods for establishing the atomic structure of organic compounds. In 1938, Moore received his doctorate for a thesis on the characterization of carbohydrates and benzimidazole derivatives. It was proved in it that the products of this reaction, several benzimidazoles, can be easily isolated in the form of stable crystalline substances that will allow the identification of different monosaccharides .

With the completion of a doctoral dissertation by Stanford Moore in 1939, it became clear that his future would be connected with biochemistry . Moore joined the Bergmann scientific group, where he was involved in work on one of the main tasks of the laboratory - structural chemistry of proteins . Of particular interest was the development of methods for the gravimetric assessment of the amino acid composition of proteins using selective precipitators. This approach was given a new impetus two years before, when William Stein began working in the laboratory and showed that aromatic sulfonic acids have properties suitable for this purpose. Stein and Moore focused their initial efforts on two sulfonic acid reagents - 5-nitronaphthalene-2-sulfonic acid for glycine and 2-bromtoluene-5-sulfonic acid for leucine - and showed that good results can be obtained with hydrolysis products of egg albumin and fibroin silks. ^[1] Work was well underway, but when the country was at war in late 1941, research was stopped. With the outbreak of war , a special study was carried out in the laboratory of Bergmann for the Office of Scientific Research and Development (UNIR). Their task was to study the physiological effect of booster war gas ( mustard gas , mustard nitrogen ) at the molecular level, with the hope of developing drugs that could be used to overcome the effects of these compounds on the human body. The rationale for the work was that effective protective measures to prevent the effects of these toxic compounds, as well as the ability to retaliate by the United States and its allies, would prevent the use of chemical poisons. While Stein worked with Bergmann and his colleagues conducted research in New York, Moore enlisted in 1942 for administrative work at the National Defense Research Committee at the Office of Scientific Research and Development (UNIIR), directing the work of universities and industry to study the biological effects of chemical warfare agents. His base was in Washington, but he traveled freely to Dumbarton Oaks , where the office of the National Defense Research Committee was located. Later (in 1944) Moore was appointed to the staff of the Department for the Coordination of New Developments of the Chemical Service, which was managed by William A. Noyez Jr. The results of the service were published in a book that saw the light after the war, Stan also contributed to it by writing an article (in collaboration with V. R. Kerner) ^[2] on the psychological mechanisms of exposure to chemicals. When the war ended, Moore served in the Hawaiian Islands Military Operations Research Unit.

After the war ended, Herbert Gasser , director of the Rockefeller Institute , offered William Stein and Stan Moore a place in Bergmann's former department, making it possible to continue work on the amino acid analysis that they had begun before the war. Scientists resumed working together in 1945, starting with the study of distribution chromatography as a method for determining the sequence of amino acids in proteins.

Using column chromatography

Moore and Stein chose the column chromatography method as a “starting point” and they needed a suitable micromethod for studying amino acids in a column solvent. To this end, William and Stan studied the reaction of ninhydrin ^[3] , a color reagent known since its discovery in 1911 and interacting with all amino acids. They found that a recoverable precipitate of the product can be obtained when the reaction is carried out in the presence of a reducing agent, initially it was divalent tin chloride. To track changes in the separation performed in the starch column , the solvent was contained in small fractions of the same volume; they were treated with ninhydrin under reducing conditions, and the resulting colored substances were measured by spectrophotometry . The concentration of colored substances in each fraction was indicated opposite the fraction number to obtain the so-called filtrate concentrate curves. The space under each peak of such curves showed the number of amino acids in the sample.

Fraction Collector

Initially, the fractions were collected manually, but a tool was quickly developed and constructed in which each drop of filtrate from the column changed the angle of refraction of the light beam illuminating the photocell, thereby acting on the recorder. Drops were collected in spectrophotometric tubes. When a certain number of drops was collected, the turntable automatically substituted a new tube. Although this tool was not the first fraction collector, it became the prototype of commercial tools that soon appeared in laboratories around the world. Thanks to these changes, it became possible to improve the chromatographic processes themselves. The original faction collector that they developed is still in excellent working condition at the Caspary Hall.

In the methods described in 1949, three approaches were required to determine all amino acids in a protein hydrolyzate. Wilman and Stan described the use of the method for determining the composition of beta-lactoglobulin and serum albumin. ^[4] For the experiment in three approaches, less than 5 mg of protein was required, and this, taking into account the standard error of less than 5 percent, is a significant achievement of that time. Understanding what a huge role this methodology can play in biochemistry, Wilman and Stan spent a lot of time in detail describing all the steps necessary for the successful application of the methods developed by them in other laboratories. Although starch columns have become a real sensation in protein chemistry, they have some limitations. First of all, this is the slow flow rate of the substance in the columns (one full analysis of the protein hydrolyzate required two weeks). Moreover, a new column had to be prepared for each research approach, and the cleavage process was very dependent on the presence of salts in the sample.

Using ion exchange chromatography

Due to the existing drawbacks of column chromatography methods, Wilman and Stan decided to turn to ion exchange chromatography ^[5] , which used polystyrene resin, as an alternative. Scientists quickly managed to efficiently separate all the amino acids in the protein hydrolyzate in just one approach thanks to elution with sodium citrate and acetate buffers with increasing pH and concentrations at different temperatures, but it was also necessary to standardize the appearance of the columns. Finally, all difficulties were overcome, and reproducible resins appeared. The successful development of the ion-exchange methodology not only significantly reduced the time spent on analysis, but also carried out a reliable analysis of amino acids contained in physiological fluids: urine ^[6] , plasma, and protein-free tissue extracts. The methods used have yielded significant results in the discovery and evaluation of new components of these fluids. At the same time, the potential of ion exchange chromatography for the separation of peptides and proteins developed. It was soon discovered that certain stable proteins - bovine pancreatic ribonuclease and chymotrypsinogen, as well as chicken egg lysozyme - are effectively chromatographed on IRC-50, a resin based on polymethacrylic acids . Elution of these proteins from the exchanger took place with a change in pH and ionic power, in full accordance with the assumptions made. Later, successful fractionation of the histone from the thymus gland of the calf was carried out.

For analysis, Moore and Stein selected a small enzyme, ribonuclease ^[7] , which they had previously studied, discussing whether knowledge of its structure would allow understanding its enzymatic activity. This work was carried out in parallel with Christian Anfinsen and his colleagues, but the approaches of the two laboratories were different and they acted in the scientific field more likely as allies than rivals. A study of the structure of ribonuclease began with a sample of an oxidized protein that was selectively hydrolyzed with trypsin, a protein-cleaving enzyme. The resulting peptide compound was separated by ion exchange column chromatography in much the same way that amino acids were cleaved before. The structure of these peptides showed that the entire amino acid sequence of ribonuclease (124 amino acid residues) was established. To determine the nature of these peptides, the oxidized enzyme was then hydrolyzed by chymotrypsin, a proteolytic enzyme of a different form from trypsin, to produce a second batch of peptides, which were also separated using sulfonated polystyrene . Due to the well-known selective qualities of trypsin and chymotrypsin, widely studied years earlier by Bergman and his colleagues, the order of arrangement of trypsin peptides in the polypeptide chain was established. Confirmation was obtained from another batch of peptides isolated from a pepsin hydrolyzate.

Automatic Amino Acid Analysis

When the work continued, it became obvious that the development of research was hindered by the limited level at which amino acid analysis was performed. With the methods that were then in use, the experiment alone required almost three days and several hundred spectrophotometric readings. So, in 1956, work began on the creation of automatic amino acid analysis. It began only after a comprehensive improvement of the tools used, so the method itself was published in 1958. With the resins that were then available, the analysis time was reduced to 24 hours, and the allowable sensitivity reached 0.5 micromoles. Subsequent development led to a reduction in analysis time by an average of an hour and an increase in sensitivity by two orders of magnitude. The importance of the knowledge of protein chemistry discovered through Moore and Stein cannot be overestimated. The original automatic amino acid analyzer ^[8] , described in 1958, and now it is in working condition, it still stands in the same laboratory at Rockefeller University in which it was assembled.

The complete covalent structure of ribonuclease was published in 1963 - the first such study of the enzyme. Then it was decided to investigate the inhibition of ribonuclease activity by iodoacetate. In a series of studies in which a change in reaction at different pH values ​​was accompanied by amino acid analysis, it was proved that the suppression of activity at pH 5 is the result of carboxymethylation either on nitrogen-1 histidine-119, or on nitrogen-3 histidine-12, but not both sides of the same ribonuclease molecule. Inhibition of activity at lower pH was detected due to reactions with methionine ; at a higher pH, when reacting with lysine-41. In this case, it could be assumed that histidine-12 and −119 are close to each other on the active side of ribonuclease. This assumption, which turned out to be the key in subsequent studies of ribonuclease, was further confirmed in other laboratories using x-ray analysis. Due to this, it became possible to interpret kinetic studies and work on nuclear magnetic resonance , which led to a detailed explanation of the mechanism of action of the enzyme. The work on ribonuclease was universally recognized as the 1972 Nobel Prize in Chemistry for Moore, Stein, and Anfinsen. After that, the Mura-Stein laboratory expanded, and other research works began to be carried out in it: determination of the amino acid sequence of pancreatic deoxyribonuclease, study of the reaction of cyanate ions when interacting with proteins; pepsin structural studies; mechanism of action and structure of streptococcal proteinase; studies of the sequence and active side of T1 ribonuclease; the selection of 2 ', 3'-cyclic nucleotide, 3-phosphohydrolase and its inhibitor; studies of ribonuclease inhibitors, as well as many studies of modifications of pancreatic ribonuclease. Moore and Stein's collaboration continued at Rockefeller University, even after Wilman Stein contracted crushing paralysis in 1969. With the exception of the war years (1942-1945), Moore was absent from the Rockefeller Institute for only one year - 1950. He spent six months in Brussels , Belgium , opening a laboratory devoted to amino acid analysis, and the second six months in England , in Cambridge , sharing a laboratory with Frederick Senger and working on studying the sequence of insulin amino acids. Stan felt that this year, spent in Europe, is important for his development as a scientist and his future work in the international scientific field.

Moore acted in the community of biochemists as an editor, and as an officer of the American Society of Biochemists, and as the head of the Organizing Committee of the International Congress of Biochemistry, held in New York in 1964. The congress was an outstanding event thanks to the organization of scientific presentations and the hospitality of Moore. During the Congress, Stan invited 8-10 guests daily for breakfast and lunch: so scientists could meet with colleagues in a relaxed atmosphere. He continued this practice for the next 15 years at international congresses and annual meetings of the American Society of Biochemists. Only his shaky health interrupted this tradition. Stanford Moore devoted his whole life exclusively to science. He was never married, avoided everything that did not concern science and scientists.

In the last two years of his life, when his health deteriorated, Moore lived with the awareness of his illness - amyotrophic lateral sclerosis . He passed away in his apartment on August 23, 1982, not far from his favorite laboratory at Rockefeller University, where he spent so many successful and fruitful years. It should be noted that no method or tool developed by Moore and Stein has been patented . They did not think about personal gain. Moreover, Stan Moore was little interested in his own property, his small office and bachelor apartment were furnished with minimal amenities. Stan’s affection for Rockefeller University and his devotion to biochemistry were reflected in his testament, in which he announced that his property “should be used as a donation to the salaries and scientific expenses of biochemistry researchers”. As Stan wrote in a letter to the President of the University, Joshua Lederberg, which was delivered after his death: “I want (despite the modesty of my abilities) to help young students as they once helped me.”

• Professor of Rockefeller University (1952-82)
• President of the American Biochemical Society (1966-67)
• Honorary Member of the Belgian Biochemical Society
• Chairman, Biochemistry Section, National Academy of Sciences (1969-72)
• Member of the American Academy of Arts and Sciences (1960-82)
• Doctor Emeritus, Faculty of Medicine, University of Brussels, 1954
• Honorary Doctor of the University of Paris, 1964
• University of Wisconsin Ph.D., 1974

Rewards

(with William Stein)

• Chromatography and Electrophoresis Award from the American Chemical Society (1972)
• Richard Medal, American Chemical Society (1972)
• Linderstrom-Lang Medal, Copenhagen (1972)
• Nobel Prize in Chemistry (1972).

1. ↑ Moore S, Stein.WH, Bergmann M. The isolation of I-serine from silk fibroin. // J. Biol. Chem .. - 1941. - Vol. 139. - P. 481-482.
2. ↑ Moore S, Kirner WR The physiological mechanism of action of chemical warfare agents. // Chemistry (Science in World War II). - 1948. - P. 288-360.
3. ↑ Moore S, Stein.WH Photometric ninhydrin method for use in the chromatography of amino acids. // J. Biol. Chem .. - 1948. - Vol. 176.- P. 367-388.
4. ↑ Moore S, Stein. W.H. Amino acid composition of β-lactoglobulin and bovine serum albumin. // J. Biol. Chem .. - 1949. - Vol. 178.- P. 79-91.
5. ↑ Moore S., Hirs CHW, Stein WH Isolation of amino acids by chromatography on ion exchange columns; use of volatile buffers. // J. Biol. Chem .. - 1952. - Vol. 195. - P. 669-683.
6. ↑ Moore S., Tallan HH, Stein WH 3-Methylhistidine, a new amino acid from human urine. // J. Biol. Chem .. - 1954. - Vol. 206. - P. 825-834.
7. ↑ Moore S., Hirs CHW, Stein WH The amino acid composition of ribonuclease. // J. Biol. Chem .. - 1954. - Vol. 211. - P. 941-950.
8. ↑ Moore S., Spackman DH, Stein WH Automatic recording apparatus for use in the chromatography of amino acids. // Anal.Chem .. - 1958. - Vol. 30. - P. 1190-1206.

• Stanford Moore - Biographical Memoirs of the National Academy of Sciences
• Stanford Moore, William H. Stein Chromatography Annual Review of Biochemistry, Vol. 21: 521-546
• Stanford Moore on William Stein
• Stanford Moore: Some personal recollections of his life and times
• Marshall, Garland R; Feng Jiawen A, Kuster Daniel J (2008). "Back to the future: Ribonuclease A." Biopolymers 90 (3): 259-77. doi: 10.1002 / bip.20845. PMID 17868092 .
• Hirs, CH (January 1984). "Stanford Moore. Some personal recollections of his life and times. " Anal. Biochem. 136 (1): 3-6. doi: 10.1016 / 0003-2697 (84) 90301-4. PMID 6370037 .
• Bernhard Kupfer: Lexikon der Nobelpreisträger. Patmos Verlag, Düsseldorf 2001, ISBN 3-491-72451-1
• Brockhaus Nobelpreise - Chronik herausragender Leistungen. Brockhaus, Mannheim 2004, ISBN 3-7653-0492-1

• Stanford Moore and William Howard Stein
• Nobel Prize in Chemistry, 1972 (English)