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MOSES KUNITZ December ~ 9, ~ SS 7April 20, ~ 9 78 BY ROGER M. HERRIOTT MOSES KUNITZ iS best remembered for his isolation and crystallization of a half-dozen enzymes and precursors. This work tract two important effects. First, the variety of the enzymes he isolated and the clarity of his evidence that the enzymes were proteins convinced those who tract reservect judgment on the earlier reports of Sumner, Northrop, Cald- well, et al. Second, his reports of the crystallization of ribo- nuclease and clesoxyribonuclease, which appeared just as the functions of nucleic acids were beginning to be explored, proviclec! information on the high specificity of these en- zymes. This information made them valuable tools for other researchers in their purification or the assignment of a bio- logical function to either RNA or DNA, the two types of nucleic acid. - Moses Kunitz was born on December 19, 1887, in SIon~m, Russia, where he was educated before emigrating to the Uniter! States. In 1909, he took up residence in New York City. Entering the Cooper Union School of Chemistry in 1910, he gracluatec! with a bachelor of science degree in 1916. In the fall of that year, he enterer! the Electrical Engi- neering School of Cooper Union, where he studied until 1919 when he transferred to the Columbia University School of Mines Engineering anti Chemistry. In 1922 he marricu- 305

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306 BIOGRAPHICAL MEMOIRS later! as a graduate student in Columbia's Faculty of Pure Science, which awarded him a Ph.D. degree in biological chemistry in 1924. Kunitz derived much of his formal education in graduate science from evening classes he attendee! while working full time as a technical assistant in Jacques Loeb's general physi- ology laboratory at the Rockefeller Institute for Medical Re- search. Loeb quickly recognized Kunitz's fine work habits ancT encourages! his educational development. When Kunitz re- ceived his doctorate, Loeb secured his appointment to the staff. He also colIaboratec] with Kunitz in studies of protein- ion equilibria and related phenomena, and many of the orig- inal measurements on this subject appear in Kunitz's cloctoral thesis ant! early publications. Upon Loeb's death in 1924, John H. Northrop was ap- pointed his successor. He invited Kunitz to continue with his fundamental studies of viscosity, swelling, and the effect of certain salts on the properties of proteins. Northrop joiner! Kunitz in many of these investigations a happy, productive collaboration that was to last for more than thirty years. Northrop and Kunitz mover! to the Princeton branch of the Rockefeller Institute in 1926, and soon after the move, their interest shifted to the isolation of proteases. Northrop's choice of pepsin and Kunitz's choice of trypsin for these stucI- ies were due in part to the commercial availability of these substances. Although crystals of trypsin were obtained in 1931, the procedure was long and teclious, and the yielcl was low. In 1933 Kunitz devised a better approach. Preliminary experiments revealed that unlike the common structural tis- sue components, trypsinogen, the precursor of trypsin in beef pancreas, was soluble and stable in cold, quarter-normal, sulfuric acid. This information led him to clevelop a unique method of extracting trypsinogen and several other precur- sors ant} enzymes.

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MOSES KUNITZ 307 Variations of the fractions in Kunitz's assays soon revealed the presence of another protease precursor and enzyme. Be- cause the new protease had strong milk-clotting action (a property not held by trypsin), Kunitz termed the precursor chymotrypsinogen and the enzyme chymotrypsin. In a rela- tively short time, Kunitz crystallized both chymotrypsinogen ant! trypsinogen and, soon after, the active enzymes them- seIves. Solubility studies that Northrop anc! he had cleveloped shower! these four proteins to be homogeneous. Kunitz also found conversion of trypsinogen to trypsin to be autocata- lyticthat is, tryspin catalyzed the conversion. Trypsin also converted chymotrypsinogen to chymotrypsin, the kinetics in this case being first order. Kunitz's extreme care in all of his experiments frequently led him to discoveries that the average worker might well have missed. Two instances illustrate this point. Kunitz inves- tigatec3 a slow change in the activity of a stores! preparation of chymotrypsin that he believed to be stable. In the course of the work, he isolated two new autolysis products, still pro- teolytically active, which he named beta and gamma chymo- trypsins, designating the original enzyme alpha. Again, when his trypsinogen preparation became active in acid solution- a result contrary to his earlier studieshe discovered that his stock HC! solution was contaminates! with a mold that liber- atect a protease that hac! brought about the activation. He isolatecl the mold an(1 then the mol(1 protease. He then used the protease to convert trypsinogen to trypsin in an acid me- dium, obtaining a cleaner preparation of trypsin than was possible by any previous procedure. The presence of substances inhibitory to trypsin in the original pancreatic extracts, and in certain soybean meal frac- tions, led Kunitz to the crystallization of a polypeptide inhib- itor from the pancreas and a protein inhibitor from soybean. Isolation of the inhibitor from the pancreas answered a.ques-

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308 BIOGRAPHICAL MEMOIRS tion Kunitz had already posed: Why does trypsinogen re- main inactive in pancreatic tissue when the pH is optimal for its activations Kunitz's interests, however, were far broader than merely the isolation of enzymes or inhibitors. In each instance, he studied the interaction of the inhibitor with the enzyme and isolated the complexes, studying their stoichiometry and other properties. One of his finest papers details the study of the kinetics and thermodynamics of the reversible dena- turation of the soybean trypsin inhibitor. From 1939 to 1940, Kunitz worked to isolate ribonuclease from beef pancreas. He found it to be one of the smallest proteins and extremely stable even to boiling. This enzyme liberates only pyrimidine mononucleotides from ribonucleic acids. The isolation of desoxyribonuclease came ten years later, after Maclyn McCarty had obtained its partial purifi- cation. Very low (nanogram) levels of this enzyme destroyed the pneumococcal transforming activity of Avery, MacLeod, and McCarty's DNA preparations a finding that led many investigators to believe that DNA carried hereditary deter- minants. With the onset of World War Il. when the laboratory's attention was focused on government projects, Kunitz was asked to isolate hexokinase, thought to be highly sensitive to the action of a poison gas. He isolated the hexokinase in crys- talline form, but had to isolate three other crystalline proteins before the hexokinase crystallized. The following anecdote reveals Kunitz's remarkable fac- ulty for crystallizing proteins. Another laboratory had de- voted considerable effort to the isolation of a plant protein of great interest to the Department of Defense, but the in- vestigator had been unable to crystallize the protein. A pack- age of the material was sent to Kunitz with the request that he attempt to crystallize it. The package arrived late one

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MOSES KUNITZ 309 afternoon. Kunitz ctissolvec! some of the cIry powder in water anc! placed small aliquots in a series of test tubes to which he acl(lecl drops of ctilute HCI, increasing the number of drops in each successive tube. A precipitate soon began to appear in the micIdIe of the series, ant! Kunitz hell! a turbid tube to the light from the window, remarking, after a few moments, "It looks granular." He placer! the tubes in the refrigerator, and the next morning several tubes had crystals of what proved to be the active protein. It is unfortunate that more beginning investigators clid not get the chance to work near Kunitz in the early years. Margaret McDonald and this author were certainly enricher! by our long association with him. After his return to New York in 1952 and the conversion of the Rockefeller Institute to its present university status, Kunitz was named professor emeritus and continued to work claily in the laboratory. Many staff and students then tract an opportunity to see how the master workocI. Kunitz's papers, moclels of scientific reporting, also illus- trate his reliance on the results of broad experimentation rather than on preconceived notions. His procedures of ex- posing proteins to strong acid or high temperatures unique at that time were avoided by most investigators. There is no more appropriate testimony to the esteem in which Kunitz was held by his peers than the comments of John Northrop, which were included in a review of Kunitz's work when he was awarded the Car} Neuberg Medal in 1957: "Dr. Kunitz possesses to a rare degree the abilities of a re- search worker of the first rank in his chosen field imagi- nation, ingenuity, persistence, great technical skill, mathe- matical facility, and a thorough theoretical knowledge. It is not surprising, therefore, that he has been able to solve al- most every problem he has attempted. Some of them are of great importance. The isolation and crystallization of ribo-

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310 BIOGRAPHICAL MEMOIRS nuclease, hexokinase, and deoxyribonuclease placed the pro- tein nature of enzymes, in general, on a firm experimental foundation. In addition, the nucleases have been invaluable tools in the elucidation of the chemistry of the nucleic acids, those remarkable substances that appear to be the very 'stuff of life'." Moses Kunitz was a modest, gentle, considerate person who loved his work and his family. His association with the Rockefeller Laboratories spanned a period of fifty-seven years. He died April 20, 197S, in Philadelphia, Pennsylvania. He is survives! by a daughter, Roslyn Albert, and a son, Jacques Kunitz.

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MOSES KUNITZ 311 HONORS AND DISTINCTIONS Moses Kunitz was associated with The Rockefeller University for almost sixty years (1913-1972~. He was elected associate member in 1940, member in 1949, and professor emeritus in 1953. Kunitz was awarded the American Society of European Chemists and Pharmacists (New York City) Carl Neuberg Medal in 1957. He was elected to the National Academy of Sciences in 1967 and received an honorary degree from The Rockefeller University in 1973. He was a member of the American Association for the Advancement of Science, the Society for Experimental Biology, the American So- ciety of Biological Chemists, and the Society of General Physiolo- gists.

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312 BIOGRAPHICAL MEMOIRS SELECTED BIBLIOGRAPHY 1923 With I. Loeb. Valency rule and alleged Hofmeister series in the colloidal behavior of proteins. I. The action of acids. l. Gen. Physiol., 5:665 -91. With l. Loeb. Valency rule and alleged Hofmeister series in the colloidal behavior of proteins. II. The influence of salts. l. Gen. Physiol., 5:693-707. 1924 A cell for the measurement of cataphoresis of ultramicroscopic particles. l. Gen. Physiol., 6:413-16. With J. Loeb. The ultimate units in protein solutions and the changes which accompany the process of solution of proteins. I. Gen. Physiol., 6:479-500. Valency rule and alleged Hofmeister series in the colloidal behavior of proteins. III. The influence of salts on osmotic pressure, membrane potentials, and swelling of sodium gelatinate. J. Gen. Physiol., 6:547-64. With I. H. Northrop. The combination of salts and proteins. I. I. Gen. Physiol., 7:25 - 38. 1925 With I. H. Northrop. An improved type of microscopic electroca- taphoresis. I. Gen. Physiol., 7:729-30. 1926 With I. H. Northrop. The swelling and osmotic pressure of gelatin in salt solutions. I. Gen. Physiol., 8:317-37. With I. H. Northrop. The combination of salts and proteins. II. A method for the determination of the concentration of com- bined ions from membrane potential measurements. I. Gen. Physiol., 9:351-60. An empirical formula for the relation between viscosity of solution and volume of solute. J. Gen. Physiol., 9:715-25. With J. H. Northrop. The swelling pressure of gelatin and the mechanism of swelling in water and neutral salt solutions. J. Gen. Physiol., 10: 161-77.

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MOSES KUNITZ 1927 313 Hydration of gelatin in solution. I. Gen. Physiol., 10:811. With I. H. Northrop. The swelling of isoelectric gelatin in water. II. Kinetics. I. Gen. Physiol., 10:905 - 26. 1928 With J. H. Northrop. Preparation of electrolyte-free gelatin. J. Gen. Physiol., 11:477-79. With }. H. Northrop. Combination of salts and proteins. III. The combination of Curly, MgCl2, CaCl2, AlCl3, LaCl3, KC1, AgNO3, and Na2SO4 with gelatin. I. Gen. Physiol., 11:481-93. With H. S. Simms. Dialysis with stirring. I. Gen. Physiol., 11:641- 44. Syneresis and swelling of gelatin. I. Gen. Physiol., 12: 289-312. 1929 With I. H. Northrop. Fractionation of gelatin. I. Gen. Physiol., 12:379-90. With I. H. Northrop. The swelling of gelatin and the volume of surrounding solution. I. Gen. Physiol., 12:537-42. 1930 Elasticity, double refraction, and swelling of isoelectric gelatin. I Gen. Physiol., 13:565-606. With I. H. Northrop. Solubility curves of mixtures and solid solu- tions. I. Gen. Physiol., 13:781-91. 1931 With i. H. Northrop. Swelling and hydration of gelatin. I. Phys. Chem., 35: 162-84. With I. H. Northrop. Isolation of protein crystals possessing tryptic activity. Science, 73:262-63. 1932 With J. H. Northrop. Crystalline trypsin. I. Isolation and tests of purity. J. Gen. Physiol., 16:267-94. \Vith J. H. Northrop. Crystalline trypsin. II. General properties. J. Gen. Physiol., 16:295 -311. With J. H. Northrop. Crystalline trypsin. III. Experimental pro-

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314 BIOGRAPHICAL MEMOIRS cedure and methods of measuring activity. I. Gen. Physiol., 16:313-21. 1933 With J. H. Northrop. Isolation and properties of crystalline tryp- sin. Ergeb. Enzymforsch., 2: 104 - 17. With I. H. Northrop. Isolation of a crystalline protein from pan- creas and its conversion into a new crystalline proteolytic en- zyme by trypsin. Science, 78:558-59. With I. H. Northrop. Uber die Wirkung des kristallisierten Trypsin auf Penta-glycyl-glycin, trim alanyl-~ alanin und Tetra-dl-alanyl dl-alanin. Fermentforschung, 13 :597-600. 1934 With M. L. Anson and l. H. Northrop. Molecular weight, molecu- lar volume, and hydration of proteins in solution. I. Gen. Phys- iol., 17:365-73. With l. H. Northrop. Inactivation of crystalline trypsin. l. Gen. Physiol., 17:591-615. With l. H. Northrop. Autocatalytic activation of trypsinogen in the presence of concentrated ammonium or magnesium sulfate. Science, 80:190. With I. H. Northrop. The isolation of crystalline trypsinogen and its conversion into crystalline trypsin. Science, 80:505-6. 1935 With J. H. Northrop. Crystalline chymotrypsin and chymotrypsi- nogen. I. Isolation, crystallization, and general properties of a new proteolytic enzyme and its precursor. I. Gen. Physiol., 18:433-58. A method for determining the rennet activity of chymo-trypsin. }. Gen. Physiol., 18:459-66. With H. Hotter and I. H. Northrop. Spaltung von Clupean durch verschiedene Trypsinpraparate. Z. Physiol. Chem., 235: 19-23. 1936 With J. H. Northrop. Pepsin, trypsin, chymo-trypsin. In: Handbuch der biologischen Arbeitsmethoden, ed. E. Abderhalden, pp. 2213- 24. Berlin: Urban. With I. H. Northrop. Die Isolierung von kristallisiertem Trypsi- nogen und dessen Umwandlung in kristallisiertes Trypsin. In:

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MOSES KUNITZ 315 Handbuch der biologischen Arbeitsmethoden, ed. E. Abderhalden, pp. 2461 - 64. Berlin: Urban. With I. H. Northrop. Isolation from beef pancreas of crystalline trypsinogen, trypsin, a trypsin inhibitor, and an inhibitor- trypsin compound. J. Gen. Physiol., 19:991 - 1007. 1938 With I. H. Northrop. Solubility of proteins as a test of purity; the solubility of chymo-trypsin and chymo-trypsinogen. C.R. Trav. Lab. Carlsberg Ser. Chim., 22:288-94. Formation of trypsin from trypsinogen by an enzyme produced by a mold of the genus Penicillium. ]. Gen. Physiol., 21:601-20. Formation of new crystalline enzymes from chymotrypsin. Isola- tion of beta and gamma chymo-trypsin. J. Gen. Physiol., 22:207-37. With J. H. Northrop. Solubility curves of pure proteins and of mix- tures and solid solutions of proteins. Cold Spring Harbor Symp. Quant. Biol., 6:325-30. 1939 Effect of the formation of inert protein on the kinetics of the au- tocatalytic formation of trypsin from trypsinogen. I. Gen. Phys- iol., 22:293-310. Formation of trypsin from crystalline trypsinogen by means of en- terokinase. J. Gen. Physiol., 22:429-46. Purification and concentration of enterokinase. I. Gen. Physiol., 22:447-50. Isolation from beef pancreas of a crystalline protein possessing ribonuclease activity. Science, 90: 112-13. Kinetics of the formation of chymotrypsin from crystalline chy- motrypsinogen and of trypsin from crystalline trypsinogen. En- zymologia, 7:1-20. 1940 Crystalline ribonuclease. l. Gen. Physiol., 24: 15-32. 1941 With M. R. McDonald. The effect of calcium and other ions on the autocatalytic formation of trypsin from trypsinogen. ~ Gen. Physiol., 25:53-73. Kristallisierte Ribonuklease. In: Die Methoden der Fermentforschung,

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316 BIOGRAPHICAL MEMOIRS vol. 2, ed. E. Bamann and K. Myrback, pp. 1940-41. Leipzig: Thieme. 1945 Crystallization of a trypsin inhibitor from soybean. Science, 101:668. 1946 With M. R. McDonald. Isolation of crystalline hexokinase and other proteins from yeast. i. Gen. Physiol., 29: 143-47. Crystalline soybean trypsin inhibitor. J. Gen. Physiol., 29: 149-54. With M. R. McDonald. An improved method for the crystallization of trypsin. J. Gen. Physiol., 29: 155-56. A spectrophotometric method for the measurement of ribonu- clease activity. I. Biol. Chem., 164:563-68. With M. R. McDonald. Crystalline hexokinase (heterophospha- tase). Method of isolation and properties. i. Gen. Physiol., 29:393-412. 1947 Crystalline soybean trypsin inhibitor. II. General properties. I. Gen. Physiol., 30:291-310. Isolation of a crystalline protein compound of trypsin and of soy- bean trypsin inhibitor. J. Gen. Physiol., 30:311-20. 1948 With J. H. Northrop and R. M. Herriott. Crystalline Enzymes, 2d ed. New York: Columbia University Press. Isolation of crystalline desoxyribonuclease from beef pancreas. Science, 108: 19. With M. R. McDonald. Isolation of crystalline ricin. J. Gen. Phys- iol., 32:25-37. The kinetics and thermodynamics of reversible denaturation of crystalline soybean trypsin inhibitor. I. Gen. Physiol., 32:241- 63. Crystallization of salt-free chymotrypsinogen and chymo-trypsin from solution in dilute ethyl alcohol. J. Gen. Physiol., 32:265- 69.

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MOSES KUNITZ 1950 317 Crystalline desoxyribonuclease. I. Isolation and general proper- ties; spectrophotometric method for the measurement of de- soxyribonuclease activity. I. Gen. Physiol., 33:349-62. Crystalline desoxyribonuclease. II. Digestion of thymus nucleic acid (desoxyribonucleic acid); the kinetics of the reaction. I. Gen. Physiol., 33:363 -77. 1951 Isolation of crystalline pyrophosphatase isolated from baker's yeast. I. Am. Chem. Soc., 73:1387. 1952 Crystalline inorganic pyrophosphatase isolated from baker's yeast. I. Gen. Physiol., 35:423-49. 1953 With M. R. McDonald. Ribonuclease. In: Biochemical Preparations, vol. 3, pp. 9-19. New York: John Wiley & Sons. 1957 With I. H. Northrop. The proportion of mutants in bacterial cul- tures. I. Gen. Physiol., 41: 119-29. 1960 Chicken intestinal alkaline phosphatase. I. The kinetics and ther- modynamics of reversible inactivation. II. Reactivation by zinc ions. J. Gen. Physiol., 43: 1149 - 69. 1961 An improved method for isolation of crystalline pyrophosphatase from baker's yeast. Arch. Biochem. Biophys., 92:270-72. With P. W. Robbins. Inorganic pyrophosphatases. In: The Enzymes, ed. P. D. Boyer, H. L. Lardy, and K. Myrback. pp. 169-78. New York: Academic Press. 1962 Hydrolysis of adenosine triphosphate by crystalline yeast pyro- phosphatase. J. Gen. Physiol., 45:31-46.