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Classics:
The Purification of Diphtheria Toxin by
Alwin M. Pappenheimer, Jr.
Robert D. Simoni, Robert L. Hill and Martha
Vaughan
J. Biol. Chem. 2004, 279:e4.
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http://www.jbc.org/content/279/4/e4.full.html#ref-list-1
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THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 4, Issue of January 23, p. e4, 2004
Printed in U.S.A.
Classics
A PAPER IN A SERIES REPRINTED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005
JBC Centennial
1905–2005
100 Years of Biochemistry and Molecular Biology
The Purification of Diphtheria Toxin by Alwin M.
Pappenheimer, Jr.
Diphtheria Toxin. I. Isolation and Characterization of a Toxic Protein from
Cornynebacterium Diphtheriæ Filtrates
(Pappenheimer, A. M., Jr. (1937) J. Biol. Chem. 120, 543–553)
1
All biographical material was taken from this source.
This paper is available on line at http://www.jbc.org
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Alwin M. Pappenheimer Jr. (1908 –1995) was born in Cedarhurst, New York. His father,
Alwin M. Pappenheimer, was a distinguished pathologist at the College of Physicians and
Surgeons at Columbia University. Among his father’s many contributions was his discovery of
the link between light and bone deposition, which was published in a previous JBC Classic (1).
Pappenheimer grew up in an intellectual household and was interested in science from an
early age. He entered Harvard in 1925 at the age of 17; he was among the first students to
enroll in the new biochemical sciences tutorial. Appropriately, he later returned to Harvard
and succeeded John T. Edsall, author of a previous JBC Classic (2), as Chairman of the Board
of Tutors of the Biochemical Sciences Program. After choosing graduate school rather than
medical school, Pappenheimer entered the graduate program in organic chemistry at Harvard
with James B. Conant as his advisor even though he was determined already at the time to
have a career in biological research. When he completed his Ph.D. work in 1932, in the midst
of the Great Depression, although jobs were difficult to find he was able to spend 1 year as a
postdoctoral fellow with Hans Zinsser at Harvard studying pneumococcal polysaccharides.
This was followed by a 2-year National Research Council Fellowship with Sir Henry Dale at
the National Institute of Medical Research in London working on the isolation of a bacterial
growth factor called “sporogenes vitamin.” This experience sparked his future interests in
isolation of a bacterial toxin.
In 1935, Pappenheimer returned to Cambridge, Massachusetts without a job but with the
plan in mind “to isolate a pure potent bacterial toxin and to find out what makes it so toxic”
(3).1 He discussed his ideas with J. Howard Mueller in the Department of Bacteriology at
Harvard, another author of a previous JBC Classic (4). Mueller helped him to obtain a
Bradford Fellowship at Harvard. He was provided space and some technical support at the
Jamaica Plains antitoxin and vaccine laboratory where the work reported in this JBC Classic
was done.
Attempts to purify diphtheria toxin had been frustrated because the bacterial culture
medium from which the toxin was to be isolated contained a complex mixture of protein, and
the purification of toxin free of contaminating culture components had been proven impossible.
Pappenheimer set out to devise a better defined culture medium without high molecular
weight ingredients to interfere with toxin purification. He was successful in optimizing growth
conditions and succeeded in the complete purification of the toxin, as reported in this JBC
Classic. The modification of the growth medium had ensured that it contained no ammonium
sulfate-precipitable material permitting separation and purification of the toxin by differential
ammonium sulfate fractionation. This was the first bacterial toxin purified in crystalline form
and brought Pappenheimer great international recognition.
In 1939, Pappenheimer moved to the University of Pennsylvania as an Assistant Professor
of Bacteriology but after only 2 years was recruited to the Department of Bacteriology at New
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Classics
York University (NYU) by the new chairman Colin E. MacLeod. MacLeod and Pappenheimer
built a new Department of Microbiology while the latter continued his work on bacterial
growth and toxin production. His research was interrupted by his service as an Army captain
in World War II but resumed when he returned to NYU in 1945. After MacLeod left NYU for
the University of Pennsylvania, Pappenheimer became chairman of the Department of Microbiology, but John T. Edsall soon recruited him to return to Harvard as Professor of Biology and
Chairman of the Board of Tutors in Biochemical Sciences, the program in which he had been
enrolled as an undergraduate.
After achieving his first goal with the purification of diphtheria toxin, Pappenheimer, his
students, and others worked on discovering the mechanism of its toxicity. With R. John Collier,
he showed that diphtheria toxin inhibited protein synthesis in HeLa cells and in HeLa cell
extracts (5). Then Collier in 1966 –1967 showed that elongation factor 2, EF-2, was inactivated
by diphtheria toxin in the presence of NAD and in 1968 –1969 D. Michael Gill described the
ADP-ribosylation reaction catalyzed by diphtheria toxin, which results in the inactivation of
EF-2.
Pappenheimer received many honors for his work including the Eli Lilly Award in 1942 and
election to the National Academy of Sciences in 1973. Together with his former student R.
John Collier, Pappenheimer was awarded the Paul Ehrlich Prize and Gold Medal.
Robert D. Simoni, Robert L. Hill, and Martha Vaughan
REFERENCES
1. JBC Classics: Hess, A. F., Unger, L. J., and Pappenheimer, A. M. (1922) J. Biol. Chem. 50, 77– 81
(http://www.jbc.org/cgi/content/full/277/23/e12)
2. JBC Classics: Edsall, J. T. (1930) J. Biol. Chem. 89, 289 –313; Cohn, E. J., Edsall, J. T., and Blanchard, M. H.
(1933) J. Biol. Chem. 105, 319 –326 (http://www.jbc.org/cgi/content/full/277/33/29351)
3. Lawrence, H. S. (1999) Biographical memoir of Alwin Max Pappenheimer, Jr. Natl. Acad. Sci. 77, 3–18
4. JBC Classics: Mueller, J. H. (1923) J. Biol. Chem. 56, 157–169 (http://www.jbc.org/cgi/content/full/277/25/e14)
5. Collier, R. J., and Pappenheimer, A. M. (1964) J. Exp. Med. 120, 1007–1018, 1019 –1039
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Alwin M. Pappenheimer, Jr. Photo courtesy of the National Library of Medicine.