Ann Rev. Biochem 1979. 48:I-tl
copvrist 0 1979 by Annual Reviews Inc All rights reserved

A "PURE" ORGANIC CHEMIST'S

9 12000

DOWNWARD PATH: CHAPTER 2-

THE

YEARS

AT P.

AND

S.*

Michael Heidelberger

Emeritus Professor of Immunochemistry, College of Physicians and Surgeons.
Columbia University, New York, New York 10032 and Adjunct Professor
of Pathology (Immunology), New York University School of Medicine,
New York, New York 10016

The World of Science . . . is the purest democracy on earth, a brotherhood from which
poverty bars no one-neither the color of skin nor religious belief. Intelligent devotion
to the pursuit of scientific truth, and competent effort, or support of effort, toward this
end. automatically enrolls one in the great company of the "Fifth Estate." One may 6nd
in this real "League of Nations" some of the most inspiring of human associations . . .
Here may be encountered some of lie's greatest opportunities for service to humanity and
certainly some of its greatat compensations. Happy are its devoted

Charles Fuller Baker,
Dean of the College of Agriculture,
University of the Philippines
In The Philippine Agriculturalist, 14455 (1926)

In 1928 the Department of Practice of Medicine of the College of Physicians
and Surgeons of Columbia University moved with the Presbyterian Hospi-
tal from 70th Street and Park Avenue to the newly built Medical Center
on the site of the Yankees' baseball park at 168th Street and Broadway.
Walter W. Palmer was, at that time and for many years later, chairman of
the department. One of his close friends and advisors was Henry D. Dakin,
an ex-English chemist of "chloramine T" and Dakin pad fame, and they
had collaborated in a study of thyroidal function. Partly as a result of

*Chapter 1 of this munt appeared in the Annual Review of Microbioloo, Volume 31,


                                            1

1977.

066-41 54/79/0701-OOO1$01 .MI

2 HEIDELBERGER

Dakin's influence and accomplishments, Palmer was determined to have a
chemist in his newly expanded department and succeeded in obtaining
one-half million dollars for that purpose from Edward S. Harkness, a
principal benefactor of the Center. Thus was the Harkness Research Fund
established, and my lucky earlier acquaintance with "Bill" Palmer in Van
Slyke's laboratory at the Rockefeller Institute led to my choice as the first
full-time chemist in a Department of Medicine. With the appointment as
Associate Professor of Medicine went the title, Chemist to the Presbyterian
Hospital, with purely consultative duties.
Medicine at the Center was taught and practised by a closely knit group
of clinicians with widely divergent interests in research. Its leaders were
Palmer himself, who studied the thyroid gland with Alexander Gutman and
Ethel Benedict (later, Mrs. Gutman); Alphonse R. Dochez, concentrating
at that time on streptococcal infections with Alvin F. Coburn. Franklin M.
Hanger, and David Seegal; Robert F. Loeb, interested in a wide range of
metabolic disorders; and Franklin M. Stevens, an allergist. Every Tuesday,
after lunch, there was a departmental meeting in Palmer's spacious office
at which he opened the proceedings with a discussion of problems of general
interest or with medical gossip when there were no problems. Visitors were
presented and anyone was free to voice suggestions or complaints or to
bring up a topic of interest. Interactions with other departments were
frequent; for example, Fordyce (Johnny) St. John, a Professor of Surgery,
came in one day to protest the burden placed on his department by opera-
tions which could have been prevented by earlier medical intervention. The
weekly meetings were continued by Robert Loeb when he succeeded Palmer
on the latter's mandatory "retirement" to become Director of the Public
Health Research Institute of the City of New York. I stress these meetings
because, in another department at the Center, there was only one depart-
mental meeting in the entire twenty-seven years I spent at P. and s. We also
had afternoon teas in Dochez's and Hanger's offices, during which stimulat-
ing ideas were tossed about. For these teas we contributed small sums or
a monthly cake.
I was given two laboratories at the northwestern end of the department's
(8th) floor. offered an office in the main stem of the hospital, near Dr.
Palmer's, I chose instead a narrow architectural afterthought leading of€
from my personal laboratory past the powerhouse stack and designed to
bring that feature out to the main building line. Although intolerably hot
in summer, it was cozy in winter and saved long walks back and forth. I
was soon joined by Forrest E. Kendall, who had just received his PhD in
organic chemistry at the University of Illinois with Professor W. A. Noyes.
It was a surprise, when Kendall arrived, to find that he had only one hand.
The other had been cut off by a threshing machine on the family farm, but

A CHEMIST'S DOWNWARD PATH 3

in spite of his handicap he had earned enough to put his younger brothers
and sisters through college. In the eight years that we worked together, the
absent hand was never missed. I had brought from Mt. Sinai Hospital, as
technician, Check M. So0 Hoo, a California-trained Chinese who served
faithfully and admirably with me for thirty-five years at P. and S. and at
the Institute of Microbiology, until a series of heart attacks ended his life.
The Harkness Fund also providxl us with a laboratory helper and assis-
tance for washing glassware.
During the last years at the Rockefeller Institute and the year at Mt. Sinai
Hospital I had been reading Jules Bordet's huge, novelesque Trait6 d'lm-
munologie and Arrhenius and Madsen's Immunochemisty, a brave at-
tempt in 1907 to apply principles of physical chemistry to the immunology
of the time. It appeared to me that there was a crying need to determine
the true nature of antibodies and that until this was done there could be no
end to the polemics and uncertainties that were plaguing immunology. It
was evident, also, that the purely relative methods in use, giving titers or
their reciprocals, were inaccurate, often misleading, and incapable of per-
mitting a decision as to whether antibodies were really globulins or sub-
stances of unknown nature adsorbed to these proteins. To a chemist, it
appeared reasonable to assume that the principles of analytical microchem-
istry could be applied to the estimation of antibodies in units of weight, a
prerequisite to the determination of their nature. This had been attempted
by Hsien Wu et al (la) but not carried to a conclusion.
Actually, the time was propitious for the development of the absolute
method we envisaged. Felton had just devised a simple procedure for partial
purification of antibodies in antipneumococcal horse sera by precipitation
with the so-called euglobulins on dilution with large volumes of water and
solution of the sediment in physiological saline (2). And if antibodies should
indeed be proteins, the ideal initial antigen for their estimation would be the
nitrogen-free, specifically precipitating capsular polysaccharide of type I1 or
type 111 pneumococcus, which I had isolated with Oswald T. Avery at the
Rockefeller Institute several years before (lb). Any nitrogen in a specific
precipitate, therefore, would be derived from the antibody if conditions
could be found to eliminate nonspecific, adsorbed proteins.
Realization of such a quantitative analytical micromethod became the
immediate task for Kendall and me. I felt, though, that I had to justify
Palmer's insistence on having a chemist in his department, so that we were
soon collaborating with Bill on a study of thyroglobulin (3) and with
Dochez's group on fractionation of the antigens of hemolytic streptococci
(4, 5). In addition, the novel presence of organic chemists in a medical
department often brought microbiologists, surgeons, and others to our
laboratory to borrow chemicals or obtain suggestions on overcoming un-

4 HEIDELBERGER

foreseen difficulties. Our horizons were immeasurably widened and there
was scarcely a day without excitement of one kind or another.
Antibodies to type I11 Pneumococcus, purified according to Felton, gave
precipitates with the nitrogen-free type I11 pneumococcal capsular polysac-
charide which were centrifuged down. The Supernatants were analyzed for
nitrogen, and this value, subtracted from the original amount of total nitro-
gen taken, gave a measure of antibody nitrogen by difference. Varying
proportions of antigen and antibody were studied and we attempted to
explain the results according to the law of mass action, much as other
chemical precipitations could be accounted for quantitatively. Shortly after
our publication (6), John R. Marrack, whose Chemkty of Antigens and
Antibodies (7) soon attracted wide notice, came to the laboratory, having
made the trip from England as ship's physician on a small Cunard liner.
He objected to our assumptions that there were only two antigen-antibody
complexes and that equilibria existed, because we had made no study of the
kinetics. This resulted in an enduring friendship and caused Kendall and
me to shift our emphasis, without abandoning the mass law, in the final
development of a quantitative theory of the precipitin reaction (8).
Realizing that analyses by difference were applicable only to solutions of
purified antibody, we added normal serum and began analyzing the precipi-
tates. When well-washed in the cold with 0.9% saline, these precipitates
yielded results identical to those previously obtained. We could then use
whole sera and determine their content of precipitable antibody in units of
weight.
This was all very well for antibodies to polysaccharides, but what about
those elicited by the vast numbers of protein antigens? To solve that prob-
lem we had to distinguish between antigen nitrogen and antibody nitrogen.
`5N had not yet been discovered and radioactive isotopes had not been
tamed for use in biology. We therefore had recourse to dyes. By coupling
tetrazotized benzidine on one side with R-salt, a naphtholdisulfonic acid,
and on the other side with crystalline hen's egg albumin, we produced an
Easter-egg-like purplish protein antigen. This was treated rather drastically
until it no longer precipitated anti-egg albumin. Injected into rabbits, it gave
rise to antibodies that formed pink to red precipitates with the antigen,
depending upon the proportions used. Washed precipitates were dissolved
with alkali, rinsed into a colorimeter cup and compared, visually, with
alkaline solutions of the dye. Photoelectric colorimeters were not yet devel-
oped. The solutions of the precipitates were rinsed quantitatively into
micro-Kjeldahl flasks. Total nitrogen minus antigen-dye N gave antibody
N (9). When we had worked out the course of this reaction, we were ready
to study that of a colorless protein, crystalline egg albumin (IO). Later, with
H. P. Treffers, now Professor of Pathology at Yale, and a young B. Davis,

A CHEMIST'S DOWNWARD PATH 5

whose short career was ended by poliomyelitis, we went on to study the
immunochemical effects of phosphorylating the protein. The denaturation
of egg albumin was also investigated with Catherine F. C. MacPherson (1 1)
(who helped develop the method for estimation of antibodies in human sera,
then studied the polysaccharides of several types of Hemophilus influemae
at Babies Hospital, went on to E. G. D. Murray's department at McGill,
and is now an authority on proteins of the brain at the University of Western
Ontario), the deamination of egg albumin with Paul H. Maurer (12a, 12b)
(now for many years Professor of Biochemistry at Jefferson Medical), and
the cross-reaction between hen's egg and duck's egg albumins (13) with
Abraham G. Osler (who became Professor of Microbiology at Johns Hop-
Ens, spent many years in research at the Public Health Research Institute
of the City of New York, and has just gone to the University of California
at San Diego).
Quite reasonably, some of our colleagues were worried about soluble
antigen-antibody complexes in the egg albumin system, but as we had
shown that they did not occur measurably and as we claimed only the
estimation of precipitable antibody, our data appeared valid. Most welcome
confirmation of our analyses came by an entirely independent physical-
chemical method (14). Even this did not convince all "hard-core" im-
munologists, for as late as 1939 a new edition of a standard text insisted that
we were "doubtless measuring non-specific nitrogen" instead.
Development of a quantitative theory of the precipitin reaction taxed our
ingenuity, but the experiments we had done led inevitably and inexorably
toward the interaction of multivalent antigen with multivalent antibody (8).
We had partially hydrolyzed the type I11 polysaccharide and shown that
the larger fragments could precipitate portions of the antibodies, so that the
intact substance was obviously multivalent. From the proportions of anti-
body to antigen in precipitates from protein systems, it seemed reasonable
to assume that a protein such as egg albumin must have at least five or six
combining groups, so that our theory was not limited to polysaccharide
antigens. As for the antibody, we knew from the beginning that precipitates
in the region of antibody excess could combine with more antigen; there-
fore, two or more combining groups did not seem unreasonable. My friend,
Felix Haurowitz, however, with true scientific skepticism, insisted for many
years that antibodies need not be more than univalent, but he finally capitu-
lated. We always remained friends in spite of this disagreement as to theory,
and we argued in our correspondence and at meetings with as much relish
as we experienced when playing sonatas together, for Felix was both a
superb scientist and an excellent pianist. For many years, Waldo E. Cohn
organized and played cello at evening sessions of chamber music at the
annual meetings of the Federation of American Societies for Experimental

6 HEIDELBERGER

Biology. Felix and I frequently took part in these. Another critical friend
was Linus Pauling, who believed that the valence of antibodies could not
exceed two. However, I attended a lecture of his at Brooklyn Polytechnic
and, with great glee, detected a trivalent antibody on one of his lantern
slides, inserted to give a specific precipitate the proper degree of compact-
ness. Linus also, with Dan Campbell and David Pressman, criticized our
assumptions as "arbitrary and unlikely," which surely was their privilege.
With a Merent set of assumptions, however, they arrived at an equation
practically identical with ours (15). This, of course, pleased us immensely.
Later, our simple equation for a quantitative theory was shown to be a
special case of a more general theory (1 6) and this, in turn, was found to
be a special case of a still more general theory requiring calculations by a
computer (17). This was progress, but for immunologists needing simple,
quantitative explanations and calculations in many, but not all, precipitat-
ing systems, our formulation and equation remained practical and useful.
Moreover, the theory permitted a number of valid predictions, as appears
later.
While Forrest and I were polishing up the precipitin reaction, our first
graduate student, Elvin A. Kabat, arrived. He had received his BS cum
Zuude at City College and was to work as a laboratory helper while studying
for the PhD degree. Within a few weeks, this young whirlwind had read H.
Gideon Wells's and Arrhenius's books and all of our papers and wanted to
know why we hadn't adapted our quantitative method to bacterial aggluti-
nation. "We've been too busy," we said: "You go ahead with it," and he
did.
Studies of the agglutination of microorganisms had been hampered, not
only by the sole use of titers for measurement, but also by overemphasis of
the effects of electrical charge and by lack of recognition that the combining
ratios of bacteria and antibodies could vary greatly in different systems.
Because of such differences, standard textbooks contained statements like
"anti-SulmoneZZu sera (titers up to 1 : l,OOO,OOO) are stronger than anti-
pneumococcal (titers up to several thousand)." Had this comparison been
valid, most anti-Salmonella animals would have died of heart failure owing
to the viscosity of their blood. Our plan was to wash killed type-specific
pneumococci until the washings no longer gave tests for capsular polysac-
charide. This was to avoid confusion of agglutinins with precipitins. A
measured volume of the washed cells could then be added to a known
volume of homologous antiserum. Agglutination occurred, as sufficient
type-specific polysaccharide remained on the cells, and the added antibody
nitrogen could be measured. Elvin worked out the proper conditions for
accuracy, and bacterial agglutination was found to be a precipitin reaction
at the cellular surface and subject to the same theory and similar quantita-
tive expression (1 8).

A CHEMIST'S DOWNWARD PATH 7

An immediate practical result was the termination of the still ongoing
dispute as to whether agglutinins and precipitins were identical or different.
Though not as virulent as that over unitarian or pantheism, the ineffectual
thought and effort given to it retarded progress. With our genuinely quanti-
tative methods it was easy to show that, with respect to a single antigen and
its homologous antibodies, precipitation and agglutination were functions
of the same fraction of antibody (19). As protection had already been found
to parallel the antipolysaccharide, three independent manifestations of the
action of antibodies were shown to be due to the same protein molecules.
There was another intensely practical result when Hattie E. Alexander,
microbiologist of the Babies Hospital at the Center, asked for help to
improve the potency of the rabbit anti-H. influenzae b sera with which she
wished to treat influenzal meningitis in infants. Quantitative agglutinin
estimations that precisely measured the results of various dosages and
routes of injection of the bacilli into rabbits soon resulted in sera with up
to ten times the former content of agglutinins and precipitins for the poly-
saccharide of type b. Injection of the new sera into infants with influenza1
meningitis cured most of them (20).
Confident of our methodology, we turned to our original quest: to lind
out whether or not antibodies were actually modified globulins. Again, luck
favored us, for we were joined for several months by Torsten Teorell,
presently (1978) Emeritus Professor of Physiology at the University of
Uppsala. Torsten had been working with W. J. V. Osterhout at the Rocke-
feller Institute and had a consuming interest in the biological functions of
electrolytes. We put him to work on the effects of varying concentrations
of salts on the precipitin reaction with the aid of his own modification of
the micro-Kjeldahl technique. An increase in sodium chloride diminished
precipitation, but all added polysaccharide was still precipitated in the
region of antibody excess. This, we soon realized, offered an approach to
the isolation of pure antibody: a thoroughly washed precipitate formed in
the region of antibody excess in 0.9% saline should liberate only antibody
when equilibrated in 0.5M or M NaC1. This was all very well on a micro-
Kjeldahl scale, at which nonspecific proteins could easily be washed out
within the limit of error of the method. Our first large-scale preparation,
however, was only about 70% pure, and it took Kendall, Kabat, and me
two years before the 6rst batch of analytically pure antibody was obtained
(21a, 21b). This was typical globulin and provided the last link in the chain
of evidence that antibodies were actually globulins. With this secure knowl-
edge of the chemical nature of antibodies, one could now proceed to the
study of their biosynthesis, cellular origin, and control. Visiting Denmark
the following summer, I tried to interest August Krogh, a leading physiolo-
gist, in undertaking such a study, but he was fully occupied with other
problems. Other Scandinavians, notably Mogens Bjiirneboe and Astrid Fa-

8 HEIDELBERGER

graeus, however, stimulated by the new findings, soon implicated the
plasma cell in the formation of antibodies (22a, 22b). These workers not
only started cellular immunology on its piodigious expansion but also
contributed much to its development.
While the quantitative studies were in progress, Florence Rena Sabin
came to the Rockefeller Institute with radical ideas and experiments on the
nature of tuberculosis. A warm friendship soon developed, and Florence
was often at our home, particularly when we also invited Oswald T. Avery.
My first wife, Nina Tachau, and I also went to the Cosmopolitan Club as
her guests or to her home, where Florence was almost as proud of her skill
in broiling steaks as of her scientific accomplishments. At her urging and
with a grant from the National Tuberculosis Association, the complex
mixtures of polysaccharides and proteins of various strains of mycobacteria
were studied by means of fractionations meticulously carried out by Arthur
E. 0. Menzel (23a, 23b) and later by Sulo A. Karjala.
In 1934 and 1936 I was given partial John Simon Guggenheim Memorial
Fellowships to work with The Svedberg's ultracentrifuge in Uppsala for
periods of about six weeb, as I did not wish to be absent longer from the
laboratory. The first time, I took samples of purified thyroglobulin along
and prepared others with Swedish material. To our great surprise, this
protein secreted by the thyroid gland was found to be of high molecular
weight (24). On the second trip, purified equine antibodies to pneumococcal
polysaccharides were centrifuged and also found to have high molecular
weight (25), confirming Kendall's and my findings with the Northrop cell
and in accord with independently arrived at results by ultrafiltration (26a,
26b). This time Nina and I were there in May and part of June, when we
could enjoy the explosive Swedish Spring and its twenty-four hour shades
of brightness. When there were not too many calculations to be done in the
evening we would drive along an east-west road, enjoying colorful sunsets
toward the north and watching them merge into equally beautiful sunrises.
Svedberg had assigned his codeveloper of the ultracentrifuge, Kai 0. Ped-
ersen, to most visitors, to teach them the many precautions, controls, and
actual use of the complicated, oil-driven machine with its terrifyingly nu-
merous gauges and its optical system perfected by Ole Lamm. Kai, a gentle,
self-effacing man of immense competence, had been nursing numerous
visiting scientists along for some years, to the detriment of his own research.
The many papers published with his help contained only brief acknowledg-
ments for his efforts. As my time was limited, he practically gave up his
work while I was there. We and our wives became fast friends and have
often exchanged visits on both sides of the Atlantic. For his essential part
in the work, I naturally made him a co-author of the resulting publications,
a precedent which happily encouraged subsequent visitors to do the same.

A CHEMIST'S DOWNWARD PATH 9

By 1937 Elvin Kabat had completed two projects worthy of the PhD
awarded for one of them. Thanks to a fellowship from the Rockefeller
Foundation, he, too, went to Uppsala to work with Arne Tiselius and Kai
Pedersen on the electrophoretic and ultracentrifugal properties of antibod-
ies, including purified samples from various species of animals' which we
sent over (27a, 2%). This research confirmed our analytical data on anti-
body content, as already noted, and even more 6rmly established the protein
nature of antibodies and demonstrated their variations in molecular size.
Goodner & Horsfall had also shown that antipolysaccharide in rabbit
antipneumococcal sera was of relatively low molecular weight, and had
reported good clinical results with these sera (28). This led us to immunize
rabbits with type I, 11, or I11 pneumococci, and, with Joseph C. Turner of
our department as clinician, to devise a simple method of partial purifica-
tion of the antibodies for use in the Presbyterian Hospital (29). Cases of
pneumonia due to I, 11, or I11 received an intravenous infusion of 400-600
mg of the appropriate antibody. Nurses were instructed to bring me a few
cc of the patient's blood half an hour later. Serum was drawn off and tested
with homologous capsular polysaccharide. A positive precipitin reaction
showing excess antibody usually resulted, but one severely infected type I11
patient required five bottles of antibody solution before an excess was estab-
lished and recovery ensued. Under this regime deaths from these types of
pneumonia were practically abolished at the Hospital as was also sem
sickness. Soon afterward, the sulfa-drugs and penicillin came into use and
typing and Serum therapy were abandoned.
Having quantitated specific precipitation and agglutination, we took on
the problem of complement (alexin), so important for diagnosis and im-
mune lytic action. Was it a substance, as Ehrlich and his followers main-
tained, or was it merely a colloidal state of freshly drawn serum, as the
school of Bordet insisted? Interest in this question was stimulated by the
arrival of Alfred J. Weil from Germany and Otto G. Bier from Brazil, both
of whom were familiar with the peculiarities of complement. An adequate
series of controls soon made it possible to show that complement-containing
sera added appreciable weight to specific precipitates formed with rabbit
antipolysaccharide or antiprotein (30). Complement was therefore a sub-
stance or a series of substances (four components were then known) and
Ehrlich's ideas were vindicated. Simultaneously, Pillemer, Ecker, Oncley,
and Cohn purified the first of the four components (31), and arrived at the
same conclusion. Further quantitative studies in the laboratory at P. and

'A turkey that had received injections of formalinized pneumococci was ultimately roasted
by a Hospital chef and became the main dish of a laboratory luncheon. The formalin and
pneumococci did not affect its flavor.

10 HEIDELBERGER

S., carried out with Manfred M. Mayer, Graciela Leyton of Chile, and
Abraham Osler, established optimal conditions for the estimation and more
efficient use of complement, and showed that, in many immune systems, its
"fixation" ran parallel to specific precipitation.
Quantitative immunochemical methods also provided a check on meta-
bolic studies with heavy nitrogen. Because of the rapid entry of 15N into
proteins in intact animals, Schoenheimer concluded that preformed peptide
bonds opened and closed easily (32). Collaborating with his group, Treffers
and I injected an 15N-fed rabbit with killed type I11 pneumococci and
antibodies to type I polysaccharide from another rabbit. Quantitative analy-
ses of the circulating actively forming and passive antibodies at varying
intervals showed that only the anti-I11 molecules being synthesized were
capable of taking up lSN. The simultaneous presence, with lSN, of indepen-
dent, immunologically specific markers thus settled an important aspect of
the metabolism of proteins in the living animal (33). The central idea for
this work, also quantitative studies of hemolysins, of antibodies to proteins
raised in horses (in part with Jules Freund and Robert C. Krueger), and
friendly assistance to foreign visitors, were contributions made by Henry P.
Treffers during four postdoctoral years in the laboratory.
A foreign visitor not previously mentioned was Pierre Grabar, who ar-
rived in 1937 as a Rockefeller Foundation Fellow, at the suggestion of
And& Boivin of Strasbourg. Pierre, a physical chemist, had only shortly
before become interested in immunochemistry but developed into its lead-
ing European exponent on returning to the Pasteur Institute in Paris. Then
there was Bertil Josephson, of Stockholm, whose interest in immunochemis-
try grew out of his specialization in the functions of the kidney. Still another
was Sverre Dick Henriksen, a microbiologist from the laboratory of T.
Thjiitta in Oslo. Sverre could not return to the Rikshospital because of the
invasion of Norway by the Germans, but was helped by a Rockefeller
Foundation fellowship until he and his wife, Aase, departed for medical
service in the Little Norway camp in Canada. Lifelong friendships devel-
oped with all of these visitors and their families.
Meanwhile, in part with D. L. Shrivastava, another Rockefeller Founda-
tion Fellow and later Assistant Director of the Central Drug Research
Laboratory at Lucknow ad an expert on cholera, we did a quantitative
study of homologous and cross-precipitation in the pneumococcal type I11
and type VI11 systems (34a, 34b). This not only disclosed five different kinds
of heterogeneity in the equine antibodies used, but also enabled us to predict
the most likely arrangement of the sugars in the type VI11 substance. There
was also a practical sequel. A paper on oxidized cotton made it obvious to
me that this linear polymer of cellobiose had been converted into analogs
of types I11 and VI11 polysaccharides by conversion of many -CHIOH

A CHEMIST'S DOWNWARD PATH 11

groups to -COOH. One could predict from our quantitative theory that
substances with multiple identically or similarly linked sugars or amino
acids should give cross-precipitation in appropriate antisera. I wrote for
samples of oxidized cotton and found at once that their neutralized solu-
tions precipitated antipneumococcal I11 and VI11 sera heavily (35). Our
surgeons were then using oxidized cotton pads as a hemostatic and as a
packing for wounds, for they could safely be left inside and would slowly
disappear. By means of a rapid precipitin test of a patient's serum or urine
with equine antipneumococcal type 111 serum, I was able to tell the sur-
geons, almost to the hour, how much time elapsed before the pads were
wholly absorbed.
Then came the Second World War and theoretical problems were slowed
or shelved and immediate concerns of the military were given priority. Two
of our projects were highly secret and both illustrated the damaging effects
of scientific secrecy. The first, with Forrest Kendall and L. A. Julianelle,
was to protect and cure animals infected with anthrax, an agent our enemies
were supposed to be planning to use. Julianelle found that for mice., penicil-
lin, just then available, was the answer. Secrecy prevented Julianelle from
publishing this and also lost him the benefits of priority when microbiolo-
gists at the Mayo Clinic, working outside of governmental auspices, an-
nounced the same finding in The New York Times. The second secret
project concerned ricin, the enormously toxic principle of the castor bean
which, it was feared, might also be used as a weapon. Elvin Kabat and I
further purified samples of ricin, and colleagues in two other laboratories
actually crystallized it. Suddenly, secrecy as to the chemical studies was
lifted because of an incautious general`s speech. I telephoned my friends in
the project, proposing that we all publish our data in the same number of
the same journal. They agreed, so we quickly wrote our paper, in which
their independent work was mentioned, and sent it to Washington. Clear-
ance was speedily granted, but alarming reports caused secrecy to be
clamped down again before our colleagues' papers were written. This
created the ridiculous situation that our publication (36) disclosed results
that those who had obtained them were forbidden to mention.
Two nonsecret projects, however, proceeded normally. Because of a con-
tinuing large series of pneumococcal pneumonia during several years at a
training camp for aviators at Sioux Falls, South Dakota, it became advisable
to find out whether or not human subjects could be protected by immuniza-
tion with purified capsular polysaccharides of the causative types. Felton
had attempted this during the Great Depression by immunizing many
thousands in camps of the Civilian Conservation Corps. His massive experi-
ment was ill-fated, however, because healthful, outdoor living conditions
led to almost no pneumonia in any of the camps, unimmunized or immu-

12 HEIDELBEROER

nized, and mouse protection tests of sera of vaccinated persons varied from
titers (alas!) of 0-2,000,000, an uninterpretable spread. I asked for volun-
teers from the entering classes at P. and S. in 1942 and 1943. Most of the
students agreed to be injected with the polysaccharides of types I, 11, and
V and were wonderfully faithful in appearing for injections and bleedings,
even though the latter often meant going without lunch during the hectic
wartime compression of the medical course into three years. For a few
exquisitely sensitive individuals it was fortunate that we first injected only
about one third (15-20 pg) of the total dose, waiting 24-48 hours before
adding the remainder if no reaction, or only a slight one, occurred.
Strangely enough, in no case was a marked reaction due to known previous
infection with pneumococci of types I, 11, or V. Maximal values of precipitin
in serum were reached in two to six weeks and a good response to one type
did not guarantee an equal result with the others. The antibodies, unlike
those to diphtheria toxoid, for example, were evoked at remarkably perma-
nent levels, diminishing only slightly during months and even years (37).
But were the volunteers protected against types I, 11, and V? Our busy
students would not have enjoyed being challenged with virulent pneumo-
cocci, but luck favored us. A paper by Barry Wood had appeared oppor-
tunely (38), in which rats sprayed 12 hr before with virulent type I
pneumococci were saved by 0.02 cc of a rabbit antipneumococcal I serum
but not by 0.002 cc. We analyzed this antiserum and calculated that 0.2 cc
gave a concentration of antibody in the blood of Wood's rats about three
times that of the average of our volunteers. Assuming that if three times our
average could cure, one-third as much should protect, I recommended to
the Surgeon General that vaccination be tried at Sioux Falls. We decided
to inject polysaccharides of types I, 11, V, and VII, as the resident microbi-
ologists had found these types in about 60% of the pneumonias. The camp's
population was randomly marshaled into two lines of 8,500 each. Those in
one line were injected with 1 cc of saline as control subjects. Those in the
other line were given 1 cc wntaining 50-70 t(g of each of the polysaccha-
rides. These had been prepared by us, by E. R. Squibb and Sons under
Tillman D. Gerlough's direction, and by Augustus C. Wadsworth and
Rachel Brown. As seen in Table 1, within two weeks there were no more
pneumonias caused by the four types among those vaccinated. Even the
nonvaccinated were partially protected because, as the microbiologists
found, the vaccinated personnel no longer carried these pneumococci in
their noses and throats. Pneumonias due to pneumococcal types not in the
vaccine remained equal in both groups, which showed the strict specificity
of the protection (39). The entire study, so beautifully organized and moni-
tored in the field under Colin M. MacLeod's direction showed that epi-
demics of pneumococcal pneumonia in closed populations could be ter-

A CHEMIST'S DOWNWARD PATH 13

Table 1  Interval between injection and the development of pneumonia in immunized
and nonimmunized subject+

Number of cases of pneumonia

Types I, 11, V, VI1

Types I, 11, V, VI1

All other types

All other types in

Interval
-~ in immunized in nonimmunized in immunized nonimmunized

Weeks subjects subjects subjects subjects

1
2
3
4
6
8
10
12
14
16
16+

Total

2
2
0
0
0
0
0
0
0
0
0

4

-

0
3
3
2
2
2
1
0
2
3
8

26

-

1
5
I
8
6
3
4
2
2
2

16

56

-

1
3
5
12
7
4
4
4
1
4
14

59

-

aReprinted from J. Exp. Med. 1945. 82:453.

minated within two weeks after vaccination with the polysaccharides of the
causative types. As many of the protected subjects had shown levels of
antibody too low for our relatively insensitive precipitin technique to detect,
it was evident that very little antibody could prevent the droplet infection
by a few pneumococci that presumably initiates the disease.
Our fourth wartime project was camed out with Manfred M. Mayer and
Myron A. Leon. The latter came from the Bronx High School of Science
at the age of 16, was drafted into the Navy at 18, was reassigned to the
project through the good offices of Admiral H. W. Smith, and is now
Professor of Immunology and Microbiology at Wayne State University in
Detroit. We had earlier prepared malarial vaccine from the hemolyzed red
cells of volunteers among the malaria-infected sailors who had been admit-
ted to the Marine Hospital on Staten Island. Manfred's wife, Elinor, a
pianist, suggested that we try curing victims of malaria between relapses
with vaccines made from their own parasites. This was done in about fifteen
patients with mildly encouraging results. As the hospitals of the armed
forces were filling up with malarial patients from the Solomon Islands and
elsewhere in the Pacific, I proposed a trial of our vaccine to the Surgeon
General of the Army. Better proof of efficiency was demanded, however,
so I went to the Bureau of Medicine of the Navy. An entire ward of about
200 patients was quickly assigned to the project at St. Albans Hospital in
Queens, New York, under the medical supervision of Commander W. A.

14 HEIDELBERGER

Coates. He explained the need for, and use of, blood from the most highly
parasitized cases during relapses, and these men gave their blood liberally
in spite of their discomfort. During the six months of the test our laboratory
did little else than prepare malarial vaccine, and at the end the vaccine-
treated and two control groups showed exactly the same rate of relapses
(40). By-products of scientific value, however, were an electromagnetic
method of concentrating the relatively few parasitized red cells in infections
due to Plasmodium malake (41) and an understanding of the triple nature
of complement-fixation in malarial blood (42).
Postwar inflation soon made the Harkness Research Fund inadequate
and recourse was had to the Rockefeller Foundation and the Office of Naval
Research (ONR), precursor of the National Science Foundation (NSF).
When I asked how the Navy could possibly be interested in one of our
projects on an ONR grant, the reply was: "DO the research as you think
best and the Navy will look out for its own interests." This liberal attitude
was continued in the NSF when Alan Waterman left ONR to be Director
of NSF.
During all these years I had been making sporadic attempts to learn more
about the capsular polysaccharides of pneumococci: for example, that of
type IV with Kendall, and types I, 111, V, and XVIII with Harold Marko-
witz, now with the Mayo Clinic, The polysaccharide of type I, Neufeld's
"typical" pneumococcus, was taken up several times, but the instability of
its hydrolytic products proved baf€ling. Happily, I have interested Bengt
Lindberg in this unusual amphoteric polygalacturonate. Now that comer-
cial quantities are being prepared one may expect that he will solve its !he
structure, as he has with types I1 and XIV.
Unlike the problem with type I, another major investigation had been
slowly expanding: the use of cross-reactions to clarify relations between
chemical structure and immunological specificity. This had begun (see lb)
with Avery's intuitive feeling that analogs of pneumococcal polysaccharides
must exist "free in Nature." His hunch was quickly justified by the cross-
reaction of gum arabic and its products of partial hydrolysis in anti-
pneumococcal type I1 serum and the analogous precipitation by the
polysaccharide of Friedlander's bacillus B (Klebsiella K2). The cross-reac-
tion of pneumoccal types 111 and VI11 has already been mentioned. Also,
during a consultantship with Merck and Co., of which Per K. Frohlich was
Director of Research, I had looked into their synthetic polyglucoses as
possible blood-extenders and found that, like the dextrans, certain fractions
precipitated heavily with antipneumococcal sera of several types. Then
glycogens of various origins were found to react in this way (43). To our
astonishment, the paper describing this was rejected by the Journal of
BioZogicaZ Chemistry as not being biochemical! Since many gums of plants

A CHEMIST'S DOWNWARD PATH 15

and other polysaccharides with nonreducing lateral end-groups of D-galac-
tose precipitated antipneumococcal type XIV serum, one could predict that
the capsular polysaccharide of this type would also contain this structural
feature. I spent a summmer in the organic chemical laboratories at the
University of Birmingham, England, learning techniques of chromatogra-
phy and methylation from Maurice Stacey (who had been sent on a wedding
trip mini-sabbatical to my laboratory in 1936 by Sir Norman Haworth) and
S. Alan Barker. We verified the prediction (44). Other early uses of cross-
reactions were the demonstration that an acidic impurity in the galactan of
bovine lungs contained D-glucuronic acid (45) and that gum arabic was a
mixture from which a fraction of quite different composition could be
precipitated by antipneumococcal type I1 serum (46). Samples began to
come in from many countries and it was often possible, by means of
rapid cross-precipitation, in which the initial qualitative tests were fre-
quently followed by quantitative estimations, to inform the sender of the
nature, position, and/or linkage of one or more sugars in the product-
information which might require weeks or months by purely chemical
means.
In 1938 I first met Selman A. Waksman. We served on a planning
committee for the International Congress of Biochemistry held in New
York in 1939. My first wife, Nina, mother of my son, Charles, was on the
Women's and Hospitality Committees, and many lasting friendships, espe-
cially with the foreign visitors, resulted from her activities as well. The
hurried departure of the English, French, and German biochemists on the
outbreak of World War I1 cast a heavy pall over the last days of the
Congress.
From 1943 to 1946 Warren Weaver was organizing some eighty scientific
talks sponsored by the US Rubber Company and transmitted over the
Columbia Broadcasting System during the intermissions of Sunday concerts
of the Philharmonic Orchestra of New York. It was, I am sure, Weaver's
insistence that resulted in the rare circumstance of scientists being paid
entertainers' fees-$500 for an eleven-minute talk. My subject was Resis-
tance to Infectious Disease. Although my wife, Nina, was dying of cancer,
as a writer she worked with me over every word, insisting on simplicity and
clarity and helping shorten long sentences. The vivid memories of her aid
required intense self-control when I gave the talk several months later,
nervous as I also was with the realization that a million or more listeners
might hear me. Perhaps they did, for this last message with her help, so
frequent with earlier papers as well, brought letters from as far away as Los
Angeles and Saskatchewan.
In December 1946, I was invited to the first postwar international scien-
tific symposium, to be held in Paris to commemorate the 50th anniversity

16 HEIDELBERGER

of the death of Pasteur. The twenty-one hour flight in a nonpressurized
DC-4 across the Atlantic, via Goose Bay and Shannon, was a true adven-
ture. The hotel in Paris was allowed to have heat for only an hour in the
morning and another in the evening, but we were given an adequate supply
of food stamps. The resurgent spirit of the French, after their enormous
losses, was inspiring, and when a minister of education spoke proudly of
French science, he was rebuked by Jules Bordet in an impassioned address
stressing its international rather than national quality. Another early post-
war international congress on microbiology was held in Copenhagen in the
summer of 1947 and I was on our delegation to it. Selman Waksman was
given an award and donated the quite large monetary portion for a Danish-
American exchange fellowship. At the "banquet," by chance I sat next to
Gabrielle Hoerner, a statuesque Alsatian pathologist who had come along
as secretary to J. E. Morin, Professor of Microbiology at Laval University
in Quebec. (I was much touched when, the following winter, Professor
MOM and a carful of students drove through a heavy snowstorm to attend
my French lecture in Professor Armand Frappier's Department at the
University of Montreal.) The cursory table talk with Gabrielle eventually
narrowed down to music and my impending visit to Paris, where Gaby was
intending to visit a "musical sister." An invitation resulted, and I was
embarrassed, being only an amateur, to find that the "musical sister" was
the leading Wagnerian soprano of the Paris opera, Germaine Hoerner. The
three of us, and Gaby's eventual husband, Robert Vogt, became good
friends and I owed many he sessions at the Ogra and participation in
chamber music with French musicians to these accidentally acquired
friends.
Through pure chance I was twice President of the American Society of
Immunologists. The society's original constitution strangely provided that
the elected president assume the vice-presidency after his presidential year.
Elected president in 1947, I was vice-president in 1948 when the member-
ship decided to change to the more usual progression. This automatically
made me president again in 1949. My presidential addresses were: "Science,
Freedom, and Peace," and its variant, "Ivory Pawn in the Ivory Tower,"
stimulated by activities novel for me up to that time.
Nina had been Chairman on Foreign Policy for the New York City
League of Women Voters and active in the Speakers' Bureau of the Ameri-
can Association for the United Nations. After her death, her friends in the
latter, lacking funds to hire experts and knowing I was going to Europe,
asked me to try to carry on her work in the US delegation to the World
Federation of United Nations Associations. There were extraordinary peo-
ple in our group: among them, its leader, Clark Eichelberger, the UNA'S
executive secretary, Charles Marburg of Baltimore, who had been in the
State Department, and Cyril Bath of Cleveland, who had begun as a laborer,

A CHEMIST'S DOWNWARD PATH 17

had reorganized the Mexican railroads into a viable system, and in 1946 was
a manufacturer of machine tools with an intensely loyal force of skilled
workers. At the meeting in Prague, I acquired two warm friends in the
Norwegian group, and because I knew French fairly well I was able, at a
meeting of the French, English, and US delegations, to eliminate differences
in meaning that had prevented agreement on two resolutions. These actu-
ally were voted on by the General Assembly of the United Nations that
winter, and although they were too international to get enough votes from
the nationalistic delegates, it was thrilling to know that my first dive into
international politics had created even a ripple. Another year at the plenary
sessions, I sat next to a pharmacist, Asare, one of two Ewe tribesmen from
Togoland who had traveled through the native villages, talking about the
United Nations and obtaining small sums for the journey to Geneva. They
told an affecting story of a village that had spent years collecting money to
build a bridge, but gave them the entire amount, considering their mission
even more useful. Asare became an important official after Ghana's inde-
pendence and sent a young student, Samuel Essandoh, to the United States
for premedical studies and the medical course at P. and S., during which
he was president of his class for one year. At the same meeting, in Geneva,
were Evelyn Fox and Mary Dingman, who had been active there in the
YWCA during the war, and we became fast friends. And later still, in order
to finance a trip to the first meeting of the UNAs in Asia, I lectured in Hong
Kong, Tokyo, Osaka, Kyoto, Hokkaido, and Bangkok, aided by the Rocke-
feller Foundation. Eleanor Roosevelt was a member of our delegation at the
meeting in Bangkok, and I learned from her how to prepare a resolution
on world peace that would be acceptable to our UNA. It was a joy for me
and for my second wife, Charlotte Rosen, to be accepted by her as friends.
In the meantime, Duncan A. Machess, of the Rockefeller Institute,
unable to get the New York Academy of Sciences to agree to convene small
meetings of experts to consider unsolved problems in the sciences, had been
holding several highly successful midweek meetings of that type at an inn
on Long Island under the auspices of the National Acadeqy of Sciences.
As the knowledge of complement was in a rather primitive state, Duncan
was willing to add a conference on it to his series, with about twenty
participants. All of us contributing to the study of complement were there
in the autumn of 1950. We spend the first evening telling the others, among
them physical chemists and enzymologists who had scarcely heard the
word, what we knew, or thought we knew, of complement. The remaining
two days were devoted to discussing the things that needed to be known and
done, with highly stimulating and useful results (47).
In 195 1 Linus Pauling, presently still very much alive, was forbidden by
his physician to go to the Indian Science Congress to which he had been
invited. As his substitute, I departed for India by air on Christmas eve and

18 HEIDELBERGER

spent six busy weeks in that f~inat~g country as part of an international
group that also included Erwin Brand, a biochemist of our Medical Center
who was wise in the ways of amino acids and proteins and who stimulated
the development of a photoelectric polarimeter for their study. Another
invited foreigner was Arthur Stoll, of Basel, who had been Willstatter's first
assistant when I studied in Zurich, and whose company, Sandoz, was
equipping a new, air-conditioned animal house at the Central Drug Re-
search Laboratory in Lucknow. In Calcutta, where the congress was held,
and wherever we went afterward on our tour through India, Stoll was met
by a Swiss consul general or commercial attach6 and was usually housed
in a governor's mansion. By contrast, our gove~ent left the amenities of
hospitality entirely to our hosts, who did their very best under the direction
of a young man named Gon@ves from the Ministry of Science who hailed
from Goa, looked very Indian, and said his family had been Christians for
800 years. The general lectures of the Congress were held in an enormous
open "pandal," or tent, which seated 8000 because of complaints that the
general public was excluded in the original plans. As a result, entire fami-
lies, from grandparents to small children, listened patiently to our technical
talks. These were made difficult by the belated reverberation of our words
from loud speakers at the back of the tent. I was greatly impressed by the
fine research our Indian colleagues were doing, often with not-too-good
facilities, but with a slowly increasing supply of excellent locally manufac-
tured apparatus. Impressive, too, was a hospital in Bombay run by Col. A.
A. Bhatnagar, with its high standards of sanitation and care of patients. In
Calcutta we foreigners were given a dinner in the governor's palace and the
privilege of indi~du~ talks with Neb, who had actively furthered the
development of the sciences in India. He and I, however, did not talk
science but found a congenial topic in criticism of the policies of John Foster
DuUes. During our tour we were kept so busy giving lectures that when we
reached Delhi I refused to give any more until I had seen the Taj Mahal.
It was arranged that the 3. B. S. Haldanes, Brand, and I would be taken
early in the morning to Agra by car, but at the appointed time the Haldanes
had gone off birding and were left behind after much delay. The sad story
of Shah Jahan and his beloved Mumtaz, and the glorious beauty of the
monument to her were a tremendous emotional experience.
After the Congress I stayed on to complete a pet project. In the old days,
Avery would say: "If we only had an elephant instead of rabbits, we'd have
plenty of antiserum." I did not want to miss this chance to study the
immunological properties of elephants, potential suppliers of huge quanti-
ties of antisera. But first, at Lucknow, my friend, D. L. Shrivastava, was
having trouble with the supply of guinea pig complement for diagnostic
tests. I suggested elephant complement instead, which apparently had never

A CHEMIST'S DOWNWARD PATH 19

been tried. Serum was obtained from the nearest available elephant, a day's
trip away, and we set up tests with great excitement, only to be disap-
pointed. As for elephants and antibodies, chance and luck had intervened
again. Back in New York, the same Mrs. Vally Weigl, through whom I met
my present wife (see lb), had been giving piano lessons to the wife of a
former maharajah. A young friend of the maharani, sister of the first secre-
tary of the Maharajah of Mysore, came to hear us play, and we became
friends through our interest in the United Nations. When the younger lady
learned of my approaching visit to India and my desire to immunize ele-
phants, she notified her brother. The result was that in Mysore and Ban-
galore I was quartered in the Maharajah's No. 1 guest house and given an
appointment to play one of the clarinet sonatas of Brahms with the Mahara-
jah, an excellent pianist. However, only a brief, cordial interview resulted,
owing to the Maharajah's sudden violent toothache. When he heard that
I could not stay for the usual weekend illumination of the fountains in the
beautiful park below the massive dam that was bringing electricity to the
farms of Mysore, he ordered a special midweek illumination and throngs
came for the unusual event. It was difficult to imagine that Thomas E.
Dewey, then Governor of New York State, would do the same for a trou-
blesome visiting scientist. The Maharajah also made two animals available,
one of which, a work elephant at the edge of the jungle, was willing to lie
down on command of his mahout. After several attempts the elephant was
injected through its massive ear vein and the thin skin behind the ear with
a few grams of human gamma globulin which I had brought along, thinking
an antiserum to it might be of use in India. When we went for a bucket of
blood some days later, the mahout and forester in charge would only let us
draw about 5 cc, fearing we might kill the animal and the mahout would
be jobless! This. although it was customary to calm obstreperous bull ele-
phants by taking ten liters of blood. Anyhow, the 5 cc sufficed to show that
one elephant, at least, was a good producer of anti-human globulin.
Whether or not this has ever been followed up I do not know. What did
follow, however, was a practical joke played by my young Indian friend who
had been so helpful. Because of the limited success of the project, she said,
she had arranged to have three elephants sent from Mysore. Seriously
alarmed because of her previous efficiency, I was able to get the New York
Zoo in the Bronx to agree to receive them. When the three elephants
arrived, it was in a cardboard box, and they were exquisitely carved out of
rosewood!
This brings me nearly to the end of my twenty-seven happy and busy
years at P. and S. Mandatory retirement from Columbia in 1956 was
looming ahead, and for some time Selman Waksman, who had founded and
funded the Institute of Microbiology at Rutgers University with his royal-

20 HEIDELBERGER

ties from streptomycin, had been asking me to start an immunochemical
group in his institute. From 1954 on, my associate, Dr. Otto J. Plescia, and
I had been giving occasional lectures at the Institute of Microbiology.
"Retirement" to the laboratories there was humanely facilitated by Colum-
bia in 1955, at Professor Elvin Kabat's suggestion, by giving me a year's
terminal leave, with salary, so that I would not have to resign and could
receive the proud title of Emeritus Professor of Immunochemistry. As no
one wanted our specialized equipment or antigens and antibodies, we as-
sembled a truckload and with it my small staff and I transferred to New
Brunswick. Thus ends Chapter 2; within two weeks we were working again
as smoothly as if we had never moved.

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