Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research,
April 15, 1944
In this report to the Rockefeller Institute in April 1944, Avery and McCarty detailed the research that culminated in the
publication of their article with Colin MacLeod in the Journal of Experimental Medicine, which revealed their discovery of
the "transforming principle" of pneumococcus. Avery and McCarty also provided a brief outline for future research
in the laboratory.
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1944-04-15 (April 15, 1944)
Scientific Report to the Corporation and the Board of Scientific Directors of the Research Institute 32 (1943-44): 131-35
Original Repository: Rockefeller Archive Center
Reproduced with permission of the Rockefeller Archive Center.
Studies on pneumococcus. The results of earlier work on the chemical nature of the substance inducing transformation of pneumococcal
types more briefly summarized in the last report to the Board. This year a paper dealing in more detail with the chemical
properties and biological specificity of the active principle was published in the February issue of the Journal of Experimental
medicine. Further work is now in progress on certain aspects of the problem, the elucidation of which, it is hoped, may provide
additional evidence in support of the belief that a nucleic acid of the desoxyribose type is the fundamental unit of the transforming
agent. In order to establish the validity of this concept and to acquire a fuller knowledge of the biochemical factors involved
in the phenomenon of transformation in vitro, the following problems are under investigation at the present time: (1) the
isolation and purification from animal tissues of the enzyme capable of depolymerizing desoxyribonucleic acid; (2) the reversible
inactivation of the transforming principle by known chemical substances.
1. Desoxyribonucleodepolymerase. In the paper referred to above it was pointed out that among various crude preparations
of enzymes tested only those that proved capable of depolymerizing authentic samples of desoxyribonucleic acid were found
to destroy the biological activity of the transforming substance. This was true irrespective of the presence of other enzymes
such as proteases, phosphatases, or esterases and regardless of whether the preparations were obtained from pneumococcal cells,
from the blood serum or organs of different mammalian species. The fact that enzyme preparations which depolymerize desoxyribonucleic
acid of animal origin also inactivate the transforming substance and the additional fact that the enzymes involved in both
of those reactions are inactivated at the same temperature and are inhibited by sodium fluoride are presumptive evidence that
the transforming principle itself is a nucleic acid of the desoxyribose type. Consequently, it seemed of prime importance
to isolate this enzyme in pure form, free from other enzymes, in order to prove that the loss of transforming activity is
actually due to a single enzyme acting specifically upon the bacterial nucleate which is believed to be identical with the
transforming substance. If under these conditions depolymerization resulted in loss of biological activity, it would prove
beyond reasonable doubt that the nucleic acid and the transforming agent are one and the same substance.
Preparations of desoxyribonucleodepolymerase extracted from commercial pancreatin and purified by fractional precipitation
with MgSO4 have been found to possess high activity in concentrations as low as 1 [mu]gm./cc. The activity of the enzyme
is measured by its capacity to reduce the viscosity of highly polymerized deoxyribonucleic acid isolated from calf thymus.
Similarly, minute amounts of enzyme protein have been shown to bring about rapid and complete inactivation of the transforming
substance. Dr. McCarty has enveloped quantitative methods for the measurement of enzyme activity.
Magnesium and manganese have been found to activate the depolymerase. The fact that Mg serves as activator is in accord with
the finding that fluoride inhibits the action of this enzyme. In the case of other enzymes which are activated by Mg, the
inhibition by fluoride is attributed to a combination of magnesium, fluoride and phosphate which removes the magnesium ions
from participation in the enzyme action. Because of the fact that citrate is known to form a soluble complex with calcium,
it seemed possible that a similar reaction might occur with magnesium and that consequently citrate might serve to inhibit
the action of the enzyme. This was found to be the case, and marked inhibition of depolymerase activity can be effected in
the presence of citrate. Similarly, the inactivating effect of depolymerase on the transforming substance is inhibited by
In the present attempts to purify the depolymerase it has not as yet been possible to free the preparation completely of all
traces of a trypsin-like enzyme. The proteolytic action of the latter results in considerable loss in activity of the depolymerase
during the preparative procedures. Methods for overcoming this difficulty and selective means for separating the depolymerase
in pure form are under investigation. Although tryptic enzymes are present, there is ample evidence that they are not responsible
for inactivation of the transforming principle, since it has been shown that neither crystalline trypsin nor chymotrypsin
has any demonstrable effect on transforming activity. Moreover, these proteases are not inhibited by fluoride or by citrate.
2. Reversible inactivation of the transforming substance. In the course of isolation and purification of the transforming
agent it was found that pneumococcal extracts treated with ascorbic acid completely lost their transforming activity. A more
detailed study of the nature of this inactivation is now being carried out using highly purified preparations of the transforming
principle. A solution containing 3 mg./cc. of active substance, of which 0.02 [mu]gm. suffices to induce transformation,
is complete1y inactivated after contact for 5 minutes with ascorbic acid in 0.01 M concentration. Traces of copper which
are known to catalyze the auto-oxidation of ascorbic acid have likewise been found to catalyze its action on the transforming
agent. A concentration of 0.001 M ascorbic acid, which by itself brings about only partial inactivation, causes complete
loss of activity in the presence of minute traces of copper.
Hopkins and Morgan have shown that reduced glutathione completely prevents the oxidation of ascorbic acid even when the latter
is catalyzed by copper ions. As little as 0.001 M concentration of glutathione has been found to protect the transforming
principle from inactivation in the presence of 0.01 M ascorbic acid. Thus, it appears that the oxidation of the ascorbic
acid is a necessary step in the inactivation of the transforming agent. More significant is the fact that transforming substance
which has been completely inactivated by ascorbic acid can be quantitatively reactivated by subsequent treatment with glutathione.
Thus, the reaction is a reversible one apparently dependent on the oxidation and reduction of biologically important groups
in the nucleic acid molecule.
A variety of other oxidants, such as ferricyanide, cytochrome C, flavine phosphate, and a-tocopherol phosphate, tested under
similar conditions, do not affect the activity of the transforming substance. On the other hand, iso- and glucosscorbic acid,
which possess little or no vitamin C activity, are just as effective in bringing about the inactivation of the transforming
substance as is ascorbic acid itself. The inactivation induced by these chemical analogues can be prevented when the reaction
is carried out in the presence of glutathione. Among other substances tested, catechol has been found to behave like ascorbic
acid in producing reversible inactivation of the transforming substance. It is noteworthy that catechol, ascorbic acid and
its derivatives all possess in common dienol groups, that their action is catalyzed by copper ions, and that their effects
can be reversed by glutathione. The loss of activity induced by these agents can be restored by the action of other sulfhydryl
compounds such as cysteine and thioglycolic acid, although under these conditions reactivation is quantitatively not so complete
as in the case of glutathione.
It is hoped that a study of the reversible inactivation of the transforming substance will afford a clue to the nature of
the chemical groupings essential to its biological activity. A survey of the agents capable of causing the inactivation and
reactivation, definition of conditions under which the reactions occur, and a search for manifestations of the change in the
molecule other than that of loss of biological activity (e.g. change in ultraviolet absorption spectrum) may lead to identification
of the particular groups involved and their relation to the biological activity of the molecule as a whole.
Avery, O. T., MacLeod, C. M., and McCarty, M., Transformation of pneumococcal types induced by a desoxyribonucleic acid fraction
isolated from Pneumococcus Type III. J. Exp. Med., 1944, 79, 137.