Studies on the pneumococcus transforming substance. In the last report to the Board two phases of the work in progress on
the transforming substance were discussed. First, in an effort to provide additional evidence that the transforming substance
is itself a nucleic acid of the desoxyribose type, a study of the enzyme which depolymerizes native desoxyribonucleic acid
was undertaken. Secondly, the reversible inactivation of the transforming substance by compounds of known chemical composition
was studied with a view to obtaining evidence concerning the nature of the chemical groupings essential to its biological
activity. Both of these lines of investigation have been pursued.
Desoxyribonuclease (McCarty). The primary objective in the study of this enzyme has been to obtain it in a purified form
as free as possible from other enzymes, and to determine the action of the purified enzyme on active preparations of pneumococcal
transforming substance. As mentioned in the last report, the major obstacle to attaining this objective was the fact that
the enzyme is rapidly inactivated by proteolytic enzymes. Pancreas is the richest source of desoxyribonuclease, but large
amounts of proteolytic enzymes are present in extracts of this organ, and are carried along in fractionation procedures in
sufficient quantities so that the nuclease preparations are very unstable in solution. By application of the methods devised
by Northrop and Kunitz for the isolation of tryptic enzymes from pancreas, this difficulty has been largely overcome.
As in the procedure of Northrop and Kunitz, fresh beef pancreas is ground and extracted in the cold with 0.25 N H2SO4. Although
the tryptic enzymes are present in this acid extract, fractionation is much sharper than in the case of watery pancreatic
extracts, and separation is readily achieved. Desoxyribonuclease is precipitated from the acid extract at 0.4 saturation
with ammonium sulfate and at this concentration the tryptic enzymes are left in solution. The material obtained at 0.4 saturation
with ammonium sulfate is subjected to further salt fractionation and finally dialyzed and dried from the frozen state. The
dried enzyme is stable and highly active. Tests for the presence of other enzymes reveal that lipase, phosphatase and ribonuclease
are entirely absent, and that proteolytic enzymes are present only in very small amounts. On the other hand, the activity
of the purified enzyme in depolymerizing desoxyribonucleic acid isolated from calf thymus is extremely high. As little as
0.01 microgram of the enzyme brings about a rapid fall in the viscosity of 5.0 cc. of a suitably buffered solution of the
nucleate. This effect, as described previously in the case of crude enzyme preparations, is activated by magnesium ion and
inhibited by citrate.
The effect of purified enzyme preparations was tested on the biologically active desoxyribonucleic acid fraction obtained
from Pneumococcus Type III. When the pneumococcal nucleate is used as substrate, the enzyme in concentrations of less than
0.01 of a microgram per cc. causes not only loss of viscosity, as observed in the case of calf thymus nucleate, but also a
rapid and irreversible loss of transforming activity. It now seems certain that the biological specificity of the desoxyribonucleic
acid is an inherent property of the intact molecule in its native, highly polmerized form, and that its structural unity cannot
be broken down without loss of transforming activity. In view of the high activity, relative purity and selective specificity
of desoxyribonuclease, these results afford strong confirmatory evidence that the transforming substance is a nucleic acid
of the desoxyribose type.
A practical application has been made of the inhibition of desoxyribonuclease by citrate. In the method previously described
for preparing the transforming substance from type III pneumococci, the bacteria1 cells were heated at 65 degrees C, for 30
minutes immediately after collection. This procedure was necessary in order to inactivate the desoxyribonuclease contained
in the cells so that the transforming agent would not be enzymatically destroyed in the course of preparation. Extraction
of transforming substance from the heated cells was known to be incomplete and much active material remained in the residual
cells. It has recently been found that by lysing the pneumococcal cells with desoxycholate in the presence of 0.1 M sodium
citrate, complete dissolution of the cells may be obtained and loss of transforming substance is prevented by virtue of citrate
inhibition of the desoxyribonuclease. Work now in progress indicates that at least a five-fold greater yield of the active
desoxyribonucleic acid fraction is obtainable by this procedure.
Reversible inactivation of the transforming substance (McCarty). The inactivation of transforming substance by ascorbic acid
and prevention and reversal of this effect by sulfhydryl compounds was considered in the last report. Subsequent work has
resulted in a better understanding of the mechanism of inactivation by ascorbic acid. Catechol, hydroquinone, phenanthrene
hydroquinone, and p-phenylenediamine have been found to have an analogous action on the transforming substance. As in the
case of ascorbic acid, the inactivating effect of these compounds is catalyzed by cupric ion, and is prevented and reversed
by sulfhydryl compounds. All of the compounds mentioned undergo a similar type of autoxidation, and the evidence indicates
that inactivation of the transforming substance is linked with the autoxidation of the inactivating agent. Exclusion of oxygen
by carrying out the reaction in an evacuated Thunberg tube completely nullified the inactivating action of these compounds.
Thus, molecular oxygen is apparently necessary in the reaction.
In view of the fact that peroxides are known to be formed in the course of aerobic, copper catalyzed autoxidation of ascorbic
acid, hydroquinone, etc., the possibility arose that peroxides were responsible for the inactivating effect. That this is
apparently the case is shown by the fact that minute amounts of crystalline catalase completely protect the transforming substance
from inactivation by ascorbic acid. Furthermore, the addition of hydrogen peroxide to a solution of transforming substance
results in inactivation, but the amount required is considerably in excess of that which would be liberated in the course
of autoxidation of the minimally effective concentration of ascorbic acid. It is thus suggested that it is not hydrogen peroxide
itself, but some other form of peroxide, susceptible to catalase action, which is responsible for the oxidative inactivation
of the transforming substance.
No specific information has been obtained as to the nature of the groups in the molecule of transforming substance which are
affected in oxidative inactivation. The fact that this form of inactivation is readily reversed by sulfhydryl compounds suggests
that SH groups are present in the transforming substance, and that when these are oxidized to the S-S form, loss of activity
results. There is, however, no additional evidence to support this suggestion. On the contrary, iodoacetic acid, which has
a specific affinity for sulfhydryl groups, has no effect on the activity of the transforming substance. Attempts to demonstrate
changes in the oxidized transforming substance other than that reflected by loss of biological activity have so far been unsuccessful.
For example, preliminary studies reveal no alteration in the characteristic ultra-violet adsorption spectrum of the pneumococcal
desoxyribonucleate as the result of treatment with ascorbic acid. Furthermore, the oxidized nucleate is depolmerized by the
specific enzyme, desoxyribonuclease, at the same rate as the native substance.
It is of interest that the two types of inactivation described - that is, enzymatic depolmerization and reversible oxidation
- are not without practical significance in the techniques employed in transformation. Pneumococcal cells contain a desoxyribonuclease
which is released from the cell into the surrounding medium. Furthermore, hydrogen peroxide is formed by the pneumococcus
and can be shown to accumulate in the medium during growth under aerobic conditions. The transformation test is carried out
in the presence of actively growing pneumococcal cells, and therefore during the course of the reaction it is possible for
both desoxyribonuclease and hydrogen peroxide to appear in the medium and destroy the transforming substance before it has
had a chance to act. However, experience has shown that certain cultural conditions can be established which tend to suppress
the liberation of enzyme and peroxide into the medium during the initial phase of interaction between the transforming substance
and the rapidly growing cells. For example, small inocula of young, actively growing R cells are used, with the result that
there is much less tendency to early autolysis and consequent release of desoxyribonuclease. In addition, as a result of
the presence of R antibodies in the medium, the cells aggregate and are sedimented early in the course of the test, and subsequent
growth proceeds at the bottom of the tube under relatively reducing conditions. In this way the formation of hydrogen peroxide
may be inhibited. In the light of these facts it is interesting to observe that the cultural conditions found requisite and
empirically used in the transforming test as originally devised can now be largely explained in biochemical terms of certain
known enzymatic and metabolic processes of the pneumococcal cell. In addition, however, there still remains an unidentified
factor, present in the serum, which is also essential in the induction of transformation. The nature of the serum factor
is now under investigation.
McCarty, Maclyn, Reversible inactivation of the substance inducing transformation of pneumococcal types, (Abstract) J. Clin.
Invest., 1944, XXIII, 942.