STUDIES ON OXIDATION AND REDUCTION BY
          PNEUMOCOCCUS.

V. THE DESTRUCTION OF OXYHEMOGLOBIN BY STERILE EXTRACTS
             OF PNEUMOCOCCUS.

BY JAMES M. NEILL, PH.D., AND OSWALD T. AVERY, M.D.
(From the Hospital oj The Rockefeller Institute for Medical Research.)

(Received for publication, December 12, 1923.)

In the fust of this series of papers suspensions of living pneumococci, which
during growth had been protected from the action of air by anaerobic cultivation,
were shown to possess the property of uniting with molecular oxygen to form
peroxide when exposed to air under conditions unfavorable for cell multiplication
(1). The three succeeding papers have dealt with the formation of peroxide (2),
the reduction of methylene blue (3), and the oxidation of hemotoxin (4) by sterile
extracts of pneumococci under conditions excluding the participation of living or
intact cells in these oxidative reactions.

The present paper is concerned with another activity of reduced
extracts of pneumococci: the destruction of oxyhemoglobin and the
formation of methemoglobin.  The principal object of this study is
the attempt to obtain more knowledge concerning the mechanism of
the destruction of oxyhemoglobin by pneumococcus, and to correlate,
if possible, the nature of this phenomenon with the other known
oxidation-reduction activities of pneumococcus extracts.

EXPERIMENTAL.

Methods.

Bacteriological.-The sterile extracts of pneumococci were prepared by the
methods described in a preceding paper (2).  Sterile broth extracts of unwashed
pneumococci were used in the following experiments unless otherwise stated.  All
of the extracts used were proved sterile by cultural and animal tests.  These ex-
tracts were prepared from anaerobically grown pneumococci and during their
preparation were protected from the action of air by being kept under a heavy
Vaseline seal. Extracts which have been stored in sealed tubes protected from air
are referred to in the text as reduced extracts.

757


7.58    OXIDATION AND REDUCTION BY PNEUMOCOCCUS. V

Chemical.-The crystalline hemoglobin was prepared from horse blood by
Heidelberger's method (5). The hemoglobin crystals were dissolved in 0.1 M
phosphate solution (pH 7.5) and the solutions passed through Berkefeld filters.
The oxyhemoglobin solutions from laked washed red cells were prepared without
crystallization from sterile blood. The spectroscopic examinations were made
with, a prism spectroscope. The oxyhemoglobin analyses were made by the
oxygen capacity method of Van Slyke (6), with the apparatus and technique
described by Van Slyke and Neil1 (7). In determining the "total hemoglobin"
the calorimetric method of Stadie (8) was used.

Destruction of Oxyhemoglobin by Steerile Extracts of Pneumococci.

Although a few investigators have demonstrated the production of methemo-
globin by sterile extracts and culture tiltrates of pneumococci, others have been
unable to obtain this reaction in the absence of the living organism.  The literature
on this subject has been reviewed by Butterfield and Peabody (9), Cole (lo),
Stadie (Ii), Schnabel (12)) and others.

In the present study, sterile reduced extracts of unwashed pneu-
mococci prepared as described, have been found to induce marked
and rapid pigment changes when added to blood or to hemoglobin
solutions.  Methemoglobin production is evident in 30 minutes in
tests in which 0.05 cc. of the extract is used. The greenish, dis-
colored areas formed by pneumococci growing on blood agar plates
can also be produced in the complete absence of living cells by placing
a small portion of the sterile extract in a depression cut in a fresh
blood agar plate.

Quantitative measurements show that the amount of oxyhemo-
globin destroyed by these sterile extracts is quite significant. The
addition of 0.5 cc. of the extract which has not previously been
exposed to air, to 5 cc. of rabbit or human blood, causes a destruction
of between 200 and 300 mg. of oxyhemoglobin within a few hours
at 37oC.

Destruction of Oxyhemoglobin to Products beyond Methemoglobin.

The intensity of the reaction and the marked pigment changes
induced by increasing amounts of active extract indicated that in
many instances the destructive process may yield degradation pro-
ducts still lower than methemoglobin. To obtain quantitative evi-
dence of this action, measurements were made of the "total hemo-


JAMES hf. NEILL AND OSWALD T. AVERY       7.59

globin" (oxyhemoglobin, hemoglobin, and methemoglobin) in solui
tions to which large and small amounts of active extract had been
added.

The data obtained in a number of experiments furnished quantita-
tive evidence that sterile extracts of pneumococci, if present in high
concentration, not only convert oxyhemoglobin to methemoglobin,
but destroy these blood pigments to still lower degradation products.
In low concentration the action of these same extracts is limited to
the conversion of oxyhemoglobin to methemoglobin.

Oxyhemoglobin Destruction by Hydrogen Peroxide in the Presertce and

Absence of Catalase.

Hydrogen peroxide is a chemical agent which is known to effect many oxidations
in much the same manner as do living cells. iis the destruction of oxyhemoglobin
by sterile pneumococcus extract is in all probability an oxidation reaction, it
seemed of interest to compare the effect on hemoglobin of the reaction brought
about by this chemical agent with that induced by the sterile bacterial preparation.
This comparison acquires especial interest from the fact that a peroxide, having
the reactions of hydrogen peroxide, is known to accumulate in media during the
aerobic growth of pneumococcus. However, the following experiments are intro-
duced here, not to emphasize the importance of hydrogen peroxide as such, but
rather to compare the action of an active extract with that of a known oxidizing
agent in the presence and absence of blood catalase.

InJluence of Catalase in Protecting Oxyhemoglobin from the Action of Hydrogen
Peroxide.-The crystalline hemoglobin used in these experiments was almost
entirely devoid of catalase. On the other hand, the solutions of hemoglobin pre-
pared directly from I'aked corpuscles were rich in blood catalase.  From these two
sources, therefore, it was possible to prepare solutions of equal hemoglobin con-
centration, which differed widely in their content of blood catalase. In previous
experiments the presence or absence of catalase was found to have little or no effect
on the oxyhemoglobin-destroying power of the reduced extract of pneumococcus,
since practically identical results were obtained with solutions of oxyhemoglobin
from l&cd cells containing abundant catalase and with solutions of crystalline
hemoglobin containing only traces of catalase. Quite different results, hovvever,   .
might be expected if preformed hydrogen peroxide ("Dioxogen") were added
directly to these two ty-pos of hemoglobin, since catalase is known to destroy
hydrogen peroxide.

Experiments were made to contrast the hemoglobin-sparing action
of catalase in the presence of hydrogen peroxide with the lack of this
protection in the presence of reduced extract of pneumococcus; and


760    OXIDATION AND REDUCTION BY PNEUMOCOCCUS. V

to compare the type of change in oxyhemoglobin brought about by
the action of hydrogen peroxide with that induced by pneumococcus
extract.  Because of lack of space the detailed protocols of these
experiments are omitted.

As might be expected, the presence of catalase greatly inhibits
the'action of hydrogen peroxide upon hemoglobin.  It was found that
hemoglobin, prepared from the crystalline product containing only
traces of catalase, was entirely destroyed to colorless products by
amounts of hydrogen peroxide which failed to produce any detectable
change in solutions of the same concentration of hemoglobin having
abundant catalase.  The influence of catalase in protecting oxyhemo-
globin from the action of hydrogen peroxide is in striking contrast
to the almost complete indifference to catalase of the oxyhemoglobin-
destroying power of reduced extracts of pneumococcus. However,
in spite of this marked protective action of catalase, large amounts
of hydrogen peroxide can destroy small amounts of hemoglobin with
the production of methemoglobin.

Comparing the type of change in blood pigment effected by this
chemical agent with that induced by bacterial extracts, reactions of
a quite similar nature were found to occur in both instances. In-
creasingly large amounts of pneumococcus extracts caused an in-
creasing and progressive degradation of hemoglobin to products
beyond methemoglobin.  Similar relations have been found to hold
true for hydrogen peroxide.  Moreover, in the presence of an excess
of preformed hydrogen peroxide, the successive phases through which
the reaction proceeds simulate most closely those brought about
by the action of the bacterial extract upon hemoglobin.

  These relations may be interpreted as evidence that the active
substance involved in hemoglobin destruction by pneumococcus
extract is an oxidizing agent which induces the same general type of
reaction as does hydrogen peroxide. However, in the presence of
blood catalase, very large amounts of preformed hydrogen peroxide
are required; while the hemoglobin-destroying agent in pneumococcus
extract is apparently indifferent to the action of catalase.


JAMES M. NEILL AND OSWALD T. AVERY       761

Inj?uence of Heat on the Hemoglobirt-Destroying Activity of Sterile
        Extracts of Pneumococcus.

The peroxide-forming and methylene blue-reducing activity of a
sterile broth extract of unwashed pneumococci is completely de-
stroyed by heating the extract at 65oC. for 10 minutes (3). In a
number of experiments the heat stability of the methemoglobin-form-
ing function of pneumococcus extract has been tested and com-
pared with the heat sensitiveness of the system responsible for the
formation of peroxide in the same extract.

If an extract is heated at a constant temperature (55oC.) for grad-
ually increasing periods of time, from 10 to 60 minutes, there results
a gradual and progressive loss in what may be termed the total
oxidizing and reducing power of the extract; less peroxide is formed,
methylene blue is more slowly and less completely decolorized, and
the rate and degree of hemoglobin destruction decrease.  Moreover,
not only is the activity of these three functions destroyed at the
same rate at 55oC. but the destruction of each shows the same marked
acceleration between 60" and 65oC. These relationships, together
with the nature of the processes induced, indicate that these prop-
erties are functions of the same or closely related systems in the
extract.

Activation of the Hemoglobin-Destroying Power of Sterile Extracts of
            Washed Pneumococci.

In the experiments thus far recorded in this paper, sterile broth extracts of
unwashed pneumococci have been used exclusively. The results recorded are
sufficient evidence of the marked hemoglobin-destroying activity of extracts of this
type. It will be recalled that extracts of washed cells in phosphate solutions have
been found to contain potential but incomplete oxidation-reduction systems,
and that extracts of this type form peroxide (2) and reduce methylene blue (3)
only if they are activated or completed by the addition of cell washings, meat
infusion, or yeast extract. In view of the close relationships which seem td exist
between these various functions, it might be expected that hemoglobin destruction
by washed cell extracts would also be dependent upon the presence of these com-
plementing substances.


762     OXIDATION AND REDUCTION BY PNEUYOCOCCUS. V

Analysis of Table I reveals the interesting fact that an extract
of washed pneumococci is unable by itself to destroy hemoglobin,
and that this same extract when activated or completed by the
addition of certain complementary substances rapidly converts the
oxyhemoglobin to methemoglobin.  It is evident that yeast extract
and muscle infusion complete some otherwise deficient system in
the washed cell extract and thus activate the function upon which
oxyhemoglobin destruction depends.  These relations are analogous
to the completion in the same extracts of the peroxide-forming and
methylene blue-reducing systems.  In an earlier study on the for-

TABLE I.

Activation of the Oxyhemoglobin-Destroying Power oj Sterile Extracts oj Washed

Salin;;s\t;ct of  PO4 solution. I !
                   Meat infusion.   Yeast extract.
pneumococci.

cc.

0.6
0.6
0.6
-

cc.

0.6
-

-
-

-

`C.

-

0.6
-

0.6
-

cc.
-

-

0.6
-

0.6

-

          HbOz destruction
HbO? solution.    and MetHb
          formation after
           2 hrs. at 37oC.

cc.

0.6         -

0.6        +
0.6        +
0.6         -

0.6         -

mation of methemoglobin by pneumococcus extracts Avery and
Cullen' found that an acetone-ether extract of muscle infusion or
serum contained substances which served to activate the methemo-
globin-forming system of washed bacterial extracts. Although in
the present study no attempt has been made to identify further the
nature of these substances, it is possible that they may be related
to the acetone-ether soluble substances which Meyerhof (13) has
shown to be important in the autoxidation processes of tissue cells.
However, it is not unlikely that meat infusion broth and yeast ex-
tract contain other types of autoxidizable substances.

Wnpublished observation.


JAMES M. NEILL AND OSWALD T. AVERY       763

Comparison of the Amount of Oxyhemoglobin Destroyed by Hydrogen
   Peroxide a,nd by Reduced and Oxidized Extracts of
               Pneumococcus.

Throughout these studies the extracts used have been prepared from anaerobi-
cally grown cultures of pneumococci and care has been taken to exclude air during
the processes of extraction. Sterile broth extracts of unwashed cells prepared
under these precautions and preserved under seal are referred to as reduced
extracts.  Whenever a reduced extract of this type is exposed to air, oxidation
products are formed which in turn react upon other constituents of the extract.
Extracts in which these changes have been induced by exposure to air are spoken
of as oxidized.  In preceding papers (3, 4) marked differences have been shown
to e.xist in the activity of the same extract in the reduced and oxidized form.

It seemed of interest to determine the effect of oxidation upon the
hemoglobin-destroying activity of an extract. For purposes of
comparison, the amount of oxyhemoglobin destroyed by an excess of
preformed hydrogen peroxide is contrasted with the amount of
blood destruction induced by the pneumococcus extract.  This com-
parison is pertinent since peroxide is formed whenever an extract
passes from the reduced to the oxidized form.  Moreover, it furnishes
quantitative proof of the fact that while blood catalase seriously
impairs the action of hydrogen peroxide on hemoglobin, it exerts
little or no effect on the hemoglobin-destroying activity of reduced
extracts of pneumococcus.

Sterile broth extract of pneumococcus which had been preserved in the reduced
form was divided; an aliquot portion of the reduced extract was exposed to the
air at 20oC. for 7 hours. This oxidized extract contained demonstrable amounts
of peroxide, which had formed during the process of oxidation. The hydrogen
peroxide consisted of titrated dilutions of `LDioxogen" in concentration of 1.12 Y
and 0.28 M solutions.  Sterile, freshly defibrinated rabbit blood in which the red
cells were hemolyzed by freezing and thawing was added in 2 cc. amounts to six
tubes; to each of the first two, 3 cc. of a known dilution of hydrogen peroxide
were added; in the next two, 0.4 cc. of reduced and oxidized extract was placed
respectively.  The last two served as controls.  In each instance the total volume
was made up to 6 cc. by the addition of broth, phosphate solution, or water as
required. All tubes were incubated at 37oC. for 6 hours, and then the oxyhemo-
globin content of the series was determined. One of the control tubes containing
diluted blood was held at 2'C. until the end of the experiment as a control of the


764    OXIDATION AND REDUCTION BY PNEUMOCOCCUS. V

initial HbO,. The loss of .HbOS in the control during incubation at 37oC. was
subtracted from the total loss of HbO, in all samples. The results are given in
Table II.

In terms of the amount of hemoglobin destroyed, Table II pre-
sents two striking contrasts; first, the disparity of action between
reduced pneumococcus extract and preformed hydrogen peroxide,
and second, the extreme difference in the activity of the reduced
and oxidized extract. The &rst contrast is strikingly brought out
by assuming, for the purposes of comparison, that the hydrogen
peroxide formed during oxidation of the reduced extract is actually
concerned per se in the destruction of the blood pigment. If the

TABLE II.

Comparison of the Amount of Oxyhemoglobin Destroyed by Hydrogen Peroxide and
      by Reduced and Oxidized Extracts of Pneumococcus.

sterile
defibrinated
rabbit blood.

cc.

2
2
2
2

2 I -

Pneumococcus extract.

Reduced.

OSiidized.

cc.         cc.        f?dhO&       w.
-         -       0.015       25
-         -       0.008       14
0.4         -       0.072       121
-       0.4       0.003       5
-         -          -        -

production of one molecule of Hz02 requires the utilization of one
molecule of O,, then it would be necessary for 0.4 cc. of the reduced
bacterial extract to utilize 75,300 c.mm. of oxygen to produce 3.36
millimols of hydrogen peroxide, an amount equal to that added in
the experiment. However, the addition of this quantity of hydrogen
peroxide resulted in the destruction of only 2.5 mg. of oxyhemoglobin.
On the other hand, 0.4 cc. of the reduced extract destroyed 121 mg.
of hemoglobin, approximately five times as much as that destroyed
by 3.36 millimols of hydrogen peroxide.  These figures do not exclude
the possibility that hydrogen peroxide as such may function as the
active agent in the destruction of hemoglobin by pneumococcus.
However, if oxyhemoglobin destruction under these conditions is
attributable to the action of the hydrogen peroxide formed by the


JAMES M. NEILL AND OSWALD T. AVERY       765

extract, then these figures furnish a striking example of the greater
activity of this agent at the moment of its formation than when
subsequently added to the system in a preformed state.

The second striking contrast revealed in Table II is the extraor-
dinarily marked difference in activity exhibited by the same extract
in the reduced and oxidized form.  The reduced extract in amounts
of 0.4 cc. destroyed 121 mg. of hemoglobin, while a like quantity of
the oxidized extract destroyed only 5 mg., an amount so slight that
it may be ignored, being not more than twice the experimental error
involved in the analytical technique.

When a reduced extract of pneumococcus undergoes oxidation, its
hemoglobin-destroying activity is rapidly lost. After 30 minutes
aeration the extract contains demonstrable amounts of peroxide and
shows a certain decrease in the rate of methemoglobin formation
when added to blood. The loss in activity of the methemoglobin-
forming system is progressive with increasing oxidation until at the
end of 2 to 3 hours exposure to air, complete and final loss of the
methemoglobin-forming power of the extract has occurred.  During
oxidation, peroxide accumulates in the extract but the peroxide
formed in the oxidized extract if subsequently added to blood is
unable by itself to effect any change in the pigment in the presence
of blood catalase.

The destruction of hemoglobin, with the resulting formation of
methemoglobin by sterile extracts of pneumococcus, is apparently
a function of the oxidation-reduction systems contained in the ex-
tracts. An extract in which oxidation has occurred is subsequently
incapable of convert&g oxyhemoglobin to methemoglobin.

On the other hand, an extract in which the active substances are
protected from oxidation retains its potency for months, even if
stored at 37oC. These facts indicate that methemoglobin formation
is a process which accompanies and is dependent upon the initial
oxidation of some constituent of the extract.  Moreover, in the for-
mation of methemoglobin, it is necessary that the oxyhemoglobin
be present in the system during the oxidation of the extract; if an
extract is completely oxidized before the addition of oxyhemoglobin
the reaction does not occur in the presence of catalase.


766     OXIDATION AND REDUCTION BY PNEUMOCOCCUS. V

Comparison of the Action of Reduced and Oxidized Extract of Pneumo-
     coccus on Oxyhemoglobin in the Presence and
         Absence of Blood Catalase.

In a preceding experiment it was shown that when hydrogen peroxide (reagent)
is added to oxyhemoglobin, the pigment changes which result are determined by
and dependent upon the balance between the concentration of the chemical agent
and the amount and activity of the blood catalase present.  In solutions of crys-
talline oxyhemoglobin containing little or no catalase, methemoglobin was rapidly
formed by amounts of hydrogen peroxide which produced no change in catalase-
containing solutions of red cells. In the latter instance the catalase in decomposing
hydrogen peroxide served to protect the oxyhemoglobin, while in the former case
the absence of catalase exposed the blood pigment to the direct action of hydrogen
peroxide itself. On the other hand,, reduced pneumococcus extract effects the
transformation of oxyhemoglobin to methemoglobin in the presence as well as in
the absence of catalase. This difference of behavior suggests that the mechanism
of the latter reaction is more complex than the simple interaction between hydro-
gen peroxide and oxyhemoglobin, This assumption would be further strengthened
if it could be shown that the peroxide which accumulates as a final product of the
oxidation of pneumococcus extract were quite as readily destroyed by the catalase
of the blood as is hydrogen peroxide when added directly as a chemical reagent.
In order to test the validity of this point the following experiment was carried out.

Oxyhemoglobin containing only faint traces of catalase was obtained by pre-
paring 0.4 mnr solutions of the crystalline product in phosphate solution (pH 7.5).
Oxyhemoglobin containing abundant catalase was obtained by the use of defi-
brinated blood undiluted or diluted tenfold in phosphate solution. -4 portion
of the same extract as that used in the reduced state was exposed to air for 12 hours
at 20oC. The oxidized extract at the end of this period and before oxyhemoglobin
was added, gave a strong peroxide reaction.  Portions of the oxidized and reduced
extract were heated 10 minutes at 75oC. to inactivate the labile constituents of the
extract. The oxidized extract after heating still gave a marked peroxide reaction.
0.1 cc. portions of heated and unheated extract, both reduced and oxidized, were
added to 0.9 cc. of the catalase-free and catalase-rich solutions of oxyhemoglobin
in the order presented in Table III. Spectroscopic examinations were made after
4 hours at 37oC.

This experiment shows that when reduced extract is added to
defibrinated blood methemoglobin is formed in spite of the presence
of catalase.  Furthermore, the same pneumococcus extract if allowed
to oxidize with the consequent production of hydrogen peroxide fails
to induce methemoglobin formation when subsequently added to
blood because of the capacity of the catalase present to destroy this


JAMES M. NEILL AND OSWALD T. AVERY       767

agent.  That the hydrogen peroxide formed in the oxidizedextract
is active in the absence of catalase is easily shown by the facility
with which it destroys crystalline hemoglobin.  Moreover, to insure
against the participation in this reaction of any active agent other
than the hydrogen peroxide, the oxidized extract was completely
inactivated by exposure to 75oC. for 10 minutes, without any effect
upon its action on crystalline hemoglobin.  Oxidized extract in which
the final products of oxidation have accumulated behaves toward
oxyhemoglobin, under these circumstances, precisely like a dilute

TABLE III.

Comparison of the Action of Reduced and Oxidized Extract of Pneumococcus trpon
    Oxyhemoglobi?t in the Presence amd 11 bsence of Blood Catalase.

Pneumococcus extract.

         LMethemoglobin formation.
Presence of
peroxide in   Oxyhemoglobin
extYact before ,frc,m ,a&, ,,,&   Solutions of
adding HbG.            crystalline
         (abundant   oxyhemoglobin
          Cat&SC).    (no catalase).

Reduced.

Unheated.
Heated 10 min. at 75oC.

Oxidized.

Unheated.
Heated 10 min. at 75oC.

solution of hydrogen peroxide; that is, it is unable to form methemo-
globin in the presence of blood catalase and is no more heat-labile
than is hydrogen peroxide itself.

Obviously, catalase can protect hemoglobin against the action of
oxidized extract containing the final product, H,O,; but it offers no
protection when the hemoglobin is present in the system during the
oxidation of the extract. A probable explanation of the difference
in activity under these two sets of circumstances is that during the
process of oxidation of a pneumococcus extract certain intermediary
products are formed which like hydrogen peroxide are strong oxidiz-
ing agents, but unlike this substance are not destroyed by catalase.
If hemoglobin is present during the formation of these intermediary
bodies, it is in turn oxidized by these substances.  Presumably when
the process of oxidation of pneumococcus extract has been completed,


768   OXIDAiION AND REDUCTION BY PNEUMOCOCCUS. V

these higher peroxides are converted into simple hydrogen peroxide
the action of which, when subsequently added to oxyhemoglobin,
is conditioned by the presence or absence of blood catalase.

InJluence of Hydrogen Peroxide on the Oxyhemoglobin-Destroying

    8     Activity of Pneumococcus Extract.
When a reduced extract of pneumococcus is exposed to the air it

suffers a rapid and progressive loss of its ability to form methemoglo-
bin. During this oxidation hydrogen peroxide is formed and it
was previously suggested that part of the loss of activity may be due
to the destructive action of this oxidation product upon the extract.
Evidence of the probable importance of the accumulated peroxide
as a factor in the destruction of the methemoglobin-forming system
in oxidized extracts has been furnished by a number of quantitative
experiments. Treatment of active extracts with reagent hydrogen
peroxide resulted in a complete loss of ability of the treated extract
to destroy oxyhemoglobin.

InfEuence of the Oxygen Tension upon Methemoglobin Formation by
       Sterile Extracts of Pneumococci.

In an earlier investigation (1914) Cole (10) observed that the degree of oxygena-
tion of the blood was an important factor in the formation of methemoglobin by
living pneumococci. To obtain further evidence of the influence of the degree of
oxygenation of hemoglobin upon methemoglobin formation it seemed desirable
to repeat Cole's experiments with sterile extracts instead of living bacteria.  In
the following experiment, therefore, a sterile pneumococcus extract was added to
and allowed to act upon hemoglobin in various degrees of oxygenation.

4.5 cc. of sterile defibrinated rabbit blood and 0.5 cc. of 0.1 M phosphate solution
(pH 7.5) were placed in each of three tonometers of 300 cc. capacity.  One of these
samples of blood was then deoxygenated by evacuation and by preliminary satura-
tion with hydrogen. Another was saturated with oxygen at 20 mm. tension and
                                                -.
the third was saturated with oxygen at 710 mm.  As a control 5 cc. of blood simi-
larly diluted to which phosphate solution instead of extract was added served
as control at atmospheric tension.  After the preliminary saturations, sterile
reduced extract in amounts of 0.8 cc. was added to each specimen of blood, pre-
cautions being taken to prevent any oxidation of the extract or any change in the
composition of the gas during the manipulation.

The tonometers containing mixtures of sterile pneumococcus extract and of
blood at different degrees of oxygenation, were then rotated in a water bath at


JAMES hi. NEILL AND OSWALD T. AVERY       769

38oC. for `2 hours. The formation of methemoglobin in the different samples was
then determined by measurements of the loss of oxygen capacity of the blood..
The results are given in Table IV.

Table IV shows that when a mixture of reduced extract of pneu-
mococcus and deoxygenated blood was exposed to an oxygen ten-
sion of less than 3 mm., little or no methemoglobin was formed.  The
actual figures in the table are not more than twice the experimental
error and indicate that if the preliminary deoxygenation had been
complete the hemoglobin would have suffered no change under

TABLE IV.

Injluence of Oxygen Tension and Degree of Oxygenation of Hemoglobin upon the
Formation of Methemoglobin by Sterile Extracts of Pneumococcus.

Oxygen tension in
gas phase.

Degree of oxygenation of hemoglobin
(Ratio __-
   Hb:kOn).

         I

j
Action of reduced extract ofpneumococcu.

Hb

HbO,

Hemoglobin content   Hemoglobin
after 2 brs. at 37%
(oxygen capacity).  destroyed (methe-
           moglobin formed).

mm:
  0
20
710
Control.

per cent'
100
50
  0

* Approximate figures.

per ccni*         ??I%       mg. in 5.8 tt.
  0         5.35         15
50         3.90        168
100         5.24         27
           5.50

these conditions.  When the reaction was carried out at high oxygen
tensions, 710 mm., that is under conditions such that the concen-
tration of deoxygenated hemoglobin was practically negligible, only
a very small amount of methemoglobin was formed.  At an oxygen
tension of 20 mm., both oxyhemoglobin and deoxygenated hemoglobin
were present in approximately equal amounts. When the extract
acted upon blood under these conditions significant amounts of
methemoglobin were formed as shown by the marked decrease in
oxygen capacity. This oxygen tension was chosen at random and
although serving the purpose of the experiment it may not represent
the optimum for the reaction.

From the results of this experiment it may be concluded that the
methemoglobin-forming reaction of pneumococcus is inftuenced by


770   0XID;ATION AND REDUCTION BY PNEUMOCOCCUS. V

oxygen tension in two ways.  Thus, while the presence of oxygen is
necessary for the formation of the oxidizing agent which oxidized
hemoglobin to methemoglobin, the reaction does not proceed to
advantage at high oxygen tensions which exclude the presence of
hemoglobin in the deoxygenated form. As previously observed by
Cole (lo), the formation of methemoglobin by pneumococcus ap-
parently involves a preliminary deoxygenation of hemoglobin. The
present study suggests that this process of deoxygenation may have

TABLE V.

Oxygen Consumed in the Destruction of Oxyhemoglobin by Pneumococcus Extract.

Control.
Extract.

    Oxygen capacity.               Oxygen content.

0        1 hr.       2 hn.       0       1 hr.      2 hrs.

rnY       rnM       mar       ?nM      ?nM       mat
6.5       6.5      6.5      6.5      6.4      6.3
6.5       5.1      4.5      6.5      3.2      1.5

Hemoglobin destroyed by extract.        Oxygen consumed by extract.

0       1.4       2.0

0       3.2      4.8

-

a twofold function; it affords a source of oxygen to the peroxide-
forming system of the cell and in turn it leaves the hemoglobin in a
form more readily oxidized to methemoglobin by the peroxide thus
formed.

The Co?~sumption of Oxygen during the Destruction of Oxyhemoglobin
        by Sterile Pneumococcus Extract.

From the results of the preceding experiments it was inferred that
the oxygen united with some constituent of the extract to form an
oxidizing agent which oxidized deoxygenated hemoglobin to methe-
moglobin.  In view of these relations, experiments were conducted
to correlate the amount of oxygen consumed with the amount of
hemoglobin destroyed.

From the results given in Table V it appears that during the reac-
tion between pneumococcus extract and oxygenated blood, the
molecular amount of oxygen consumed is greater than that of hemo-


JAMES M. NJZILL AND OSWALD T. AVERY       771

globin destroyed.  From the previous experiment it was inferred
that deoxygenated hemoglobin represents an intermediary product
in the formation of methemoglobin. Although it is frequently im-
possible to demonstrate the presence of intermediary substances in
biological oxidations, it is interesting to observe that deoxygenated
hemoglobin accumulates in significant concentrations during this
reaction, at least in systems in which oxyhemoglobin is the source
of molecular oxygen.

DISCUSSION.

Sterile broth extracts of unwashed pneumococci actively destroy
oxyhemoglobin with the resulting formation of methemoglobin.
The rate and degree of destruction are largely dependent upon the
concentration of the reagents used.  Larger amounts of extract not
only transform oxyhemoglobin to methemoglobin but may destroy
the blood pigment to products beyond methemoglobin.  This process
seems to be a function of the same system or systems responsible for
the formation of peroxide and the reduction of methylene blue by
pneumococcus extract (3, 4).  This fact is further evidenced by the
heat sensitiveness of the mechanism involved, for all three of these
activities are destroyed when an extract is heated at 65oC.  More-
over, an extract prepared from washed pneumococci, that is the
"incomplete" type of cell extract which by itself exhibits none of these
reactions, may be activated in each instance by the addition of the
same substances, meat infusion or yeast extract.

As already pointed out in the first paper of this series (I), the
peroxide-forming activity varies with different strains of pneumo-
cocci.  These differences are reflected in the activity of the bacterial
extracts not only in the peroxide-forming function, but also in the
other oxidation-reduction processes described (24), including the
hemoglobin destruction discussed in this paper. The fact that the
sterile extracts possessing the most marked peroxide-forming activity
cause the most marked destruction of hemoglobin may be interpreted'
as further evidence that the same system is involved in these two
functions of the extracts.

In the destruction of oxyhemoglobin, as in the oxidation of pneu-
mococcus hemotoxin by sterile extracts, the type of reaction induced


772    OXIDATION AND REDUCTION BY PNEUMOCOCCUS. V

represents one in whicha substance, not itself easily oxidized by molec-
ular oxygen, is oxidized through the agency of some product formed
by the union of molecular oxygen with the reactive substance in the
extract.  It may be recalled that an active pneumococcus extract
prepared from unwashed bacterial cells, consists of two factors, each
of which is separably inactive.  One of these factors represents
certain constituents of the bacterial cell which are heat-labile and
are present in saline extracts of washed pneumococci.  Extracts of
this type are "incomplete" and non-reactive with molecular oxygen.
The other constituents which are essential to the complete oxidation-
reduction system may be furnished from sources other than those
of bacterial origin.  Meat infusion and yeast extract contain thermo-
stable substances which, although themselves inactive, serve to com-
plement the potentially active but otherwise deficient system of the
washed cell. With these distinctions in mind, the process involving
the change of oxyhemoglobin by an active reduced extract of pneu-
mococcus may be schematically represented as consisting of the
following intermediary reactions.  If C be allowed to represent the
thermolabile cell constituents including the bacterial enzymes and RH
the easily oxidizable thermostable substances, then the active system
in the broth extract in the reduced state would consist of [C + RH];
and

ROOH + Hh -+ MetHb + other oxidation products.

The first intermediary step involves the deoxygenation of the
oxyhemoglobin.2 That a source of oxygen in the system is essential

2It is equally in keeping with the experimental data to interpret the methemo-

globin-forming reaction in a manner somewhat more analogous to Conant's
interpretation of the action of potassium ferricyanide on hemoglobin solutions.
Reduced hemoglobin, oxyhemoglobin, and molecular oxygen are in equilibrium
in the system. Upon the addition of reduced pneumococcus extract the molecular
oxygen unites with constituents of the extract. The oxidizing agent thus formed
oxidizes reduced hemoglobin to methemoglobin. This reaction, by removing
either (or both) reduced hemoglobin or molecular oxygen, causes more oxyhemo-
globin to dissociate until there are established the final equilibria between the
oxidizing agent, reduced hemoglobin, and methemoglobin, and between oxyhemo-
globin, reduced hemoglobin, and molecular oxygen.


JAMES hf. NEILL AND OSWALD T. AVERY       773

for the initiation of the reactions is clearly shown by the fact that
reduced extract in the presence of reduced hemoglobin does not form
methemoglobin.  The oxygen furnished by the oxyhemoglobin unites
with the reactive substance of the extract to form a peroxide (ROOH).
This peroxide in turn oxidizes the reduced hemoglobin to methe-
moglobin.  The necessity of oxygen for the initiation of the reaction
and the demonstration of hydrogen peroxide in extracts which under
the influence of ordinary oxygen have changed from the reduced to
the oxidized form, suggest that hydrogen peroxide as such may be
the active agent in the oxidation of hemoglobin. However, the
fact that hydrogen peroxide may be detected as an end-product in
oxidized pneumococcus extract does not necessarily imply that other
active, but transient products may not be formed in the earlier stages
of the reaction.  Indeed, there are reasons to believe that the actual
agent in the oxidation of hemoglobin under these conditions may be
a higher, organic peroxide, a precursor of hydrogen peroxide. One
reason for this view is the fact that if blood is present during the
oxidation of a reduced extract, the active substance that is formed
is wholly unaffected by blood catalase, transforming hemoglobin in
the presence as well as in the absence of this enzyme.  On the other
hand, if pneumococcus extract is completely oxidized before it is
added to oxyhemoglobin, methemoglobin is formed only in the ab-
sence of catalase.  In this respect an oxidized extract behaves toward
hemoglobin merely as does a dilute solution of hydrogen peroxide.
These differences are brought out in those experiments in which the
comparative action of a reduced and oxidized extract on oxyhemo-
globin was studied in the presence and absence of catalase.

The conception that methemoglobin formation by pneumococcus
involves the preliminary deoxygenation of oxyhemoglobin was sug-
gested by Cole (10).  The present study supports this interpretation
and is in accord with the recently published evidence (Conant (14))
that the transformation of hemoglobin to methemoglobin is a true
oxidation in the electronic sense, while the conversion of hemoglobin
to oxyhemoglobin is merely an oxygenation.  Conant interprets the
transformation of oxyhemoglobin to methemoglobin by the action
of ferricyanide as a reaction involving the oxidation of previously


774    OXIDATION AND REDUCTION BY PNEUMOCOCCUS. V

deoxygenated hemoglobin.  The actual oxidation process, which
gives methemoglobin, Conant believes to consist in the oxidation of
the ferrous iron of deoxygenated hemoglobin to the ferric form.  This
reaction may be brought about by a number of oxidizing agents.
The present study suggests that the oxidizing agent in the case of
pneumococcus is a peroxide of bacterial origin.

From the experimental evidence available, it is inferred that in
the formation of methemoglobin by pneumococcus, the initial reaction
involves the deoxygenation of oxyhemoglobin and the union of the
free oxygen with some autoxidizable substance of the cell and that a
peroxide thus formed serves as the actual oxidizing agent in the
conversion of the hemoglobin to methemoglobin.

SUXMARY.

1. Sterile broth extracts of unwashed pneumococci actively de-
stroy hemoglobin to methemoglobin and lower degradation products.
2. Sterile saline extracts of washed pneumococci do not by them-
selves form methemoglobin; extracts of this type may be completed
or activated by the addition of certain complementary substances
such as meat infusion and yeast extract.
3. The hemoglobin-destroying activity of pneumococcus extract is
lost by exposure to 65oC. for 10 minutes.
4. The properties of an extract upon which these blood changes
depend are related to other known oxidation-reduction functions of
the same extract.

5, Oxyhemoglobin is converted to methemoglobin only by cell
extracts in the reduced form; completely oxidized extracts are inactive
in the presence of blood.  The action of hydrogen peroxide and the
influence of blood catalase on these reactions are discussed.
6. During the reaction between oxyhemoglobin and pneumococcus
extract oxygen is consumed.

7. The mechanism of methemoglobin formation by pneumococcus
is interpreted as an oxidation process in which deoxygenation of
oxyhemoglobin and peroxide formation occur as intermediary reac-
tions. The active agent of the oxidation of hemoglobin is considered
to be a peroxide of bacterial origin.


JAMES i-if. NEILT, AND OSWALD T. AVERY       775

BInLIOCRhPI-IY.

1. Avery, 0. T., and Neill, J. M., J. fix-p. Med.,, 1924, xxxix, 347.
2. Avery, 0. `I'., and Ncill, J. Al., J. Em). Med., 1924, xxxix, 3.57.
3. Avery, 0. `I'., and Neill, J. RI., J. Evp. MUI., 1924, xxx&, 543.
4. Avery, 0. T.? and Ncill, J. fir., .T. 13.~~. Afc~i., 1924, xxxix, 745.
5. Ilcitlc~llxrgcr, nr., .I. Ijto/. C'h~m., 1922, liii, 31.
6. `I'm Slykc, II. I)., J. IJiol. Chcut., 19lS, xsxiii, 127.
7. Van Slykc, D. D., and Ncill, J. A/f ., J. Biok. Chcm., 1924 (in press).
8. Staclic, Ii. C.; J. Biol. Chcv~, 1920, xii, 237.
9. Butterfield, E. E., and I'cnbody, F. W., J. Jkp. Med., 1913, xvii, 587.
10. Cole, R., J. Esp. Med., 1914, xx, 363.
11. Stadic, IV. C., J. E.I$. AT&., 1921, sxxiii, 627.
12. Schnnbcl, A., %. Ilyg. ,I(. I~~~ctior~sI:rtr~rl:i~., 1921) sciii, 175.
13. Mcycrhof, 0.) 7'11~ I/uIw>~ .IA~o~rs, 1923.
14. Connnt, J. T3., J. Biol. Chcnr., 1923, Ivii, 401.
1.5. RL'lACOd, J. I\`., ancl C:ordon, J., J. Pc1111. md hcl., 1922, xsv, 139.