STUDIES ON THE SPONTANEOUS OXIDATION OF

CYSTEINE
III. THE METHOD OF ACTION OF CYANIDES AND CYSTINE ON
CYSTEINE OXIDATION

BY E. G. GERWE*
(From the Department of Biochemistry, Medical College, University of
Cincinnati, Cincinnati)

(Received for publication, May 27, 1931)

INTRODUCTION

Bernard (l), in 1857, investigated the physiological effect of

cyanide and the succeeding contribution of Gaehtgens (2), Gep-
pert (3), and many others indicate that in some way or other
exposure of living organisms to adequate concentrations of cyanide
is followed by a reduction in the O2 consumption and COZ pro-
duction. Cyanide is therefore considered as a respiratory poison.
That cyanides and nitriles inhibit the spontaneous oxidation of
cysteine solutions was discovered by Mathews and Walker (4)
who considered the possibility that it inhibited by uniting with
iron in the solutions, but discarded this possibility in favor of a
direct union of cyanide with cysteine since they believed that their
preparations were free from iron, Warburg (5), Wieland (6), and
Thunberg (7) make extensive use of this cyanide depression action
on cysteine oxidation in support of their theories of biological
oxidation-reduction.
Warburg believes that the cyanides inhibit respiration by
combining with the iron which, in combination with cysteine,
he believes to be the respiratory enzyme responsible for cellular
oxidations. He concludes that intracellular iron combines in
some unknown chemical way with the cyanide.
Voegtlin (8), opposing this theory, states that Warburg failed
to perform the crucial experiment; i. e., he did not demonstrate
* William S. Merrell Fellow in Biochemistry, 1928-30.
525

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 Cysteine Oxidation. III
that the inhibition of respiration produced by threshold concen-
trations of cyanide can be overcome by equivalent amounts of
iron salts. He found that previous injections of iron salts into
rats failed to antagonize minimum lethal doses of sodium cyanide.
He did note, however, that cystine, cysteine, or glutathione did
possess such action and, when injected in the proportion of 5 parts
of the sulfhydryl compound to 1 part of cyanide, the animal was
protected.
From these results he concluded that the inhibiting action of
the cyanides on respiration is due not to a conversion of intracel-
lular iron to an inactive complex substance but rather to a disturb-
ance in the equilibrium between reduced and oxidized sulfur
compounds as represented in the following relation.
RSSR ti 2RSH

He believed, as has previously been suggested by Professor

Mathews, that the toxic action of cyanides is due to chemical
reaction which takes place between the cyanide and the cystine
sulfur of tissues. Making use of polarimetric measurements on a
solution of cystine and varying amounts of cyanide, he found that
in such solutions the cystine was reduced to cysteine as indicated
by specific rotations of small negative values. Then on aeration
the specific rotation again reached a high negative figure which
indicated complete reversion to cystine. Hence, he assumed
that the reaction taking place is that of reduction of cystine to
cysteine and the subsequent conversion to the oxidized form by
aeration. The possibility that NaCN is oxidized to NaCNS is
practically ruled out, according to his evidence, as such a change
would involve the splitting off of S from the cystine. The aeration
experiments indicate that the sulfur is not split off.
He concluded that the cyanide is converted into the cyanate
according to the following equation,
R-S-S-R + Hz0 + NaCN + NaCNO + 2RSH

which does not involve the participation of molecular 02 and is

therefore applicable to anaerobic oxidation.
However, much evidence is supplied by Bodansky and Levy (9)
and others confirming much earlier work to show that cyanides
 E. G. Gerwe 527

and nitriles are converted into the thiocyanates within the body.
The former (10) has shown recently that in alkaline solutions
cystine converts cyanide into the thiocyanate in vitro to the extent
of as much as 60 per cent, but in neutral solution the reaction
occurs only to a very slight extent. Testing such solutions by a
special method he failed to detect the slightest amount of NaCNO.
It was also shown that the oral administration of KCN produced
a marked increase of the thiocyanate content of the saliva, which
he believed argues for the physiological conversion of cyanides
into thiocyanate.
Using the procedure of Mauthner (ll), he isolated from alkaline
cyanide-cystine mixtures a substance which on analysis proved to
be a-amino-fi-sulfocyanopropionic acid. This result suggests that
at least a portion of the thiocyanate is in organic combination as
the compound mentioned above. This conversion is expressed by
R-S-S-R + KCN + RSK + RSCN

thus confirming Pulewka and Winzer (12) who report that in

the reaction between cystine and cyanide a molecule of cysteine
is formed for each molecule of cystine, as determined by iodometric
titration.
Dixon and Elliot (13), attempting to determine the extent of
cyanide inhibition on the oxygen uptake of various tissues, found
the average maximum inhibition reached to be about 60 per cent.
In no case was the respiration completely inhibited by cyanides
but in some tissues and especially in yeast, the inhibition reached
90 per cent of the normal rate of oxygen uptake. In the majority
of cases 0.001 M concentration of cyanide was sufficient to produce
the maximum degree of inhibition obtainable.
From these results they conclude that the respiration of animal
tissues is made up of two parts. One, accounting for about two-
thirds of the total, is due to systems poisoned by cyanides, the
other one-third is due to systems which are stable to cyanides.
The same authors believe that cyanide-stable systems such as the
xanthine oxidase may contribute to the cyanide-stable part of
respiration. However, xanthine oxidase cannot account for the
whole of this part, since Morgan has shown that it is absolutely
absent from muscle, yet it is likely that there are a number of
other systems of this type.
528 Cysteine Oxidation. III
EXPERIMENTAL

Although it has been shown that cyanide inhibits cysteine oxida-

tion in much the same manner as it does tissue oxidation, yet in no
instance during the experiments of the author has the inhibition
been complete. Even though it has been shown by Sakuma (14),
Harrison (15), and the author (16) that pure iron-free cysteine
oxidizes only with extreme slowness when compared to that
catalyzed by metals, still no proof exists to show that cysteine is
not autoxidizable. Hence, it is very likely that cysteine itself
may be a component of the cyanide-stable system.
Harrison, who obtained oxidation rates for his pure cysteine
hydrochloride as low as 2.32 c.mm. of O2 per hour per 10 mg. of
RSH.HCl, was able to inhibit oxidation only to the extent of a
1.86 c.mm. of 02 per hour uptake in one case, and a 2.94 c.mm.of
O2 per hour uptake in another. In both experiments cyanide was
present in 0.001 M concentration.
Since cyanide is such a powerful inhibitor of iron catalysis it
would certainly seem reasonable to believe that if the residual
oxidation were due to iron, then cyanide should completely inhibit
oxidation. If there were present in a 10 mg. sample of cysteine
HCl about 0.000044 mg. of Fe, as was shown in a previous paper
(12) to be the amount necessary to account for this residual oxida-
tion if we are to assume that iron is the only active substance,
then if the solution contained cyanide to the extent of 0.001 M
concentration, there would be present l,OOO,OOO molecules of
cyanide for every molecule of iron.
A series of experiments was carried out to determine whether
cyanide could depress the oxidation rate of a specially purified
cysteine hydrochloride prepared according to a method described
by the author (17), by means of which cysteine hydrochloride
was obtained which contained less than 1 part of iron in 20
million parts of the cysteine hydrochloride. The lack of inhibiting
action in the pure cysteine is shown in Table I which gives definite
proof that cyanides do not inhibit the oxidation of cysteine when
completely freed from iron.
That the oxygen uptake in my preparation could not be due to
the oxidation of HCN to HCNO by atmospheric oxidation was
ruled out by the results of a previous experiment in which it was
shown that a pure 0.01 M HCN solution took up absolutely no
 E. G. Gerwe
measurable quantity of O2 when measured in the Barcroft appara-
tus under identical conditions.
In order to show that the cyanide would inhibit the oxidation
if it were due to iron, experiments were performed in which oxida-

TABLE I
Effect of Cyanide on Oxidation of Cysteine
-
T
02 uptake of 10 HCN added to 0% uptake after
PH Temperature mg. RSH . HCl make final con- oyanide addition
per hr. centration
-.
“C. e.mm. M c.m?n.
7.4 22 3.2 0.001 2.8
7.6 22.5 3.3 0.605 2.4
7.4 24.0 2.8 0.04 3.0
7.6 23.0 0.005 2.2
7.6 22.5 0.01 2.3
7.4 24 0.001 2.0
7.6 25 0.01 3.1
-
Average uptake. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
-
It has been shown in a previouspaper that pure iron-free cysteine oxi-
dizes at an average rate of about 2.2 c.mm. of oxygen per hour per 10 mg.
sample (16). Oxidation was measured by means of a Barcroft micro respi-
rometer.
TABLE II
Effect of Cyanides on Iron Catalysis

ori&haleo2 Fe added as F&la cat$py$; O2 HCN added to Cya~dc$&iited

make solution 2

e.mm. ml. e.mm. .4f e.wwn.

2.5 0.0610 57.0 0.01 1.9
3.2 0.0010 50.3 0.01 3.0
1.9 0.0005 21.2 0.001 2.2

The uptake rates are expressed in terms of c.mm. of oxygen per hour
per 10 mg. sample of cysteine hydrochloride.

tion rates were determined for the pure substance. Iron was then
added and the increased oxygen uptake noted. When cyanide is
added to these catalyzed oxidations the rates are inhibited only
to the extent of catalysis by the added iron. In other words, the
oxidation now goeson at the original rate. It may be noted from
 Cysteine Oxidation. III

Table II that the inhibited rates are almost identical to oxidation

rates before the iron addition.
That the cyanide in solution is relatively stable for a fairly
long period of time was shown in an experiment in which the
original uptake was 1.8 cmm. of O2 per hour per 10 mg. of cysteine
hydrochloride. Enough HCN solution was then added to make
the final concentration 0.001 M. The oxidation continued at
approximately the same rate for 10 hours, after which 0.001 mg.
of Fe was added and readings again taken. The added iron failed
to increase the oxidation showing that the added iron was imme-
diately converted into an iron-cyanide complex in which form it
could not act catalytically.
In conclusion, pure cysteine oxidizes at approximately the same
rate in the presence of varying amounts of cyanide as in its
absence. Cyanides do not inhibit completely the oxidation even
in the presence of iron and reduce the rate of uptake only to the
extent of that catalyzed by iron. Cysteine, catalyzed by iron
and oxidizing at a high rate, takes up oxygen only at a rate equal
to that of cysteine alone after cyanide is added. From these
experiments it is evident that cyanide acts by convertingironinto
a non-catalytic form and exerts no influence on pure cysteine.
Action of Cyanides on Iron Solutions
Since iron in a cysteine solution is so rapidly converted into a
complex compound in which form it no longer exerts any catalytic
effect, a study was made of the nature of the formation of a com-
plex iron-cyanide compound. When KCN solution is added to
a ferrous ammonium sulfate solution a dirty blueprecipitateforms
which on standing settles to the bottom of the flask. This consists
of ferrous hydroxide which partially oxidizes to the ferric state.
The supernatant solution on neutralization and the subsequent
addition of ferric chloride gives a heavy blue precipitate of the
well known Prussian blue, indicating the formation of K4Fe(CN)6.
When ferric chloride is added to KCN there is formed a heavy
brown precipitate consisting of ferric hydroxide. The yellow
supernatant liquid when tested with a ferrous salt gives the typical
Turnbull’s blue which confirms the presence of KDFe(CN)&.
However, when a ferric salt instead is added to this same solution
a heavy blue precipitate of Prussian blue is formed. This means
 E. G. Gerwe 531

that in addition to K3Fe(CN)6 there is also KdFe(CN)e pres-

ent. It is very improbable that KCN can reduce KaFe(CN)G to
K4Fe(CN)6. The formation of the K4Fe(CN)a is no doubt due to
an interaction between the cyanide and some Fe(OH)z formed as a
result of a reduction of the Fe(OH), by KCN according to the
following equation.
2Fe(OH)3 + KCN + 2Fe(OH)z + KCNO + Hz0

Then the Fe(OH)z combines with KCN to form K4Fe(CN)s

according to the reactions,
Fe(OH), + 2KCN -+ Fe(CN)2 + 2KOH
Fe(CN)2 + 4KCN -+ KdFe(CN)a

From the results of these reactions, which take place also at

neutral reaction, it is quite evident that this complex iron-cyanide
compound which Warburg assumes to be formed may in reality
be either K4Fe(CN)6 or a combination of K4Fe(CN)G and cysteine.
If the iron is assumed to be bound up in this manner then it must
be shown that iron added in the form of the complex cyanide does
not catalyze cysteine oxidation.
When 0.001 mg. of iron is added to cysteine in the form of either
K3Fe(CN)G or &Fe(CN)6 the oxidation rate is only slightly greater
than that of cysteine alone averaging 5.6 c.mm. of 02 per hour
whereas cysteine oxidizes at the average rate of 2.2 c.mm. of 02
per hour. An equivalent amount of iron added in ionic form as
FeCh caused an increased rate of 50 to 60 c.cm. of 02 per hour.
The slight increase over the oxidation of cysteine alone may be
accounted for by the traces of free iron which the complex cyanide
contained. It is quite evident from these experiments that the
iron in the complex cyanide cannot function as a catalyst.
If ferrous or ferric salt is added to a neutral cysteine solution
containing KCN, K4Fe(CN)B is formed as detected by the forma-
tion of Prussian blue when a small amount of ferric salt is added.
When ferric salt is added to a neutral cysteine solution it is imme-
diately reduced to the ferrous state. A consideration of the ease
with which iron is converted into the complex cyanide on the
addition of KCN both in the presence and absence of cysteine,
points to the possibility that this reaction may represent the
inhibiting action of cyanides on iron catalysis. The inability
532 Cysteine Oxidation. III

of K4Fe(CN)6 and K3Fe(CN)6 to act catalytically on the cysteine

oxidation lends confirmation to this possibility.

E$ect of Cystine on the Autoxiclation of Cysteine

As had been mentioned in a previous section Abderhalden and
Wertheimer (18) observed a definite inhibiting action of cyanides
on the oxidation of their samples of cysteine hydrochloride which
they believed to be iron-free. They attributed this inhibiting
effect to the action of cyanides on the cystine rather than the
action on any trace of iron which might have been present. Dixon
and Tunnicliffe (19) claim that cystine greatly accelerates the
oxidation of cysteine by atmospheric oxygen, and that the maxi-
mum reactivity is reached when a ratio of 2.5 parts of the disulfide
to 1 part of cysteine exists. Hence, Abderhalden and Wertheimer
attribute the slight oxidizability of the cysteine to a contamination
of cystine which they believed to be present in their samples.
Since cyanide is able to reduce cystine to cysteine, the inhibition
of the oxidation of cysteine by cyanide is due, according to them,
to the removal of the catalytic cystine thus,
R-S-S-R + KCN + Hz0 + 2RSH + KCNO

However, no such action of cystine was observed in experiments

carried out by the author. Pure iron-free cystine hydrochloride
when added to pure cysteine hydrochloride failed to increase the
oxidation rates. An increased oxidation rate due to the addition
of cystine could be explained only by assuming that the added
cystine contained traces of iron. If cystine had such catalytic
powers, then, in every experiment in which measurements were
takenover long periods of time, one would observe a steady increase
in oxidation rates as the conversion of cysteine to cystine pro-
ceeded, the rate reaching a maximum when 60 per cent of the
cysteine had been oxidized, when, according to Dixon and Tunni-
cliffe, 2.5 parts of cystine would be present forevery part of cysteine.
Instead of an increase in rate as would be the case if cystine
were acting catalytically, one really observes a rather constant
rate of oxidation and when the oxygen uptake is plotted against
time the curve is linear until near the end of the oxidation when all
of the cysteine has been converted into cystine.
 E. G. Gerwe 533
I wish to express my gratitude for the helpful criticism and
interest shown by Professor A. P. Mathews.
SUMMARY

1. The autoxidation of iron-free cystine is not inhibited by

cyanides.
2. The addition of iron greatly accelerates the oxidation of pure
cysteine but subsequent addition of cyanide to the catalyzed oxi-
dation reduces its rate to the original value.
3. Potassium cyanide readily converts ferric or ferrous chloride
to the corresponding potassium iron-cyanide compounds. Cya-
nides inhibit iron catalysis in this manner.
4. Iron added in the form of potassium ferrocyanide or potassium
ferricyanide does not accelerate oxidation.
5. Pure iron-free cystine does not catalyze cysteine oxidation,
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