NATURE VOL.
224 OCTOBER 11 1969
lest I Forget Thee, &lutalhione . • •
by
E. M. KOSOWER*
N. S. KOSOWERt
Departments of Biophysics and
Biological Ultrastructure,
The Weizmann Institute of Science,
Rehovot, Israel
THE tripeptide glutathione is an almost universal con-
stituent of functioning biological systems. Originally
discovered by Rey-Pailhade 1 in 1888, glutathione (GSH)
really found a place in biochemistry through the report
of Hopkins in 1921 (ref. 2). The correct structure,
y-Glu-Cys-Gly (I), was established somewhat later•.
NH 2 CH 2SH
I I
HOOCCHCH CH CONHCHCONHCH COOH
2 2 2 and survival. We shall briefly r eview tho chemistry of the
I thiol-oxidizing agents use d in this work and describe the
The role of GSH as a cofactor for several enzymes
(glyoxalase, maleylacetoacetato isomerase) is well estab-
lished, and GSH is a catalyst in disulphide-thiol inter-
change reactions. Evidence for other roles which are
plausible on chemical grounds (for example, free radical
trapping through hydrogen atom donation, redox buffer-
ing, chelation of heavy metals) is not abundant•-•. It
has been difficult to translate chemical and biochemical
information about glutathione into firm facts for bio-
logical system,;, largely because there has been no con-
venient method for altering the GSH concentration within
cells without side effects.
We have developed a new class of thiol-oxidizing agents
which give new possibilities for the investigation of the
role of GSH within biological systems 7 • 8 • These com-
pounds, which have the general formula RN =NCOX or
XCON = NCOX, cause a rapid, stoichiometric, oxygen-
independent oxidation of GSH to the oxidized form known
as GSSG. In many conditions, the oxidation of GSH to
GSSG is effected with few obvious permanent changes to
the cell. In all cases the capacity for reversing the oxida-
tion reaction, converting GSSG back to GSH, is retained.
Because all tho agents readily penetrate the cell, the
effects of a temporary loss of GSH on the functioning of
a biological system can be evalu ated. The choice of
reagent is made according to the requirements of a
particular study.
• Permanent address: Department of Chemistry, State University of
New York. Stony Brook, New York, 11700.
t Permanent address: Department of Medicine, Albert Einstein College of
Medicine, Yeshiva University, Dronx, New York, 10461.
117
Glutathione plays many parts in living organisms, and new metho~s
for the oxidation of this tripeptide reveal some of them. This
review of the present situation ends with some suggestions for
the future.
The effects of these thiol-oxidizing agents on several
biological systems include: (1) a marked change in the
feeding behaviour of whole organisms; (2) delay and
inhibition of the growth, division and germination of
fungal spores and bacteria; and (3) changes in membrane
integrity through exposure of the cell to certain chemical
challenges.
0Ul' results suggest that glutathione is an essential
cellular constituent, absolutely required for maintenance
interesting variety of effects which they produce in
biological systems.
Chemistry of Thiel-oxidizing Agents
The overall reaction of the thiol-oxidizing agents (Table
1) with glutathione is illustrated in equation (1) with the
example of the azoester (a). The mechanism of the
reaction has been elucidated as described elsewhere•.
Azoester (a) and other esters of the series are hydrolysed
in neutral aqueous solution, yielding carboxylic acids
which a.re quite unstable and are rapidly transformed
into a class of compounds called monosubstituted diazenes,
RN=NH (equation (2)). Monosubstituted diazenes have
only recently been observed and studied10 ,n and react
either with hydroxide ion (equation (3)) or with oxygen
(equation (4)). Reaction with hydroxide ion leads to the
corresponding hydrocarbon, usually an inert and un-
reactive species. Reaction with oxygen produces free
radieals 10 ,u which present a severe chemical challenge
to biological systems through their high reactivity. The
nature of the substituent R in the monosubstituted
diazene, RN =NH, determines the relative importance of
t h e reactions with hydroxide ion or oxygen. Electron-
withdrawing substituents (for example, the 6-purinyl
group in ester c) favour r eaction with hydroxide ion, and
thiol-oxidizing agents, which form such monosubstituted
diazenes, produce few free radicals.
If the thiol-oxidizing agent is stable towards hydrolysis,
neither the corresponding hydrocarbon nor free radicals
are produced. The properties of various thiol-oxidizing
agents are summarized in T able 1.
© 1969 Nature Publishing Group
I 18 NATURE VOL. 224 OCTOBER 11 1969

2CSH + C 6H 5N=NCOOCH 3 -->-GSSG + (for example, azoester a) provide intracellular chemical

C 6 H 5NHNHCOOCH 3
C';H 5 N-NCOOCH 3 + H 2O--,.. CH 3OH +
C6 H 5N=NCOOH--,.. C\H 5N-NH
The choice of a thiol-oxidizing agent is governed by
sen•ral criteria. Agents which are readily hydrolysed
rapidly disappear from tho svstem Lo which they are
applied. The esters (a, b and c) are thuR short l,erm ·thiol-
oxidizing agents. Tho amides d and c are, in contrast,
long term thiol-oxidizing agents, and can be used to
maintain a low intracellular GSH concentration for a
considerable time. Compounds which produce free radicals
(1) challenges to the cell.
The quantity of agent used depends on tho struc-tnre
of the agent and the nature of the biological syskrn tu
+ CO 2 (2) which it is applied. "Usually about, 50 per cent of the
ester thiol-oxidizing agents aro consumed in reaction
with protein amino groups• (equation (5)). The amide
agents (d and e) react only with GSH or other equivalently
reactive thiols. Nono of the thiol-oxidizing agents ro,1cts
appreciably with protein thiol groups at tho concentra-
tions normally used•.
C 6 H 5N=NCOOCH 3 + I'XH 2 --.- C,H,N=:NCONHJ·
+ CH})H ( P =protein, ( .5)
Biological Effects of Thiol-oxidizing Agents
A significant development in neuroehemistry has bC"cn
the finding that thiol groups and disulphides arc invoked

Table 1. PROPERTI.E;-; OF 'l'HIOLMOXIDIZT~G AGE~'T'S nrn I~TUACEJ,1.rr AR r;-;E

Reactivity H Y<lro- FrrC'
Trivial towards 1,:t,i(.' r.uliral
Co1npound for;11ula name GSH* siahi- forma-
(s<'corn.18) lity ·l" tion:;:

(a) >r-N =NCOOCH,. .AzoestPr 10 ~- +

/
,.·/

/ = - '"'
):'N =NCOOCH 3 <1

'\___/'

,~N=
NCOOCH 3
/;,: N
N : ~ Adenine ester 2 7() s
(")
I II /
'\ )-N'
'\:x H

N = NCON(CH 3).
,::~
,;· /'_N
3.oun h
'I ~ AU.euinc amide :20
(d) N
~
'\N
)-N/ H

(CH 3 ),NCON =NCOX(CH 3 ), Diamide <1 :3,00U h

The intrac,;llular reaction of each reagent with GSII h:1s be~n 1:onfir:m~d
with human reU blood cells 7 • 8 • Related compounds arc readily pr~paI' ..\(1 and
are beinf( investigat.ed by J\lrW. Correa, State University of New York, St.on)'
H.rook. The correct n01nenclature for th;s::: compo11nds is: a, niethyl phon~·l-
t11azcnccarboxylate; b, methyl 4-cyanophenvldiazcnccarluxylate; r, nlf'thyl
6-pnrinyldiazenecarb')xylate; d, fi-purinyldiazenecarboxylic ai'.i<l X ,X-di-
rnethylan1ide; r, cliazcnedicarboxylic arid bis(~, ~-1lim'.~thylamid :./.
XT, Not tesfod.
* Approxirnate half-lif~ for the rn'.t.ctio11 of the com.pound with (;~H in
aqueous solution. _pH 7·4-, with both reactants at 10-a "NL (Ratt>e: at\' <·0• 1 1~
parable with that found for a 30 per cent red hloo,l cell susnension.) Tt;,,
numbers provide an indication for use of the compounds with organi$m~,,
tissue cultures. and so on.
-, Approximate half-life for hy,lrolrsis in aqueous buffer, pH,·~.
t As ,iud~~d by the formation of ·•r.1di1'.ll-typc pnducts'" fr:nn th1~
<.liaze1w10.11.

© 1969 Nature Publishing Group

NATURE VOL. 224 OCTOBER 11 1969
in t h e functioning and activity of neurones, with several
specific demonstrations available fo r acctylcholine r e-
ceptors12-18. It was thus reasonabl e to expect that thiol-
oxidizing agents would affect the behaviour of organisms .
W e have tested azoester (a) on several organisms and
h ave fou nd marked behav iour a l responses. The most
spectacular response was that of mosquitoes, which were
strongly rep eJ!ed by tho agent. It is of some interest that
r epcllency of mosquitoe,; was 0quivalcnt to that c>f t h e
b est known repellents (for example, N,N-diethyl-o- tolu-
amid<>) . Inhib ition of sucking (equivalent to feeding) was
obs( rvcd for t icks, m edicina l leech es and tsetse fli es. It,
0
was n t>cessary to pretreat t h e tsetse flies with azoester (a)
in order to produce the inhibitio n , possibly because of the
extreme rapidity with which these fli es feed. In all cases
the inhibitions wern produced in spite of tho presence of
strong feeding stimulants in the solutions 19 - 22 • All inhi-
bitions were reversible.
The behaviour of hydra has been d escribed by Lenhoff 23 •
AzoPste.r ~a) at different concentrations affects different
phases of the behaviour of hydra: nematocyst discharge,
tentacle waving and mou t h opening.
The diversity of effects produced w ith a common r e -
age n t suggests that the common elemen t in all of t h ese
behavioural respo nses is t h e intracellular oxidation of
GSH to GSSG. FurLher investigat ion is n ecessar y to
esta blish the nature of t h e link b etween the conversion
of GSH to GSSG and the actual b ehaviour. The effec ts of
.tzoester on the behaviour of organ isms are summarizecl
in T.tble 2 (unpublished r esults of R. Gatun, H. M. Lenhoff
and ourselves).
'f,1ll]e i . EFFECTS OF AZOESTER ON THE BEHAVIOUR OF ORGANISMS
Organism Species Effect
Mosqu ito A fldCR ae11YPl-i Strongly repeilcnt•
Culex mole.stus Lethal t
Tick Ornitlwdorus tho!owni Juhibits sucking
l\Iedicin:tl
leech Hiritdo medicinalis Inhibits "tasting" a nd sucking :j:
Tsetse fl y Glossina austeni Slow inhibition of sucking
Hydra, pr;eudoliyact,i s Inhibits (i) uematocyst discharge§
H ydrn
(ii) tentacle waving
(iii) mouth opening
These are results of tmpul>lisl, ctl expsri ments carried out with R. Gahm
and H. M. J,cnhoff using azoester (a).
'' Diamidc (e) h u<l only repellency.
'j' Observed during t he use of glass enclosure for standard repe!leney test on
rabbit.
t Tliarnide (e) inhibited onl y sucking.
§ E flect (i) was observed at ve ry low conc, ntrnt ions, effects (i) and (ii) at
intermediate concentrations and all effects found at high concen trations of
azoesf.e r (ll).
An a d equate ,;upply of r edu ced glutathione is apparently
n ecessary for many of tho normal activities of cells.
Oxidation of the GSH to GSSG with in t h e Escherichia coli
cell by adenine ester (c) d elays growth for about one
divi sion, after which growth r esumes at the normal rate
(unpublished results of E . Rosenberg and ourselves).
H arris 24 has reported that both DNA synthesis (as
m easured by t hymidine incorpor ation) a nd protein syn-
t h esis (by lcucine incorporation) are t emporarily dimin-
ish ed by the oxidation of about 80 per cent of the GSH
within Ehrlich ascit,es cells w ith azoester (a) . We h ave
found (unpublished results of T. Zehavi , D. D a non and
ourseh •es) t hat protein synt,h esis a nd the r eplacement of
p olysom es by monosonrns wit hin ra bbit rcticuloeytes are
corr elated with GSH content, by the use of diamide (e).
Treatment of t,he spores of Trichoderma viride with
© 1969 Nature Publishing Group
119
azoester (a) severely r e,,._.:ds germination if carried out
at tho midpoint of the germination process, hut has much
l ess effect at its beginning o r a t its end (unpublished
r esults of D. Weinman, E. Galtm and ourselves). It is
known that the germination of N euro8pora cra8sa. is
accompan ied by an increase in the GSH content 25 • Signi-
ficant in this connexion is th e r ecent discovery of highly
p oten t ant ifungal agents (j and g} 2 " with structures v ery
simila r to several of tho t liiol-oxidizing agents listed in
T ab le 1.
xc.H,N=NC(=S)NH. xo.H,N=NC(=SO)NH.
f g
An intimate connexion b et ween cellular t.hiol content
and cell division has long been postul.tted 27 • Our result,;
suggest several reactions through which cellular thiol
cont ent might be relat,od to growth and division, but
by no means exhaust tho possibilities . It seems clear that
GSH is vital to the mainten a n ce of numy processes in
cells.
The integrity of the coll m embran e is protected b y
glutathione against chemica l ch a llen ges or oxidizing
agen ts. \Ve have obtained a beautiful illnstr ation of t hi s
point by using azoester (a) to generate free radical s n ear
t he membrane of cell s. Application of azoester (a), in
exce,:;s of that r equired to oxidize a ll the intracellul ar
GSH to CSSG, to human r ed b lood cells saturated with
carbon monoxide and suspended in a ir-satnrated buffer
leads to severe haemolysis.
The formation of fre e radicals from azoester proceeds
through hydrolysis and reaction with oxygen (equations
(2) and (4)). In a 0011 loaded w ith oxygen , such as the
oxygenated hum an rod blood cell. free radicals a r c form ed
within the cell and cause damagt• throughout the cell .
The damage is v isible in the form of H e inz bodies a nd is
not accompanied by lysis.
Treatment of a carbon monox ide -l oaded cell with excPss
azoester in the absence of oxygen .in t h e medium docs not
h ave any observable dfoet: neither Heinz bodies n or
lysis arc seon. If the carbon monoxide -loaded cell is
suspend ed in an air-saturated buffer so that an oxygen
con cen t ration gradient is set up at the membrane, free
r ad ical formation in the r egion of the membrane causes
d a mage which eventually leads to lysis.
Most, cells are oxygen-poor. Treatm ent of many different
cell types with azoester (a) , in excess of t h at reqnired to
oxidize a ll the GSH to GSSG, lead s to lysis. Among t h e
cell types examined eith er in su spension or in tissue
c ulture are human red blood cells". human epithelia l
can cer (KB) eeJ!s (unpublished r esults of G. Yagi! a nd
ourselves), Escherichia coli, human lymphocytes (un-
published results of S. Pade and ourselves), human
myeJobJasts (unpublished results of D. Plutznick and
ourselves), Ehrlich aseites tumour cells 2 4 • 28 and norma l
and glucose-6-phosphat,o d ehydrogenase -deficient fibro-
blasts (unpublished results of M . S iniscaleo and ourselves ).
The response of these cell,; is t h er efore analogous t o t h at
of the r ed blood cell loaded w ith carbon monoxide in
oxygenated m edium .
Lysis does not occur unless most of t h e GSH w ithin the
cell is oxidized to GSSG and a ch emical challenge is presen t
during t he time when the con centration of GSH is low ·
Thus treatment, of any of th e cell s we h ave listed with
quantities of azoester (a) sufficient to oxidize only 50 - 70
per cent of tho intracellular GSH has little observable
effect. Use ofthiol-oxidizing agents like adenine ester (c)
120
adenine amide (d) or diamide (e) (which do not generate
free radicals), oven in excess, causes no lysis.
We can infer that an adequate concentration of GSH
is necessary for the cell to withstand certain chemical
challenges.
Possible Applications
It seems likely that the concentration of GSH within
cells plays somfl part in the regulation of protein synthesis.
The accumulation of GSH in the cytoplasm of Yoshida
ascites tumour strains sensitive to the alkylating agent,
chlorambucil, after treatment with the agent, is consistent
,1,ith this oonclusion 29 • It is conceivable that an undflr-
standing of the role of GSH in the complex reaction
svstem which comprises protein synthesis might lead to
;n improvement in tho treatment of or in tho design of
drugs for cancer.
It is probable that chemoreception is affected by tho
intracellular concentration of GSH, but it is not yet clear
,d1ether or not this involves only the reception process
("detection"), neurone action (neurones to chemoreceptors
being perhaps more exposed to the chemical environment
than others), or both. When thiol-containing drugs are
given to man, taste acuity is abolished or decreased, and
30
1t can be returned to normal by divalent metal ions
One might hope that new insights into the behavioural
response of insects might be gained through the use of
intracellular thiol-oxidizing agents, and that new com-
pounds could be produced for the control of insects.
A common clinical problem in medicine is the occurrence
of haemolytic anaemias after administration of drugs.
This is especially frequent among those individuals who
suffer from inherited deficiencies in glucose-6-phosphate
rlehydrogenase (G6PD), and is exacerbated by the fact
that mild anaemia is already present in many cases• 1 . A
deficiency in G6PD diminishes tho intracellular supply of
)JADPH, tho reducing agent required by glutatluone
disulphide reductaso for the conversion of GSSG to G~H.
In normal conditions, red blood cells of G6PD-deficient
individuals are removed from circulation more rapidly
than the red blood cells which contain the enzyme.
Production of new cells does not quite match the demand
c:md a mild anaemia results. A diminished or negligible
c,tpacity for the regeneration of GSH is the o_nly important
consequence of the deficiency, and cells with the defect
do not survive as long as normal reel blood cells, and so
chemical challenges must arise in a natural way from
soluble components of the blood. These challenges slowly
erode the integrity of the red blood cell me1'.'1brane and
e,-entually cause it to be recognized as defective. .
If an extra challenge is posed to a system whwh 1s
already in precarious balance, the whole arrangement
collapses and massive haemolysis results. The best ~nown
example of such effects occurring on a large scale 1s m _the
preventive dosing with primaquine which was used agamst
malaria during tho Korean War (1950-52) 32 • ~he_ drug,
nr one of its metabolites, oxidizes the GSH w1thm the
red blood cell. We believe that the resulting haemolysis
arises from irreversible damage to the membrane caused
hy the drug or one of its metabolites in the absenc,, of
protective GSH. The measures which can be proposed on
i:he basis of this picture for the effect of primaquine are
rwo-fold. First,, to determine quantitatively the capacity
nf the red blood cells to regenerate GSH" and regula~e
, he drug dose so that the supply of intracellular ~SH _18
ne,-er severely lowered. Second, it might be possible m
some cases to change the molecular nature of the drug so
© 1969 Nature Publishing Group
NATURE VOL. 224 OCTOBER 1 1 1 969
that it does not offer a chemical challenge to the reel blood
cell.
The problem is by no means trivial. It has IJcen
estimated that about 3 per cent of the population of the
world is deficient in G6PD 33 • And malaria is still ,-,1a, of
t,he most widespread public health problems.
If a sublethal chronic chemical challonge is posed by tho
normal environment to red blood cells with the conseq1wnt
reduction in their life span, might llot a similar clmllenge
be made to all other cells ? lf so, glutathione is om· , ,ft ho
chief lines of defence against such challenges and thPrefure,
we may say, against one possible cornponent. of agc·ing.
We might also speculate on the importance of gluta-
thione in biological evolution. Its presence in all cells
leads readily to the idea that GSH was an early entry
into the compendium of moleculPs that constitute ln·ing
matter. It is too early to guess whether or not its appear-
ance was in the prebiotic or biotic periods. We hope that
the use of specific thiol-oxiclizing agents will enahle a
more quantitative evaluation of the function of gluta-
thione in biological systems than has been possible so far.
Much of our work was carried out in collaboration 1,·ith
several different research groups, whom we thank for
their cooperation. We are grateful for the facilities
afforded to us by the departments of biophysics (Profr,c;sor
E. Katchalski) and biological ultrastructurn (Profossor
D. D<lnon). N. S. K. is t,he recipient of a car,wr df'\:,•lop-
nrnnt award of the Nat.ional Institutes of Health. l'.S
Public Health Service. E. M. K. was a National Sc-i,•nc,·
Foundation senior postdoctoral follow.
1
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