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**New equations for redox and nano-signal transduction
**

John T. Hancock

a,

*, Radhika Desikan

a

, Steven J. Neill

a

, Andrew R. Cross

b

a

Centre for Research in Plant Science, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, UK

b

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, USA

Received 10 April 2003; accepted 4 August 2003

Abstract

Cells maintain redox potentials (E

h

) in intracellular compartments, sometimes referred to as redox environments. These potentials

are often very reducing, for example in the cytoplasm, but throughout the cell different potentials are maintained, commensurate

with the functionality of that particular part of the cell. Furthermore, within a simple cellular compartment, ‘‘hot-spots’’ of redox

poise may be maintained. However, despite this complexity, the quantiﬁcation of such redox potentials has been attempted, and

there is indeed a need to accurately assess such potentials, and to monitor how they might change with time. Changes in intracellular

potentials may control the oxidation or reduction of protein residues, such as cysteine, which would alter the conformation of those

proteins and so modulate their function. Although there are several methods for estimating the intracellular redox potential, the

most accessible technique is the measurement of intracellular concentrations of GSH and GSSG, and the calculation of E

h

using the

Nernst equation. However, using this equation shows that the E

h

imposed by the glutathione couple is dependent on the total

concentration of glutathione present, and therefore values of E

h

obtained may be erroneous. Here, we suggest new equations that

can be used to calculate the redox environments of cells.

r 2003 Published by Elsevier Ltd.

Keywords: Apoptosis; Glutathione; Nano-switches; Nernst equation; Redox potentials

1. Introduction

The Nernst equation, developed in the late 19th

Century by Walter H. Nernst, has been used for several

decades in calculations to demonstrate the thermody-

namic feasibility of electron transfer pathways. Only

recently has its use for an understanding of modulation

of protein function been appreciated, especially for its

implications for cell signalling processes. Many proteins

contain residues with side chains that can undergo redox

cycling, in which functionality is bestowed upon the

protein via the structural changes that result from

electron transfer, and not the presence of electrons

per se. Such proteins, or protein domains, usually

containing thiol groups, have been described as nano-

switches or nano-transducers (Schafer and Buettner,

2001; Cooper et al., 2002). The role of such nano-

switches can be deﬁned only if the mid-point potential

for the redox mechanism within the protein can be

determined, and if the redox state of the protein’s

environment can be measured accurately.

There are various methods of estimating the redox

environment (E

h

) in cells, including the use of micro-

injection of glutathione reductase crystals (Keese et al.,

1999), the use of engineered ﬂuorescent proteins

(Østergaard et al., 2001), or the measurement of the

concentrations of reduced and oxidized glutathione

(GSH and GSSG) in cells (Meyer et al., 2001; Ridnour

et al., 1999). These concentrations can then be used in

the Nernst equation to calculate E

h

; as discussed below.

Therefore, it is possible to determine the E

h

of cells

before and after stimulation, or during physiological

responses such as apoptosis where the intracellular E

h

has been suggested to become more oxidizing by

approximately 60 mV (Kirlin et al., 1999; Cai and Jones,

1998).

Profound changes in the concentration of reduced

glutathione have also been noted following treatments

that might induce the onset of apoptosis (Smith et al.,

2000). However, even though GSH depletion led to a

ARTICLE IN PRESS

*Corresponding author. Tel.: +44-117-344-2475; fax: +44-117-344-

2904.

E-mail addresses: john.hancock@uwe.ac.uk (J.T. Hancock),

radhika.desikan@uwe.ac.uk (R. Desikan), steven.neill@uwe.ac.uk

(S.J. Neill), scross@scripps.edu (A.R. Cross).

0022-5193/$ - see front matter r 2003 Published by Elsevier Ltd.

doi:10.1016/j.jtbi.2003.08.003

release of cytochrome c from mitochondria, apoptosis

was not an inevitable consequence (Ghibelli et al., 1999),

and therefore the exact ramiﬁcations of GSH depletion

have yet to be fully understood. Using the Nernst

equation it would be assumed that the E

h

inside the cell

will alter following glutathione depletion. However, it

might be the change in GSH concentration per se which

is critical, rather than an alteration in E

h

: Therefore,

robust equations are required to unravel the exact

nature of the role of glutathione in such processes.

Here, we derive new equations for determination of

the redox environment, which will be instrumental for

an understanding of the role of glutathione and nano-

switches in cell signalling, and will be crucial for a full

understanding of the role of redox in processes such as

apoptosis.

2. Results and discussion

A key signalling process operating in all cells involves

rapid and reversible changes in protein structure,

recognized and acted upon by the next component in a

signalling cascade. For many years phosphorylation and

dephosphorylation have been thought of as the foremost

mechanism for such rapid alterations in protein

structure, but recent suggestions also highlight changes

in the thiol status of proteins as having regulatory

importance (Cooper et al., 2002; Rhee et al., 2000;

Schafer and Buettner, 2001). A thiol group will undergo

reduction or oxidation only if its reduction mid-point

potential is appropriate to accept or donate electrons

from or to likely donors and acceptors respectively.

Therefore, the likelihood of a redox reaction of a

particular thiol group proceeding is partly due to the

three-dimensional structure of the protein and partly

determined by the solution in which the protein resides,

the latter being termed the redox environment, deﬁned

by Schafer and Buettner (2001) by the equation:

Redox environment ¼

X

E

h

½reduced species:

This is the summation of the product of the reduction

potential (E

h

) of the redox couples found in solution and

the concentration of reduced species of the redox

couples. Clearly, within any cell there will be many

redox couples contributing to this environment, many

with a very negative value for E

h

: Using the equation

above, every couple added to the sum would, in theory,

make the cell environment more negative, which clearly

can not be the case. Secondly, both the reduced and

oxidized species of couples will contribute to the E

h

imposed by the couples. Thirdly, the units derived from

the above equation do not allow easy interpretation of

the results of the calculation, being ‘‘mVmM’’ (Schafer

and Buettner, 2001). Therefore, we suggest that the

redox environment should be calculated as a mean of

the redox contributions of these couples and that the

equation should be modiﬁed to:

Redox environment ¼

P

E

h

½total concentration of couple

P

½total concentration of couple

:

Values of E

h

which are needed for the above equations

are calculated using the Nernst equation:

E

h

¼ E

m

À

RT

nF

2:303 log

½red

½ox

;

R is the gas constant (8.314 J K

À1

mol

À1

), T is the

temperature in Kelvin, F is the Faraday constant

(9.6485 Â10

4

Cmol

À1

), while n is the number of

electrons involved in the redox of the couple. E

m

is the

mid-point potential, that is, the electrical potential at

which the concentration of the reduced species and

concentration of the oxidized species are equal for any

given redox couple, and therefore the concentrations at

which E

h

¼ E

m

:

In order to calculate E

h

; a known value of E

m

is

required. Experimentally, the voltage of the solution, or

E

h

value, can be chemically modiﬁed, and the concen-

tration of reduced and oxidized species determined.

Therefore, the Nernst equation can be used to derive E

m

:

Although there are undoubtedly many couples to be

considered for the determination of the redox environ-

ment of any particular part of the cell, the one that

is used extensively is that of the glutathione couple

(Cai and Jones 1998; Kirlin et al., 1999; Schafer and

Buettner, 2001). Here the intracellular concentrations of

reduced glutathione (GSH) and oxidized glutathione

(GSSG) are measured, entered into the Nernst equation

and the value of intracellular E

h

calculated. Glutathione

exists in cells at a relatively high concentration, reported

to be 1–11 mM (Meyer et al., 2001; Smith et al., 1996),

and undergoes the redox chemistry:

GSSGþ2H

þ

þ2e

À

-2GSH:

Therefore, to calculate the contribution to the redox

environment of glutathione, the [red] function within the

Nernst equation has to be squared, giving:

E

h

¼ E

m

À

RT

nF

2:303 log

½GSH

2

½GSSG

:

Using this equation, with an E

m

value of zero, a

theoretical contribution of the reduced:oxidized ratio to

the value of E

h

can be obtained as shown in Fig. 1. It is

clear that the mid-points of the redox curves, giving a

true value for E

m

; depend on the concentration of total

glutathione. In order for the redox of the solution to

remain the same as the concentration of total glu-

tathione alters, the proportion of the GSH/GSSG in

the oxidized or reduced state would also need to alter,

following the curve shown in Fig. 1 (inset). The

proportion of GSH/GSSG for a ﬁxed redox potential

should remain the same (as the Nernst equation suggests

for most other redox couples), and not alter with

ARTICLE IN PRESS

J.T. Hancock et al. / Journal of Theoretical Biology 226 (2004) 65–68 66

concentration. Thus, in this case E

m

is not known, and

E

h

cannot be determined even if [GSH] and [GSSG] are

known. Fig. 1 shows that the dependency of E

m

on the

concentration of total glutathione is particularly pro-

nounced over the range that has been determined for

intracellular levels of glutathione, i.e., 1–11 mM (Meyer

et al., 2001; Smith et al., 1996). This suggests that in

the case of the GSH/GSSG couple the Nernst equation

returns values of E

h

which are false by a factor

of730 mV, and if E

m

can not be determined, the Nernst

equation must be modiﬁed in order to compensate for

this.

Expressing the theoretical values for E

m

over the

range 0.1–10 mM of total glutathione concentration, the

relationship shown in Fig. 2A is seen. A log

10

derivation

(Fig. 2B), shows that the change of E

m

with glutathione

concentration is linear, and the slope can now be used to

re-adjust the value of E

m

back to that predicted from

the Nernst equation. Therefore, we suggest that for the

calculation of redox environments in cells from mea-

surements of GSH:GSSG concentrations the following

equation should be used:

E

h

¼E

m

À

RT

nF

2:303 log

½GSH

2

½GSSG

þ

RT

nF

2:303 log½total glutathione;

which can be condensed to:

E

h

¼E

m

À

RT

nF

2:303

Â log

½GSH

2

½GSSG

þlog½total glutathione

and shortened further to:

E

h

¼E

m

À

RT

nF

2:303

Â log

½GSH

2

½GSSG

½total glutathione

:

The value for E

m

for the glutathione couple at pH 7

(E

m7:0

) has been taken as approximately –240 mV

(Schafer and Buettner, 2001; Kirlin et al., 1999). This

was determined using the catalysis of GSSG-reductase

(Rost and Rapoport, 1964), but was calculated at

T=40

**C, while most calculations using the Nernst
**

equation assume T=25

**C. This new derivation of the
**

Nernst equation now allows the determination of E

m

over a range of physiological temperatures and a range

of total glutathione concentrations, and can be used for

the true measurements of intracellular E

h

:

It should be noted that the calculation of E

h

should

include a factor for pH, again as a squared relationship,

although intracellular pH is not always easy to estimate.

The pH factor has been omitted here for clarity, but

needs to be added for accurate determination of the

redox environment (see Schafer and Buettner, 2001).

The accurate determination of intracellular E

h

values

in different parts of the cell and organelles is extremely

important. Only by such correct measurements can the

roles and impact of the oxidation and reduction of thiols

in the modulation of protein structures in nano-switches

be assessed. Fig. 3 shows the alteration in redox state of

both one and two electron couples, calculated using the

Nernst equation but for simpliﬁcation with an assumed

E

m

of zero. It is clear that a change of redox potential

of730 mV will have a profound effect on the ratio of

ARTICLE IN PRESS

0

10

20

30

40

50

60

70

80

90

100

-80 -70 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 70 80

mV

%

G

S

S

G

0

0 2 4 6 8 10

20

40

60

80

100

max [GSH]

%

G

S

S

G

Fig. 1. The Nernst redox curves for glutathione. The theoretical plots for the ratio of GSH/GSSG against the voltage of the solution over a range of

concentrations of maximum GSH (i.e. total glutathione in a solution), assuming that the E

m

function of the Nernst equation is zero. =10 mM;

}=8 mM; Â=6 mM; m=4 mM; ’=2 mM; E=1 mM; =0.5 mM; J=0.25 mM; n=1 mM. Inset: the alteration in GSH/GSSG ratio that

would be needed to maintain the redox of the solution as the concentration of total glutathione alters.

J.T. Hancock et al. / Journal of Theoretical Biology 226 (2004) 65–68 67

oxidized and reduced redox species and could determine

whether a nano-switch is on or off, i.e. propagating a

cellular signal or not. It has already been noted that

during apoptosis cellular E

h

becomes more positive (Cai

et al., 1998; Kirlin et al., 1999; Schafer and Buettner,

2001) and been suggested that this might be important

for the progression of apoptosis (Hancock et al., 2001).

Redox signalling may determine many cellular events,

including whether a cell lives or dies, and needs to be

studied in a robust manner.

References

Cai, J., Jones, D.P., 1998. Superoxide in apoptosis: mitochondrial

generation triggered by cytochrome c loss. J. Biol. Chem. 273,

11401–11404.

Cooper, C., Patel, R.P., Brookes, P.S., Darley-Usmar, V.M., 2002.

Nanotransducers in cellular redox signaling: modiﬁcation of thiols

by reactive oxygen and nitrogen species. Trends Biochem. Sci. 27,

489–492.

Ghibelli, L., Coppola, S., Fanelli, C., Rotilio, G., Civitareale, P.,

Scovassi, A.I., Ciriolo, M.R., 1999. Glutathione depletion causes

cytochrome c release even in the absence of cell commitment to

apoptosis. FASEB J. 13, 2031–2036.

Hancock, J.T., Desikan, R., Neill, S.J., 2001. Does the redox status of

cytochrome c act as a fail-safe mechanism in the regulation of

programmed cell death? Free Rad. Biol. Med. 31, 697–703, doi

0891-5849/01/S0891-5849(01)00646-3.

Keese, M.A., Saffrich, R., Dandekar, T., Becker, K., Schirmir, R.H.,

1999. Microinjected glutathione reuctase crystals as indicators of

the redox status in living cells. FEBS Lett. 447, 135–138.

Kirlin, W.G., Cai, J., Thompson, S.A., Diaz, D., Kavanagh, T.J.,

Jones D.P., 1999. Glutathione redox potential in response to

differentiation and enzyme inducers. Free Rad. Biol. Med. 27,

1208–1218, doi: 0891-5849/99/S0891-5849(99)00145-8.

Meyer, A.J., May, M.J., Fricker, M., 2001. Quantitative in vivo

measurement of glutathione in Arabidopsis cells. Plant J. 27, 67–78.

Østergaard, H., Henriksen, A., Hansen, F., Winther, J.R., 2001.

Shedding light on disulﬁde bond formation: engineering a redox

switch in green ﬂuorescent protein. EMBO J. 20, 5853–5862.

Rhee, S.G., Bae, Y.S., Lee S.-R., Kwon, J., 2000. Hydrogen peroxide:

a key messenger that modulates protein phosphorylation through

cysteine oxidation. Science’s Signal Transduction Knowledge

Environment, http://stkesciencemag.org/cgi/content/full/OC sigtrans;

2000/53/pe1.

Ridnour, L.A., Winters, R.A., Ercal, N., Spitz, D.R., 1999. Measure-

ment of glutathione, glutathione disulﬁde, and other thiols in

mammalian cell and tissue homogenates using high-performance

liquid chromatography separation of N-(1-pyrenyl)maleimide

derivatives. Methods Enzymol. 299, 258–267.

Rost, J., Rapoport, S., 1964. Reduction-potential of glutathione.

Nature 201, 185.

Schafer, F.Q., Buettner, G.R., 2001. Redox environment of the cell as

viewed through the redox state of the glutathione disulﬁde/

glutathione couple. Free Rad. Biol. Med. 30, 1191–1212, doi:

0891-5849/01/S0891-5849(01)00480-4.

Smith, J.D., O’Neill, A., Brady, H.R., Fitzpatrick, J.M., Watson,

R.W.G., 2000. Cellular glutathione extrusion is an integral part of

neutrophil apoptosis. FASEB J. 14, A194.

Smith, C.V., Jones, D.P., Guenthner, T.M., Lash, L.H., Lauterburg,

B.H., 1996. Contemporary issues in toxicology. Compartmentation

of glutathione: implications for the study of toxicity and disease.

Toxicol. Appl. Pharmacol. 140, 1–12.

ARTICLE IN PRESS

0

10

20

30

40

50

60

70

80

90

100

-200 -100 0 100 200

mV

%

o

x

i

d

i

s

e

d

Fig. 3. The Nernst redox curve. The theoretical plot for the

concentration of reduced species in a redox couple against the voltage

of the solution. This plot assumes E

m

=0. E: n=1; ’: n=2.

-40

-30

-20

-10

0

0 2 4 6 8 10

10

20

30

40

[GSH] mM

m

V

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

-30 -20 -10 10 20 30 0

mV

l

o

g

[

g

l

u

t

a

t

h

i

o

n

e

(

m

M

)

]

(a)

(b)

Fig. 2. The variance of glutathione E

m

. (a) Variance from 0 mV of the

calculated E

m

for glutathione against the concentration of total

glutathione (maximum concentration of GSH in the solution). (b) A

linearized log

10

plot for the variance of calculated E

m

for glutathione

against the concentration of total glutathione.

J.T. Hancock et al. / Journal of Theoretical Biology 226 (2004) 65–68 68

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