The catalysed NADH reduction of resazurin to resorufin

Article abstract of DOI:10.1039/a806431h

Details are reported on the mechanism whereby NADH can be used for the reduction of resazurin 1 to give the fluorescent product resorufin 2, a process requiring the use of a catalyst, such as N-methylphenazinium methosulfate 3.

Full text of DOI:10.1039/a806431h

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The catalysed NADH reduction of resazurin to resorufin
a
b
b
c
Luis P. Candeias, Donald P. S. MacFarlane, Sean L. W. McWhinnie, Nicola L. Maidwell,
c
c
b
Carl A. Roeschlaub, Peter G. Sammes* and Rachel Whittlesey
a
Gray Laboratory Cancer Research Trust, Mount Vernon Hospital, Northwood, Middlesex,
UK HA6 2JR
Chemistry Department, Brunel University, Uxbridge, Middlesex, UK UB8 3PH
Molecular Probes Unit, Chemistry Department, School of Physical Sciences,
University of Surrey, Guildford, Surrey, UK GU2 5XH
b
c
Received (in Cambridge) 14th August 1998, Accepted 28th September 1998
Details are reported on the mechanism whereby NADH
can be used for the reduction of resazurin 1 to give the
fluorescent product resorufin 2, a process requiring the use
of a catalyst, such as N-methylphenazinium methosulfate
occurring at pH 6.5. The overall reduction reaction is shown in
eqn. (1).
ϩ
ϩ
NADH ϩ 1 ϩ H → NAD ϩ 2 ϩ H O
(1)
2
3
.
catalyst
The mechanism of this catalysis has been studied. When
NADH and NAD(PH) are important biochemical vectors and
measurement of intracellular turnover of these reagents is often
used to indicate cellular viability and activity. NAD(P)H con-
ϩ
NADH and PMS 3 are mixed, a rapid reaction occurs in
ϩ
1
which NADH disappears, to form NAD , with concomitant
formation of the unstable hydrophenazine derivative 4. Since
centrations can be monitored by direct fluorescence but, since it
6
ϩ
NADH mainly acts as a two-electron reductant, it is assumed
is rapidly turned over [re-oxidised to NAD(P) ], absolute levels
that, in this reaction, the hydrophenazine 4 is formed directly.
In our studies, no sign of any long-lived intermediates such as
and 6 (Scheme 1) could be observed in the reaction between
do not necessarily reflect the level of enzymic and metabolic
activity occurring within the cell. As a consequence there is
considerable interest in using reagents that themselves are
selectively reduced by NAD(P)H with the formation of either a
coloured or fluorescent signal. Few dye precursors react directly
with NAD(P)H and, in order to overcome this problem, many
5
N
N
N
e
2
chemists have shown that certain enzymes, such as diaphorase,
+
N
can act as electron carriers in order to effect reduction. Thus,
the reduction of tetrazolium dyes, such as iodonitrotetrazo-
lium blue, by NADH is catalysed by diaphorase, to form the
intensely violet formazan dye. However the product formazans
3
CH SO –
5
3
4
NADH
H+
3
are not fluorescent. Use of the dye resazurin 1, which, when
H
N
H
pure, is only weakly fluorescent, has also been made and dia-
N
+
phorase also catalyses the reduction of this dye by NADH to
4
the strongly fluorescent product, resorufin, 2.
e
N
N
O–
4
6
N
N
+
Scheme 1
–O
Na+
O
O
–O
Na+
O
O
3
and NADH. At neutral pH, compound 4 is only sparingly
2
soluble in water but, although it can be isolated by extraction
into dichloromethane, the product is unstable to air, highly
1
H
N
7
coloured products forming. The hydrophenazine can also
be formed by careful reduction of PMS with sodium boro-
N
ϩ
hydride. Although the hydrophenazine 4 is formally ‘anti-
aromatic’ and expected to be a good reducing agent, by itself
it was found not to reduce resazurin, even after several hours, so
it is not acting as the catalytic species. Another, phenazinium-
derived, species must therefore be responsible for the catalytic
action.
+
N
N
MeOSO3-
3
4
The details of the mechanism whereby the enzyme diaphorase
acts as a catalyst do not seem to have been investigated. We
report here some progress with the use of the organic catalyst
N-methylphenazinium methosulfate (PMS , 3) as an altern-
ative to the use of enzymes for catalyzing the reduction of res-
azurin. PMS has been widely used as an electron transfer agent
in a range of enzyme assays and potentiometric titrations.
Pulse radiolysis studies were carried out in order to gain
some insight into the overall reduction processes. Radiolysis of
aqueous sodium formate solutions (0.1 M) saturated with
ϩ
Ϫ
nitrous oxide yields the formate radical anion (CO ), a one-
2
᝽
ϩ
electron donor. This species rapidly reduced PMS 3, to give a
ϩ
ؒ
new species, the reduced product PMS , 5 (Scheme 1). Different
5
spectra were obtained at pH 5 and pH 9 indicating that this
radical can be protonated, to give another new species,
Mixing NADH with resazurin 1 in a buffer at pH 5–8 gave no
reaction and no formation of the fluorescent reduction product,
resorufin 2, occurred. When the experiment was repeated in the
ϩ
PMSH᝽ , 6, assigned to the equilibrium shown in eqn. (2).
ϩ
presence of PMS 3, reduction of the resazurin 1 occurred to
ؒ
ϩ
ϩ
PMS ϩ H
PMSH᝽
(2)
form the highly fluorescent dye, resorufin 2. This change was
pH dependent, the highest conversion of resazurin to resorufin
5
6
J. Chem. Soc., Perkin Trans. 2, 1998, 2333–2334
2333

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Under acidic conditions the species 6 is stable for periods of
PMS+
H+
2 PMS+
+
PMSH
2 PMS
+
ϩ
hours and is unaffected by the presence of starting PMS , 3.
3
4
5
However, in alkaline solution (pH 9.5) it was found to undergo
a pseudo-first order reaction in which the rate was dependent on
ϩ
the concentration of the parent compound (PMS ),
2 1–
3
Ϫ1
ϩ
4
Ϫ1
Resazurin 1
kobs ≈ 7 × 10 s at [PMS ] = 100 µM and k ≈ 3 × 10 s at
NAD(P)H NAD(P)+
obs
ϩ
[
PMS ] = 1 mM. At the higher concentration a new absorption
band at ca. 710 nm was observed, suggesting the formation of a
complex 7 between PMS 5 and the parent PMS 3 [eqn. (3)].
-H2O
ؒ
ϩ
Resorufin 2
ؒ
ϩ
ؒ
ϩ
PMS ϩ PMS
[PMS ؒPMS ]
(3)
Scheme 2
5
3
7
from which it can be seen that the role of the catalyst is to act as
a one-electron carrier to resazurin.
The species 5 shows absorbance at 580 nm and the pK of the
a
The detection of NADH [or NAD(P)H] formation and turn-
over by the catalysed formation of the fluorescent product
resorufin is currently being investigated as a simple means for
enhancing the widely-used but error-prone Papanicolau test for
ϩ
species PMSH was determined by monitoring this absorb-
᝽
ance at different pH before the formation of the complex 7, to
yield the value pK = 7.72 ± 0.04 (at room temperature and
a
ionic strength ≈ 0.1 M). The equilibria (2) and (3) lead to the
13
the early detection of cervical cancer.
prediction that the apparent pK value measured after forma-
a
tion of the complex (as would be obtained by standard
methods) should be dependent on the concentration. This
would explain the conflicting pKa values of 6.8 and 5.7
Experimental
8
9
NADH, as the disodium salt and phenazine were purchased
from Sigma-Aldrich Co. Ltd, Poole, Dorset. The phenazine was
methylated with redistilled dimethyl sulfate to produce the
yellow phenazinium methosulfate, mp 158–160 ЊC (decomp.)
reported for the species 6 in the literature.
ϩ
Under the conditions used for the reduction reaction, PMS
ؒ
3
and PMSH 4 interact to form the active species PMS 5,
according to eqn. (4). Resazurin 1 was then subjected to
14
using a modified literature method. Solutions were freshly
prepared each day and stored in the dark before use. The
principal buffer used was NЈ-(2-hydroxyethyl)piperazine-N-
ethanesulfonic acid (HEPES), adjusting the pH with 0.1 mol
ϩ
ؒ
ϩ
PMS ϩ PMSH → 2PMS ϩ H
(4)
3
4
5
Ϫ3
dm sodium hydroxide or hydrochloric acid. The γ-radiolysis
reduction under pulse radiolysis conditions, using the formate
studies were conducted using the pulse radiolysis facilities
of the Gray Laboratory, using a Co source with a nominal
activity of 2000 Ci. Fluorescence measurements were made
on a Perkin-Elmer LS50B luminescence spectrometer and
resorufin fluorescence was examined using λex 545 nm and λem
583 nm.
Ϫ
60
anion, CO2 . A rapid one-electron reduction occurs. The
᝽
reduction was followed by bleaching of the resazurin absorp-
tion peak at 630 nm followed by a partial recovery of the
absorbance, which followed second order kinetics. This is
assigned to the reduction of 1 to the respective radical anion,
Ϫ
10
1
᝽
followed by its disproportionation to yield resazurin and
ϩ
resorufin, 2 [eqns. (5) and (6)]. At pH 5, addition of PMS 3 to
Acknowledgements
Ϫ
Ϫ
1
ϩ CO2 → 1 ϩ CO2
(5)
(6)
᝽
᝽
We thank the EPSRC for a research studentship (to C. A. R.)
and a research grant (N. L. M.).
Ϫ
ϩ
2
1᝽
ϩ 2 H → 1 ϩ 2 ϩ H O
2
References
Ϫ
ϩ
the intermediate species 1 leads to formation of PMSH
6
᝽
᝽
ϩ
1
R. P. Haughland, Handbook of Fluorescent Probes and Research
Chemicals, Molecular Probes Inc., Eugene, Oregon, USA, 6th edn.,
1997, ch. 16.3, pp. 381–382; J. T. Mosmann, Immunol. Methods,
and resazurin 1, indicating that the stable species PMSH is,
thermodynamically, not able to reduce resazurin. However,
when PMSH , generated radiolytically at pH 5, is added to an
excess of resazurin a relatively slow reaction occurs leading to
᝽
ϩ
᝽
1
983, 65, 55; H. Imamura, S. Takao and T. Aikou, Cancer Res.,
1
994, 54, 3620; S. W. Thom, R. W. Horobin, E. Seidler and M. R.
the formation of resorufin. The disproportionation, reaction
Barer, J. Appl. Bacteriol., 1993, 74, 433.
(
6), is therefore an important step in helping to drive the overall
2
C. H. Self, J. Immunol. Methods, 1985, 76, 389.
process forward.
The role of PMS as a catalyst for the reduction of resazurin
must therefore be in a kinetically controlled process involving
3 E. Seidler, The Tetrazolium-Formazan System: Design and Histo-
chemistry, in Progress in Histochemistry and Cytochemistry, Gustav
Fischer Verlag, Stuttgart, 1991, vol. 24, pp. 1–86.
ϩ
ؒ
4 D. B. Cook and C. H. Self, Clin. Chem., 1993, 39, 965.
the equilibria (2) and (4). The PMS radical 5 can then react
5
6
7
Cf. F. G. Halaka, G. T. Babcock and J. L. Dye, J. Biol. Chem., 1982,
257, 1458.
with resazurin 1 to form a small quantity of the radical anion,
Ϫ
1
, which then undergoes the irreversible process (6), yielding
᝽
G. Blankenhorn, in Pyridine Nucleotide Dependent Dehydrogenases,
ed. H. Sund, W. de Gruyter & Co., Berlin, 1977, pp. 185–205.
G. Davis and P. J. Thornalley, Biochem. Biophys. Acta, 1983, 724,
the fluorescent product resorufin, 2.
Since the active, reduced phenazinium species 5 can also react
7
with oxygen and the phenazinium cation is light sensitive, the
4
56.
detection of NAD(P)H is best carried out in the dark in the
presence of a relatively large amount of the phenazinium cata-
lyst. Under these conditions, the reproducible formation of the
fluorescent resorufin is observed.
8 P. S. Rao and E. Hayon, Anal. Chem., 1976, 48, 564.
9 W. Rubaszewska and Z. R. Grabowski, J. Chem. Soc., Perkin Trans.
2, 1975, 417.
1
0 W. Prutz, J. Butler and E. Land, Arch. Biochem. Biophys., 1996, 327,
2
39.
Although the radical species generated in these reactions can
interact with oxygen producing, inter alia, the superoxide
1
1 A. K. Raap, Histochem. J., 1983, 15, 977.
12 M. Nishikimi, M. N. A. Rao and K. Yagi, Biochem. Biophys. Res.
Commun., 1972, 46, 849.
3 V. Ponti, M. V. Damiani, K. Cheeseman and T. F. Slater, Chem. Biol.
Interact., 1978, 23, 281.
14 F. Kehrmann and E. Havas, Ber., 1913, 46, 341.
11
anion, we have found that the addition of superoxide ion to
resazurin does not produce resorufin, so this species is also not
involved in the reduction. This is in contrast to the known
reduction of tetrazolium salts to formazans effected by super-
1
12
oxide ions.
The overall reduction process is summarised in Scheme 2,
Communication 8/06431H
2
334
J. Chem. Soc., Perkin Trans. 2, 1998, 2333–2334