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Oxidation of phenazine methosulfate by hepatic aldehyde oxidase
Vol. 8, No. 1, 1962
BIOCHEMICAL
AND BIOPHYSICAL
OXIDATIONOF PHENAZINEM!3THWULFAZ3
RESEARCH COMMUNICATIONS
BY HEF'ATICAIDEHYDEOXIDASE
K. V. Rajagopalan and Philip
Handler
Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina
Received
May
1, 1962
Rabbit liver it
catalyzes
aldehyde oxidase exhibits
the oxidation
hyde, acetaldehyde, droxylation
of a wide variety
salicylaldehyae,
of nitrogen-containing
nicotinamide,
a dual catalytic
of alaehydes (e.g.,
pyridoxal)
quinoline and quinine (Knox, 1946).
methylene blue, 2,6-dichlorophenolindophenol,
for the highly purified
formalde-
and also catalyzes
aromatic heterocycles
zolium as well as phenazine methosulfate
specificity;
such as $-methyl-
Oxygen, ferricyanide,
cytochrome c, nitroblue
(PMS) serve as electron
enzyme (Rajagopalan, Fridovich
incubation
in the absence of a conventional
of this dye with the enzyme resulted
tetra-
acceptors
and Handler, 1962).
In the course of experiments in which PMSwas to serve as electron it was observed that,
the hy-
substrate,
acceptor aerobic
in the appearance of a red
Further examination revealed that PMS can serve not only as electron
color.
acceptor but as an oxidizable
substrate (electron
Since this dye has occasionally introduced into biological
donor) for aldehyde oxidase.
been observed to serve as a reductant when
systems as an oxidant
(Kok and Hoch, 1961), it
appeared desirable to record the present findings. Absorption Spectrum of Oxidized F%IS purified
aldehyde oxidase was prepared from rabbit
previously
described (Rajagopalan et al.,
allowed to
react
aerobically
1962).
liver
Five w of the enzyme was
with 2 x 10D5 M PMS, in 0.05 M phosphate,
PH 7.8, Containing .m5k versene-Fe3 in spectrophotometric reaction
was complete.
by the method
cuvettes until
The absorption spectrum of the red product as well
as that of PMSwas obtained with a Cary Model 14 recording 43
spectrophoto-
Vol. 8, No. 1, 1962
meter.
EIOCHEMICAL
AND BIOPHYSICAL
The absorption spectra of F%lSand its
presented in Fig. 1.
The molar extinction
at several wavelengths are listed 520 q.& on oxidation
of FMS affords
RESEARCH COMMUNICATIONS
red oxidized
coefficients
in Table I.
derivative
are
of the two compounds
The increase in extinction
the most sensitive
at
spectrophotometric
assay for aldehyde oxidase available. I(;n for PMSwas found to be 1.9 x lo4
Mj the values for IV'-methyl
nicotinamide and acetaldehyde are 2.8 x lop4 M and 1.0 x 10'" M, respectively.
Figure 1.
Absorption spectra of PMSand oxidized PMS.
TABLFII Molar extinction
coefficients
of IN5 and oxidized FMS Molar extinction coefficient (1 cm. light path) OXIDIZED PMS Fws
Wavelength nq* 230 260 284 387 520
Probable Identity
9,900 82,000 250 25,700 400
31,500 =,500
of the Oxidation Product
The red oxidation
product (I) was reduced to a leuco form [II)
each of two procedures: (a ) ju dicious addition 44
of dithionite
by
or (b) anaero-
Vol. 8, No. 1, 1962
bit
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
incubation with aldehyde oxidase in the presence of N-l-methyl nicotin-
amide as electron donor in 0.05 M phosphate, pH 7.8. troduction reduction
of oxygen resulted in reoxidation of (I) did not result
Anaerobic incubation plex data.
Initially,
In both cases, rein-
to the original
in reformation
red form.
Thus,
of phenazine methosulfate.
of PM3with alaehyde oxidase yielded rather
PMSwas oxidized to its red derivative
com-
(I) with other
molecules of FMS serving as electron acceptors in the conventional manner, resulting
in the formation of leuco PMS (III).
served as electron acceptor an equilibrium tained PMSas well as
I, II
compounds
The red compound(I)
exhibited
Subsequently, as (I)
mixture was obtained which con-
and III. several properties
described for pyocyanine (10 methyl phenazin-l-one). Michaelis
which resembled those Thus, Friedheim and
(1931) reported that pyocyanine, which is blue in alkaline medium,
is converted to a leucoform by a 2-electron however, pyocyanine is wine red; addition tion of a stable green free radical colorless
form.
reduction. of dithionite
In comparison, the oxidation
at acid PH. Addition
results in formareduced to a
product (I) derived from phena-
pH and yielded an orange-yellow solution
of a small amount of dithionite
tion which was completely bleached by a further In contrast
In acid solution,
which may then be further
zine methosulfate is red at alkaline
to the behavior of (I),
yielded a green solu-
addition
of dii&onite.
the photoxidation
product of PMS,
prepared by aerobic illumina tion of PIE in 0.05 M phosphate buffer, ccanpletely resembled pyocyanine when treated with dithionite kaline solution. and properties
Kehrmann and Cherpillod of oxidized
in the 2- and 3-positions latter
also
compoundswith those of (I)
with lo-methyl
phenazin+-one
that the electron
distribution
phenazine with oxygen functions
Comparison of the properties
indicated
(Fig. 2).
that the latter
of the
is identical
This is compatible with the fact
around carbon 3 of phenazine methosulfate re-
sembles that about the 6-carbon of N1-methyl nicotinamide, substrate for aldehyde oxidase.
in acid or al-
(1924) reported the synthesis
forms of lo-methyl respectively.
pH 7.8,
a conventional
It is noteworthy that sometumor tissues 45
Vol. 8, No. i, 1962
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
C
B
Figure 2. A - lo-methyl phenazine. B - lo-methyl phenazin-l-one (Pyocyanine). C - lo-methyl phenazin+-one.
have been found to oxidize oxidase (Dickens, 1936;
FT45to the sameproduct as obtained with aldehyde
Dickens and McIllwain,
1938).
N-methyl acridine and N-methyl (1) acridone have also been observed to serve as substrates for aldehyde oxidase, whereas pyocyanine acts solely as an acceptor. many respects,
Milk xanthine oxidase, which resembles aldehyde oxidase in does not oxidize
any of these compoundsunder the samecondi-
tions. Recently Kok and Hoch (1961) tion,
reported that PMS, contrary
acted as a source of "reducing power" in photosynthesizing
tions derived from various a&ae.
capable of conducting photophosphorylation.
tron acceptor, thesizing
prepara-
Horio and Kamen(personal communication)
have observed the reducing capacity of PMSin bacterial
ting the oxidation
to expecta-
preparations
The present data, demonstra-
of F'MSby an enzyme which can use cy-bochromec as elec-
suggest the presence of a similar
enzyme in these photosyn-
systems.
This work was supported by grant RG-91 from the National Institutes of Health.
46
Vol. 8, No. 1, 1962
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Dickens, F., Biochem. J. 2, 1064 (1936). Dickens F., and McIllwain, Biochem. J., s 1615 (1938). Friedheim, E., and Michaelis, L., J. Biol. Chem. $U, 355 (1931). Horio, T., and Karen, M. D., Personal communication. Kehrmann, F., and Cherpillod, F., Helv. Chim. Acta, 1, 973 (1924). Kok, B., and Hoch, G., in McElroy, W. D. and Glass, B., Ed., A Symposium on Light and Life, The Johns Hopkins Press, Baltimore, 1961, p. 397. Rajagopalan, K. V., Fridovich, I., and Handler, P., J. Biol. kern. a, 922 (1962) .
47
BIOCHEMICAL
AND BIOPHYSICAL
OXIDATIONOF PHENAZINEM!3THWULFAZ3
RESEARCH COMMUNICATIONS
BY HEF'ATICAIDEHYDEOXIDASE
K. V. Rajagopalan and Philip
Handler
Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina
Received
May
1, 1962
Rabbit liver it
catalyzes
aldehyde oxidase exhibits
the oxidation
hyde, acetaldehyde, droxylation
of a wide variety
salicylaldehyae,
of nitrogen-containing
nicotinamide,
a dual catalytic
of alaehydes (e.g.,
pyridoxal)
quinoline and quinine (Knox, 1946).
methylene blue, 2,6-dichlorophenolindophenol,
for the highly purified
formalde-
and also catalyzes
aromatic heterocycles
zolium as well as phenazine methosulfate
specificity;
such as $-methyl-
Oxygen, ferricyanide,
cytochrome c, nitroblue
(PMS) serve as electron
enzyme (Rajagopalan, Fridovich
incubation
in the absence of a conventional
of this dye with the enzyme resulted
tetra-
acceptors
and Handler, 1962).
In the course of experiments in which PMSwas to serve as electron it was observed that,
the hy-
substrate,
acceptor aerobic
in the appearance of a red
Further examination revealed that PMS can serve not only as electron
color.
acceptor but as an oxidizable
substrate (electron
Since this dye has occasionally introduced into biological
donor) for aldehyde oxidase.
been observed to serve as a reductant when
systems as an oxidant
(Kok and Hoch, 1961), it
appeared desirable to record the present findings. Absorption Spectrum of Oxidized F%IS purified
aldehyde oxidase was prepared from rabbit
previously
described (Rajagopalan et al.,
allowed to
react
aerobically
1962).
liver
Five w of the enzyme was
with 2 x 10D5 M PMS, in 0.05 M phosphate,
PH 7.8, Containing .m5k versene-Fe3 in spectrophotometric reaction
was complete.
by the method
cuvettes until
The absorption spectrum of the red product as well
as that of PMSwas obtained with a Cary Model 14 recording 43
spectrophoto-
Vol. 8, No. 1, 1962
meter.
EIOCHEMICAL
AND BIOPHYSICAL
The absorption spectra of F%lSand its
presented in Fig. 1.
The molar extinction
at several wavelengths are listed 520 q.& on oxidation
of FMS affords
RESEARCH COMMUNICATIONS
red oxidized
coefficients
in Table I.
derivative
are
of the two compounds
The increase in extinction
the most sensitive
at
spectrophotometric
assay for aldehyde oxidase available. I(;n for PMSwas found to be 1.9 x lo4
Mj the values for IV'-methyl
nicotinamide and acetaldehyde are 2.8 x lop4 M and 1.0 x 10'" M, respectively.
Figure 1.
Absorption spectra of PMSand oxidized PMS.
TABLFII Molar extinction
coefficients
of IN5 and oxidized FMS Molar extinction coefficient (1 cm. light path) OXIDIZED PMS Fws
Wavelength nq* 230 260 284 387 520
Probable Identity
9,900 82,000 250 25,700 400
31,500 =,500
of the Oxidation Product
The red oxidation
product (I) was reduced to a leuco form [II)
each of two procedures: (a ) ju dicious addition 44
of dithionite
by
or (b) anaero-
Vol. 8, No. 1, 1962
bit
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
incubation with aldehyde oxidase in the presence of N-l-methyl nicotin-
amide as electron donor in 0.05 M phosphate, pH 7.8. troduction reduction
of oxygen resulted in reoxidation of (I) did not result
Anaerobic incubation plex data.
Initially,
In both cases, rein-
to the original
in reformation
red form.
Thus,
of phenazine methosulfate.
of PM3with alaehyde oxidase yielded rather
PMSwas oxidized to its red derivative
com-
(I) with other
molecules of FMS serving as electron acceptors in the conventional manner, resulting
in the formation of leuco PMS (III).
served as electron acceptor an equilibrium tained PMSas well as
I, II
compounds
The red compound(I)
exhibited
Subsequently, as (I)
mixture was obtained which con-
and III. several properties
described for pyocyanine (10 methyl phenazin-l-one). Michaelis
which resembled those Thus, Friedheim and
(1931) reported that pyocyanine, which is blue in alkaline medium,
is converted to a leucoform by a 2-electron however, pyocyanine is wine red; addition tion of a stable green free radical colorless
form.
reduction. of dithionite
In comparison, the oxidation
at acid PH. Addition
results in formareduced to a
product (I) derived from phena-
pH and yielded an orange-yellow solution
of a small amount of dithionite
tion which was completely bleached by a further In contrast
In acid solution,
which may then be further
zine methosulfate is red at alkaline
to the behavior of (I),
yielded a green solu-
addition
of dii&onite.
the photoxidation
product of PMS,
prepared by aerobic illumina tion of PIE in 0.05 M phosphate buffer, ccanpletely resembled pyocyanine when treated with dithionite kaline solution. and properties
Kehrmann and Cherpillod of oxidized
in the 2- and 3-positions latter
also
compoundswith those of (I)
with lo-methyl
phenazin+-one
that the electron
distribution
phenazine with oxygen functions
Comparison of the properties
indicated
(Fig. 2).
that the latter
of the
is identical
This is compatible with the fact
around carbon 3 of phenazine methosulfate re-
sembles that about the 6-carbon of N1-methyl nicotinamide, substrate for aldehyde oxidase.
in acid or al-
(1924) reported the synthesis
forms of lo-methyl respectively.
pH 7.8,
a conventional
It is noteworthy that sometumor tissues 45
Vol. 8, No. i, 1962
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
C
B
Figure 2. A - lo-methyl phenazine. B - lo-methyl phenazin-l-one (Pyocyanine). C - lo-methyl phenazin+-one.
have been found to oxidize oxidase (Dickens, 1936;
FT45to the sameproduct as obtained with aldehyde
Dickens and McIllwain,
1938).
N-methyl acridine and N-methyl (1) acridone have also been observed to serve as substrates for aldehyde oxidase, whereas pyocyanine acts solely as an acceptor. many respects,
Milk xanthine oxidase, which resembles aldehyde oxidase in does not oxidize
any of these compoundsunder the samecondi-
tions. Recently Kok and Hoch (1961) tion,
reported that PMS, contrary
acted as a source of "reducing power" in photosynthesizing
tions derived from various a&ae.
capable of conducting photophosphorylation.
tron acceptor, thesizing
prepara-
Horio and Kamen(personal communication)
have observed the reducing capacity of PMSin bacterial
ting the oxidation
to expecta-
preparations
The present data, demonstra-
of F'MSby an enzyme which can use cy-bochromec as elec-
suggest the presence of a similar
enzyme in these photosyn-
systems.
This work was supported by grant RG-91 from the National Institutes of Health.
46
Vol. 8, No. 1, 1962
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Dickens, F., Biochem. J. 2, 1064 (1936). Dickens F., and McIllwain, Biochem. J., s 1615 (1938). Friedheim, E., and Michaelis, L., J. Biol. Chem. $U, 355 (1931). Horio, T., and Karen, M. D., Personal communication. Kehrmann, F., and Cherpillod, F., Helv. Chim. Acta, 1, 973 (1924). Kok, B., and Hoch, G., in McElroy, W. D. and Glass, B., Ed., A Symposium on Light and Life, The Johns Hopkins Press, Baltimore, 1961, p. 397. Rajagopalan, K. V., Fridovich, I., and Handler, P., J. Biol. kern. a, 922 (1962) .
47