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Effect of phospholipids, Triton X-100 and biological membranes on redox systems involving tetrazolium salt reduction. Its implications for the assay of enzymatic activities
Vol. 104, No. 4, 1982 February 26, 1982
BIOCHEMICAL
AND BIOPHYSICAL
EFFECT OF PHOSPHOLIPIDS,
TRITON X-100
AND BIOLOGICAL
ON REDOX SYSTEMS INVOLVING TETRAZOLIUM ITS IMPLICATIONS
RESEARCH COMMUNICATIONS Pages 1623-1629
FOR THE ASSAY OF ENZYMATIC ACTIVITIES.
Eddy M.Massa+
and Ricardo
December
*
+
N.Far?as
Departamento de Bioquimica de la Nutricicn, Instituto ciones Biol6gicas (INSIBIO), CONICET-UFT, Chacabuco de Tucumzn. ARGENTINA Received
MEMBRANES
SALT REDUCTION.
Superior de Investigs 461, (4.000) San Miguel
18, 1981
SUMMARY.The influence of phospholipids and Triton X-100 on the time course of chemical and enzyme-mediated reductions of a commonly used tetrazolium salt, MTT, was studied. MTT reduction was followed by the absorbance changes at 570 nm. With ascorbate as reducing agent, a 3-fold increase in the initial rates of the absorbance changes and a 24 % increase in the final absorbance values were observed in the presence of Triton X-100 micelles or phospholipid vesicles. The enzyme-mediated reduction of MTT with NADH generated by the NAD-dependent lactate dehydrogenase was also enhanced in the presence of Triton X-100, phospholipids or erythrocyte membranes. No enhancement was observed following the enzymatic generation of NADH at 340 nm in the absence of MTT.The above findings were interpreted as arising from: a) solubilization of reduced MTT in the detergent micelles or phospholipid vesicles which favors the redox reaction occurring in the aqueous fase, and b) changes in the spectral properties of reduced MTT in aqueous and lipid-like media. Tetrazolium in the assay oxidases
and all
by the
on the reduction strongly
course
of enzymatic
have
in
activating
on the
the enzyme, The studies
mediated
reductions
This
(5-7).
effect
salts
of lipid
was not
with
salt
formation (l-4). coloured
allowing
continous
spectrophotometric
or lipid-like However,
molecules salt
of NADH or NADPH is
based
formazanes
which
monitoring
of the
has been
used
on the activity
of
that
reduction
itself,
that
chemical
lipids
may
besides
considered.
described
here
of a commonly
demonstrate
used
tetrazolium
salt
*
This work was supported by a grant from the Consejo gaciones Cientlficas y T&nicas (Argentina).
+
Career Investigator of the cas y T&nicas (Argentina).
1 Abbreviations used: MTT, tetrazolium bromide; PMS, phosphatidylcholine; LDH, reductase, EC 1.1.1.27);
aceptors or
The assay
assay
the possibility
tetrazolium
electron
dehydrogenases
method
to deeply
region
reactions.
dehydrogenases a primary
reactions
the visible
the effect
used as artificial
Flavin-dependent
tetrazolium
of tetrazolium
absorb
to evaluate
are widely enzymes.
dehydrogenase
can be determined
purified
salts
of oxidative
Consejo
National
(MTT)l National
de Investigaciones
and enzymeare
directly
de InvestiCientzfi
3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyl phenazine methosulfate; DMPC, L-&-dimyristoyllactate dehydrogenase (L-lactate: NAD oxidoCMC, critical micelle concentration.
1623
0006-291X/82/041623-07$01.00/0 Copyright 0 I982 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
Vol. 104, No. 4, 1982 affected
BIOCHEMICAL
by the addition
Detergent
micelles,
of a lipidic
phospholipid
the LDH-mediated
AND BIOPHYSICAL
reduction
fase
vesicles
RESEARCH COMMUNICATIONS
to the aqueous or biological
of MTT eventhough
they
did
reaction
mixture.
membranes
enhanced
not
interact
with
the
enzyme. MATERIALS AND METHODS. L-&-phosphatidylcholine from egg yolk,DMPC, Triton X-100, NAD, PMS, MTT and LDH from beef heart (crystalline suspension in ammonium sulfate containing 1000 U/ml) were purchased from Sigma. Rat erythrocyte membranes were prepared by osmotic lysis as described by Steck and Kant (8) with the addition of 1 mM EDTA to the phosphate buffer used for washing and suspending the membranes. Phospholipid vesicles were prepared by the procedure of Batzri and Korn (9) injecting 50~1 of an ethanolic solution of phosphatidylcholine or DMPC (25 mg/ml) into 2.5 ml of distilled water at about 30" C. Freshly prepared phospholipid vesicles were used and the final ethanol concentrations in test and control incubation mixtures were 1 % or lower. The CMC of Triton X-100 was determined by the fluorescence method (10) tritrating 2 ml of 50# 8-anilino-I-naphthalene sulfonate in 50 mM sodium phosphate pH 8 with microliter quantities of the detergent. The sample was excited at 350 nm and emission was measured at 470 nm in a SLM 4048 spectrofluorometer. Chemical reduction of MTT by ascorbate was followed by the absorbance changes at 570 nm. The reactions were initiated by addition of ascorbate to the incubation mixture. Other experimental conditions are given in the corresponding figure legends. Lactate dehydrogenase was assayed at 25" C by following the increase in NADH concentration at 340 nm (reaction I) or coupling the NADdependent LDH reaction with the reduction of MTT which was followed at 570 nm (reaction I plus reactions II and III). In this later case, the hydrogen of NADH was transferred non-enzymatically to MTT in the presence of PMS as electron carrier: LDH (I) L-lactate + NAD+ w piruvate + NADH + H' (II)
NADH + PMS + H+ -
NAD+ + reduced
PMS
Reduced
PMS + formazan
(570 run)
(III)
PMS + MTT -
A ten-fold dilution of the stock qolution of LDH was done daily in 10 mM phosphate buffer containing 2 mM NAD , at the same pH (8 or 6) as that of the assay mixture. From this, further dilutions in phosphate buffer were done just before use when necessary. Other assay conditions are given in the legends to the corresponding figures. The concentrations of PMS and MTT were the same as those which have been generally used in enzymatic assays (5,7,11). Absorbance changes were monitored using a Gilford 250 spectrophotometer equipped with a thermoset temperature controller and a Gilford 6051 recorder. RESULTS. Fig. 1 shows the time course of MTT reduction by ascorbate in the absence
and presence
concentrations precipitation excess
of MTT (18 and 36p) of the
of ascorbate.
1 mM ascorbate
of the non-ionic formazan
derived
detergent were from
These MTT concentrations
in a time
period
which
Triton
X-100.
used in order their were
was neither
Low
to avoid
complete
reduction
completely too long
by an
reduced nor
by
too short
and the complete course of the reduction reactions could be easily followed. With 10 mM ascorbate, the final absorbance values in the plateau were the same as those with 1 mM ascorbate but were reached in a very much shorter time
period.
As can be seen
in Fig.1,
the presence 1624
of 0.1
% Triton
X-100
in
vol. 104, No. 4, 1982
nc
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
b
1
0
4
8
12
01
Time
16
20
0
a02
02
(min)
rriton
X-100(%)
Fig. 1: Time courses of MT reduction with 1 mM ascorbate, in the absence (---) and presence (- ) of 0.1 % Triton X-100. The reaction was carried out at 25O C in 50 mM sodium phosphate buffer pH 8. MIT concentrations were 18 and 36 JIM in curves (a) and (b), respectively. Fig. 2:Initial rates of the absorbance changes (o-o) and final absorbance values (x-x) as a function of Triton X-100 concentrations. The reaction mixture contained 50 mM sodium phosphate pH 8, 24 p MTT, 1 mM ascorbate and Triton X-100 at the concentrations shown on the abscissa. Initial rates were the tangents at t = 0 from plots of absorbance at 570 nm vs. time and final absorbances were the values in the plateau. Data were expressed relative to the value dn the absence of Triton X-100. The CMC of Triton X100 is indicated by the arrow.
the reaction
mixture
change
24
and a
produced
% increase
a 3-fold in the
increase
final
in the
absorbance
The relationship
between
of Triton
is
2. As can be observed,
of the absorbance Triton
X-100
under
our
shown in Fig. changes
agreement
and the final
concentrations
experimental with
around conditions
the value
on the
time
The absorption are
shown in Fig.3.
in the same
absence
reduction
peak
addition, in trace
X-100 at about
the absorbance (a).
Another
was completely
are
MTT in
is
at 570 nm is
about
feature into
that
to Triton
different
properties
of reduced
MTT are 1625
media
trace
after (a)
the
presents
in trace
(b).
in trace
reduced
The the complete
(b)
an In than
MTT in phosphate
fase when partitioned
different
buffer
out in
was added
chloroforme, and the absorption spectrum of this chloroformic reduced MTT (Fig. 3, trace c) coincided with trace (b). It is the spectral
X-100
shown).
respectively.
24 % higher
the lower
(12).
in phosphate
not observed is
to be 0.015%
(not
three
at
in good
similar
X-100,
It can be seen that
important
increased
was carried
detergent
rates
was determined
the spectra
when MTT reduction
620 nm which
initial
and Sb'derlund
of 0.1 % Triton
extracted
the
by ascorbate
of reduced
MTT
and the concentration
and Methods),
had an effect
and (b)
and when the
of MTT by ascorbate.
absorption
buffer
(a)
absorbance at both
values
CMC which
of MTT reduction
and the presence
of Triton
its
by Helenius
spectra
Traces
(b) was obtained
trace
presence
absorbance
vesicles
course
effects
(see Material
reported
Phosphatidylcholine micelles
these
values,
concentrations. X-10'
initial
in aqueous
with
extract of clear that and "lipid-
BIOCHEMICAL
Vol. 104, No. 4, 1982
AND BIOPHYSICAL
Wavelength
RESEARCH COMMUNICATIONS
(nm)
3: Absorption spectra of reduced MTT in different media: (a) 50 mM *ph~.~phate PH a ; (b) 50 r&J sodium phosphate pH 8 plus 0.1 % Triton X-100; (c) chloroforme. Experimental conditions were as follows. 24 JIM MTT in 50 mM sodium phosphate pH 8 was completely reduced by 10 mM ascorbate in a final volume of 9 ml from which three aliquots of 3 ml were taken. One of these aliquots was directly used for spectrum (a), another one for spectrum (b) previous addition of 30 1 of 10 % Triton X-100, and the last one was partitioned with 3 ml of ch i"oroforme and the lower fase used for spectrum (c). Spectra were scanned against their corresponding blanks without MTT, within 5 to 15 minutes following the addition of ascorbate to the solution of MTT.
like"
media , althouth
the nature
of the Triton
mediated
X-100
reduction
on the
further
spectral
experimentation
and phospholipid
vesicles
of MTT in the LDH system.
enzyme or on MTT can be easily
reaction
would
0.1 % Triton absorbance
of MT,
X-100 change
distinguished
340 nm (B).
respectively.
in the
also
In this
at 340 nm and at 570 nm (see Material
and the presence
be necessary
to define
changes. affected
direct
by following and Methods)
As shown
the enryme-
system,
in Fig.
in
assay mixture
increased
in about
affecting
the absorbance
were
obtained
the LDH the absence
4, the presence
at 570 nm (A) without
The same results
effects
70 % the
initial
change
when egg phosphatidylcholine for Triton system was
studied
affect
reduction X-100
of our
suspicion
of MTT in a fashion
micelles.
This
might
that similar
have
membrane
lipids
to phospholipid
implications 1626
in
could vesicles
the assay
at or
DMPC vesicles and rat erythrocyte membranes were substituted X-100. The influence of a biological membrane on this assay because
of
the
and Triton
of membrane-bound
Vol. 104, No. 4, 1982
BIOCHEMICAL
lime
AND BIOPHYSICAL
(min.)
RESEARCH COMMUNICATIONS
Time
(min.)
Fig. 4: Time course of MTT (A) and NADi (B) reductions in the LDH system at 25' C, in the absence (---) or the presence (-) of 0.1 % Triton X-100. dilution of LDH Reactions were initiated by addition of 10 1 of a loo-fold to 0.5 ml of incubation m$dia containing: YA), 50 mM sodium phosphate pH 8, 10 mM L-lactate, 2 mM NAD 120 ug/ml PMS and 60 pg/ml MTT; (B), the same components as in (A) except'that PMS and MTT were omitted.
enzymes
by the
between
the initial
tetrazolium
concentrations (C).
salt
rates
of DMPC (A),
At pH 8, a maximal % Triton
The effect
was much greater
DISCUSSION. reduction
Since
salt Lipids
when they
are
assays
of lipid
membranes
are
50 us/ml
frequently that
with
5O,ug/ml
in the
DMPC (Fig.
initial
performed
salt
in the presence it
molecules
rate
5A).
on tetrazolium
molecules, these
membranes
at 40 pg protein/ml.
increase
are based
and the
erythrocyte
70 % was obtained
assays
is
of
important
to
may have on the
itself.
and detergents in
can not
the test
be omitted
sample.
That
from
is
the assay mixture
the case of membrane
in the assay of membrane-bound enzymes, or detergents and lipids in test samples from purification steps following solubilization
membrane-bound enzymes
enzymes.
such as those
detergents
Lipids from
may be added
The possible
influence
tetrazolium
was used
be present
or serum.
in order
was not
salt
may also
plasma
of these
on MTT reduction with
(B) or rat
of about
with
influence
present
the LDH system
erythrocyte
or lipid-like
reduction
5 shows the relationship in
at pH 6: a 3.5-fold
many enzymatic
the direct
tetrazolium
Fig.
X-100
or rat
was obtained
amounts
determine
lipids present
X-100
and these
variable
Triton
increase
DMPC, 0.02
of MTT reduction
method.
of MTT reduction
to study variable
considered coli
their
effect
amounts
in previous
(5,6,11,13,14).
the D-LDH from Escherichia
in samples
On the other
1627
and
on the enzyme activity.
enzymatic
activations
of soluble
hand lipids
of lipids
Moreover, (7),
of
and detergents studies
in studies have been
where carried
this out
interpreted
Vol. 104, No. 4. 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
B
TOY 0 0.05
0.15 0.25 X-~COWO)
Triton 2.0
C t
1.5
0
50 150 DMPC o@ml)
1.0r 0 LO 120 200 Membmne OJg protein/ml I
250
'g 5: Relative initial rates of MTT reduction in the LDH system as a fEk;ion of DMRC (A), Triton X-100 (B) and erythrocyte membrane (C) concentrations, at pH 8 (o-a) or 6 (o-o). Assay conditions were as in Fig. 4A, except that at pH 6 a lo-fold dilution of LDH was used. Initial rates were the tangents at t = 0 from the recorded plots of absorbance at 570 nm vs. time and were expressed relative to the control values (no DMPC, Triton X-100 or erythrocyte membrane). as arising
from
phospholipids to affect
lipid-enzyme
or Triton MTT reduction
phospholipids increase
the initial
X-100.
value
effects
were
could
the reduction
of tetrazolium
that
dissolve
in organic
at concentrations
above its
into
the detergent
fase
surrounding
into
the lower
Fig.
3. Solubilization
favor fold
the redox increase
micelles these
fase
ascorbate change
larger
than
micelles.
able
as follows. (1).
CMC, reduced and will
occurring
initial
with
Formazans
absorbance
changes
In addition,
the difference
1628
above
derived compounds X-100
preferentially in the
aqueous
was completely
extracted
in the experiment
MTT in the micelles aqueous
24 % in
of Triton
absent
chloroform
in the
of about
dissolve
the same as it
when partitioned
a 3-fold
concentrations
In the presence
MTT will
of
water-soluble
be practically
j ust
and enzyme-
agent,
X-100
are sparingly
solvents
chemical
as reducing
at Triton
salts
of reduced
that
of Triton
fase. observed
X-100
Therefore,
observed
of will
the 3-
in the presence
Triton X-100 (Fig. 1) was mainly due to the decreased concentration formazan in the aqueous fase resulting from its solubilization in detergent
of those
by the presence
and an increase
obtained
micelles,
reaction in the
were
demonstrate
be explained
from
well
the concentrations
media
influenced
With
absorbance
absorbance
CMC. These
here
of MTT are directly
and Triton in
the final its
we presented
reductions
although
in the assay
itself.
The results mediated
interactions
X-100
in
of
of the the
the time
course
Vol. 104, No. 4, 1982 of the absorbance partially
AND BIOPHYSICAL
at 570 nm in the absence
arised
formazan
BIOCHEMICAL
from
the difference
in aqueous
and organic
media
24 % higher
in 0.1
(compare
difference
the absorption
of about
MTT was completely
enhanced
in the LDH system
MTT reduction erythrocyte effect
was not
observed lipid have
experimental
results
reduction of lipid arise
since
changes
from
their salt
direct reduction
reduced
Of
3).
This
absorbance
MTT than
explains
values
change
in
the
at 570 nm
above
clearly
enzymatic in the time
for
at 570 nm was also
phospholipid
vesicles It
is
or
clear
of the enzyme since
it
that
the
reduction
chemical
to the need for assays
based
course
of the
by ascorbate. in interpreting
on tetrazolium reduction
molecules
in the incubation
mixture
influence
on the enzyme activity
of
of MTT may
reduction caution
this
was not
at 340 nm. The influence
on the enzyme-mediated
given
from
of the
or chloroform
to the assay mixture.
on NAD+ reduction
point
and lipid-like
tetrazolium
added
molecules
Our findings
X-100
in Fig.
X-100,
due to activation
the same explanation
properties
by the absorbance
when Triton
assay based
and lipid-like
shown
X-100
reduced.
were
primarily
in the
of Triton
the absorbance
% Triton
in the final
followed
membranes
since
spectra
24 % observed
once all
and presence
in the spectral
at 570 nm was about water
RESEARCH COMMUNICATIONS
salt as the amount is varied
and/or
may
the
itself.
ACKNOWLEDGEMENT: We thank Dr.Roberto D.Morero fluorescence measurements in the determination his helpful discussions and continued interest
for performing the of Triton X-100 CMC and for in these studies.
REFERENCES Wahlefeld,A.W. and Michal,G. (1974) Methods of Enzymatic 1. Mollereing,H., Analysis (Bergmeyer,H.U.ed.) Vol 1, pp 136-144. Academic Press,New York. 2. Bergmeyer,H.U. and Bernt,E. (1974) Methods of Enzymatic Analysis (Bergmeyer,H.U.ed.) Vol. 2, pp. 579-582, Academic Press, New York. 3. Fried,R. and Fried,L.W. (1974) Methods of Enzymatic Analysis (Bergmeyer, H.U.ed.) Vo1.2, pp. 644-649. Academic Press, New York. 4. Abdallah,M.A. and Biellmann,J.F. (1980) Eur.J.Biochem. 112, 331-333. 5. Tanaka,Y., Anraku,Y. and Futai,M. (1976) J.Biochem. 80, 821-830. 6. Kimura,H.and Futai,M. (1978) .J.Biol.Chem. 253, 1095-1100. 7. Fung,L.W.M., Pratt,E.A. and Ho,C. (1979) Biochemistry 18, 317-324. 8. Steck,T.L. and Kant,J.A. (1974) Methods in Enzymology. (Fleischer, S. and Packer L,eds.). Vol. xXx1, Biomembranes, part A, pp. 172-180,Academic Press, New York, San Francisco and London, 9. Batzri,S. and Korn,E.D. (1973) Biochim.Biophys.Acta 298, 1015-1019. 10. Horowitz,P. (1977) J.Colloid Interfase Sci. 61, 197-198. 11. Futai,M. (1973) Biochemistry 12, 2468-2474. 12. Helenious,A. and Soderlung,H. (1973) Biochim.Biophys.Acta 307, 287-300. 13. Kohn,L.D. and Kaback,H.R. (1973) J.Biol.Chem. 248, 7012-7017. 14. Weiner,.J.H. and Heppe1,L.A. (1972) Biochem.Biophys.Res.Commun. 47,13601365.
1629
BIOCHEMICAL
AND BIOPHYSICAL
EFFECT OF PHOSPHOLIPIDS,
TRITON X-100
AND BIOLOGICAL
ON REDOX SYSTEMS INVOLVING TETRAZOLIUM ITS IMPLICATIONS
RESEARCH COMMUNICATIONS Pages 1623-1629
FOR THE ASSAY OF ENZYMATIC ACTIVITIES.
Eddy M.Massa+
and Ricardo
December
*
+
N.Far?as
Departamento de Bioquimica de la Nutricicn, Instituto ciones Biol6gicas (INSIBIO), CONICET-UFT, Chacabuco de Tucumzn. ARGENTINA Received
MEMBRANES
SALT REDUCTION.
Superior de Investigs 461, (4.000) San Miguel
18, 1981
SUMMARY.The influence of phospholipids and Triton X-100 on the time course of chemical and enzyme-mediated reductions of a commonly used tetrazolium salt, MTT, was studied. MTT reduction was followed by the absorbance changes at 570 nm. With ascorbate as reducing agent, a 3-fold increase in the initial rates of the absorbance changes and a 24 % increase in the final absorbance values were observed in the presence of Triton X-100 micelles or phospholipid vesicles. The enzyme-mediated reduction of MTT with NADH generated by the NAD-dependent lactate dehydrogenase was also enhanced in the presence of Triton X-100, phospholipids or erythrocyte membranes. No enhancement was observed following the enzymatic generation of NADH at 340 nm in the absence of MTT.The above findings were interpreted as arising from: a) solubilization of reduced MTT in the detergent micelles or phospholipid vesicles which favors the redox reaction occurring in the aqueous fase, and b) changes in the spectral properties of reduced MTT in aqueous and lipid-like media. Tetrazolium in the assay oxidases
and all
by the
on the reduction strongly
course
of enzymatic
have
in
activating
on the
the enzyme, The studies
mediated
reductions
This
(5-7).
effect
salts
of lipid
was not
with
salt
formation (l-4). coloured
allowing
continous
spectrophotometric
or lipid-like However,
molecules salt
of NADH or NADPH is
based
formazanes
which
monitoring
of the
has been
used
on the activity
of
that
reduction
itself,
that
chemical
lipids
may
besides
considered.
described
here
of a commonly
demonstrate
used
tetrazolium
salt
*
This work was supported by a grant from the Consejo gaciones Cientlficas y T&nicas (Argentina).
+
Career Investigator of the cas y T&nicas (Argentina).
1 Abbreviations used: MTT, tetrazolium bromide; PMS, phosphatidylcholine; LDH, reductase, EC 1.1.1.27);
aceptors or
The assay
assay
the possibility
tetrazolium
electron
dehydrogenases
method
to deeply
region
reactions.
dehydrogenases a primary
reactions
the visible
the effect
used as artificial
Flavin-dependent
tetrazolium
of tetrazolium
absorb
to evaluate
are widely enzymes.
dehydrogenase
can be determined
purified
salts
of oxidative
Consejo
National
(MTT)l National
de Investigaciones
and enzymeare
directly
de InvestiCientzfi
3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyl phenazine methosulfate; DMPC, L-&-dimyristoyllactate dehydrogenase (L-lactate: NAD oxidoCMC, critical micelle concentration.
1623
0006-291X/82/041623-07$01.00/0 Copyright 0 I982 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
Vol. 104, No. 4, 1982 affected
BIOCHEMICAL
by the addition
Detergent
micelles,
of a lipidic
phospholipid
the LDH-mediated
AND BIOPHYSICAL
reduction
fase
vesicles
RESEARCH COMMUNICATIONS
to the aqueous or biological
of MTT eventhough
they
did
reaction
mixture.
membranes
enhanced
not
interact
with
the
enzyme. MATERIALS AND METHODS. L-&-phosphatidylcholine from egg yolk,DMPC, Triton X-100, NAD, PMS, MTT and LDH from beef heart (crystalline suspension in ammonium sulfate containing 1000 U/ml) were purchased from Sigma. Rat erythrocyte membranes were prepared by osmotic lysis as described by Steck and Kant (8) with the addition of 1 mM EDTA to the phosphate buffer used for washing and suspending the membranes. Phospholipid vesicles were prepared by the procedure of Batzri and Korn (9) injecting 50~1 of an ethanolic solution of phosphatidylcholine or DMPC (25 mg/ml) into 2.5 ml of distilled water at about 30" C. Freshly prepared phospholipid vesicles were used and the final ethanol concentrations in test and control incubation mixtures were 1 % or lower. The CMC of Triton X-100 was determined by the fluorescence method (10) tritrating 2 ml of 50# 8-anilino-I-naphthalene sulfonate in 50 mM sodium phosphate pH 8 with microliter quantities of the detergent. The sample was excited at 350 nm and emission was measured at 470 nm in a SLM 4048 spectrofluorometer. Chemical reduction of MTT by ascorbate was followed by the absorbance changes at 570 nm. The reactions were initiated by addition of ascorbate to the incubation mixture. Other experimental conditions are given in the corresponding figure legends. Lactate dehydrogenase was assayed at 25" C by following the increase in NADH concentration at 340 nm (reaction I) or coupling the NADdependent LDH reaction with the reduction of MTT which was followed at 570 nm (reaction I plus reactions II and III). In this later case, the hydrogen of NADH was transferred non-enzymatically to MTT in the presence of PMS as electron carrier: LDH (I) L-lactate + NAD+ w piruvate + NADH + H' (II)
NADH + PMS + H+ -
NAD+ + reduced
PMS
Reduced
PMS + formazan
(570 run)
(III)
PMS + MTT -
A ten-fold dilution of the stock qolution of LDH was done daily in 10 mM phosphate buffer containing 2 mM NAD , at the same pH (8 or 6) as that of the assay mixture. From this, further dilutions in phosphate buffer were done just before use when necessary. Other assay conditions are given in the legends to the corresponding figures. The concentrations of PMS and MTT were the same as those which have been generally used in enzymatic assays (5,7,11). Absorbance changes were monitored using a Gilford 250 spectrophotometer equipped with a thermoset temperature controller and a Gilford 6051 recorder. RESULTS. Fig. 1 shows the time course of MTT reduction by ascorbate in the absence
and presence
concentrations precipitation excess
of MTT (18 and 36p) of the
of ascorbate.
1 mM ascorbate
of the non-ionic formazan
derived
detergent were from
These MTT concentrations
in a time
period
which
Triton
X-100.
used in order their were
was neither
Low
to avoid
complete
reduction
completely too long
by an
reduced nor
by
too short
and the complete course of the reduction reactions could be easily followed. With 10 mM ascorbate, the final absorbance values in the plateau were the same as those with 1 mM ascorbate but were reached in a very much shorter time
period.
As can be seen
in Fig.1,
the presence 1624
of 0.1
% Triton
X-100
in
vol. 104, No. 4, 1982
nc
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
b
1
0
4
8
12
01
Time
16
20
0
a02
02
(min)
rriton
X-100(%)
Fig. 1: Time courses of MT reduction with 1 mM ascorbate, in the absence (---) and presence (- ) of 0.1 % Triton X-100. The reaction was carried out at 25O C in 50 mM sodium phosphate buffer pH 8. MIT concentrations were 18 and 36 JIM in curves (a) and (b), respectively. Fig. 2:Initial rates of the absorbance changes (o-o) and final absorbance values (x-x) as a function of Triton X-100 concentrations. The reaction mixture contained 50 mM sodium phosphate pH 8, 24 p MTT, 1 mM ascorbate and Triton X-100 at the concentrations shown on the abscissa. Initial rates were the tangents at t = 0 from plots of absorbance at 570 nm vs. time and final absorbances were the values in the plateau. Data were expressed relative to the value dn the absence of Triton X-100. The CMC of Triton X100 is indicated by the arrow.
the reaction
mixture
change
24
and a
produced
% increase
a 3-fold in the
increase
final
in the
absorbance
The relationship
between
of Triton
is
2. As can be observed,
of the absorbance Triton
X-100
under
our
shown in Fig. changes
agreement
and the final
concentrations
experimental with
around conditions
the value
on the
time
The absorption are
shown in Fig.3.
in the same
absence
reduction
peak
addition, in trace
X-100 at about
the absorbance (a).
Another
was completely
are
MTT in
is
at 570 nm is
about
feature into
that
to Triton
different
properties
of reduced
MTT are 1625
media
trace
after (a)
the
presents
in trace
(b).
in trace
reduced
The the complete
(b)
an In than
MTT in phosphate
fase when partitioned
different
buffer
out in
was added
chloroforme, and the absorption spectrum of this chloroformic reduced MTT (Fig. 3, trace c) coincided with trace (b). It is the spectral
X-100
shown).
respectively.
24 % higher
the lower
(12).
in phosphate
not observed is
to be 0.015%
(not
three
at
in good
similar
X-100,
It can be seen that
important
increased
was carried
detergent
rates
was determined
the spectra
when MTT reduction
620 nm which
initial
and Sb'derlund
of 0.1 % Triton
extracted
the
by ascorbate
of reduced
MTT
and the concentration
and Methods),
had an effect
and (b)
and when the
of MTT by ascorbate.
absorption
buffer
(a)
absorbance at both
values
CMC which
of MTT reduction
and the presence
of Triton
its
by Helenius
spectra
Traces
(b) was obtained
trace
presence
absorbance
vesicles
course
effects
(see Material
reported
Phosphatidylcholine micelles
these
values,
concentrations. X-10'
initial
in aqueous
with
extract of clear that and "lipid-
BIOCHEMICAL
Vol. 104, No. 4, 1982
AND BIOPHYSICAL
Wavelength
RESEARCH COMMUNICATIONS
(nm)
3: Absorption spectra of reduced MTT in different media: (a) 50 mM *ph~.~phate PH a ; (b) 50 r&J sodium phosphate pH 8 plus 0.1 % Triton X-100; (c) chloroforme. Experimental conditions were as follows. 24 JIM MTT in 50 mM sodium phosphate pH 8 was completely reduced by 10 mM ascorbate in a final volume of 9 ml from which three aliquots of 3 ml were taken. One of these aliquots was directly used for spectrum (a), another one for spectrum (b) previous addition of 30 1 of 10 % Triton X-100, and the last one was partitioned with 3 ml of ch i"oroforme and the lower fase used for spectrum (c). Spectra were scanned against their corresponding blanks without MTT, within 5 to 15 minutes following the addition of ascorbate to the solution of MTT.
like"
media , althouth
the nature
of the Triton
mediated
X-100
reduction
on the
further
spectral
experimentation
and phospholipid
vesicles
of MTT in the LDH system.
enzyme or on MTT can be easily
reaction
would
0.1 % Triton absorbance
of MT,
X-100 change
distinguished
340 nm (B).
respectively.
in the
also
In this
at 340 nm and at 570 nm (see Material
and the presence
be necessary
to define
changes. affected
direct
by following and Methods)
As shown
the enryme-
system,
in Fig.
in
assay mixture
increased
in about
affecting
the absorbance
were
obtained
the LDH the absence
4, the presence
at 570 nm (A) without
The same results
effects
70 % the
initial
change
when egg phosphatidylcholine for Triton system was
studied
affect
reduction X-100
of our
suspicion
of MTT in a fashion
micelles.
This
might
that similar
have
membrane
lipids
to phospholipid
implications 1626
in
could vesicles
the assay
at or
DMPC vesicles and rat erythrocyte membranes were substituted X-100. The influence of a biological membrane on this assay because
of
the
and Triton
of membrane-bound
Vol. 104, No. 4, 1982
BIOCHEMICAL
lime
AND BIOPHYSICAL
(min.)
RESEARCH COMMUNICATIONS
Time
(min.)
Fig. 4: Time course of MTT (A) and NADi (B) reductions in the LDH system at 25' C, in the absence (---) or the presence (-) of 0.1 % Triton X-100. dilution of LDH Reactions were initiated by addition of 10 1 of a loo-fold to 0.5 ml of incubation m$dia containing: YA), 50 mM sodium phosphate pH 8, 10 mM L-lactate, 2 mM NAD 120 ug/ml PMS and 60 pg/ml MTT; (B), the same components as in (A) except'that PMS and MTT were omitted.
enzymes
by the
between
the initial
tetrazolium
concentrations (C).
salt
rates
of DMPC (A),
At pH 8, a maximal % Triton
The effect
was much greater
DISCUSSION. reduction
Since
salt Lipids
when they
are
assays
of lipid
membranes
are
50 us/ml
frequently that
with
5O,ug/ml
in the
DMPC (Fig.
initial
performed
salt
in the presence it
molecules
rate
5A).
on tetrazolium
molecules, these
membranes
at 40 pg protein/ml.
increase
are based
and the
erythrocyte
70 % was obtained
assays
is
of
important
to
may have on the
itself.
and detergents in
can not
the test
be omitted
sample.
That
from
is
the assay mixture
the case of membrane
in the assay of membrane-bound enzymes, or detergents and lipids in test samples from purification steps following solubilization
membrane-bound enzymes
enzymes.
such as those
detergents
Lipids from
may be added
The possible
influence
tetrazolium
was used
be present
or serum.
in order
was not
salt
may also
plasma
of these
on MTT reduction with
(B) or rat
of about
with
influence
present
the LDH system
erythrocyte
or lipid-like
reduction
5 shows the relationship in
at pH 6: a 3.5-fold
many enzymatic
the direct
tetrazolium
Fig.
X-100
or rat
was obtained
amounts
determine
lipids present
X-100
and these
variable
Triton
increase
DMPC, 0.02
of MTT reduction
method.
of MTT reduction
to study variable
considered coli
their
effect
amounts
in previous
(5,6,11,13,14).
the D-LDH from Escherichia
in samples
On the other
1627
and
on the enzyme activity.
enzymatic
activations
of soluble
hand lipids
of lipids
Moreover, (7),
of
and detergents studies
in studies have been
where carried
this out
interpreted
Vol. 104, No. 4. 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
B
TOY 0 0.05
0.15 0.25 X-~COWO)
Triton 2.0
C t
1.5
0
50 150 DMPC o@ml)
1.0r 0 LO 120 200 Membmne OJg protein/ml I
250
'g 5: Relative initial rates of MTT reduction in the LDH system as a fEk;ion of DMRC (A), Triton X-100 (B) and erythrocyte membrane (C) concentrations, at pH 8 (o-a) or 6 (o-o). Assay conditions were as in Fig. 4A, except that at pH 6 a lo-fold dilution of LDH was used. Initial rates were the tangents at t = 0 from the recorded plots of absorbance at 570 nm vs. time and were expressed relative to the control values (no DMPC, Triton X-100 or erythrocyte membrane). as arising
from
phospholipids to affect
lipid-enzyme
or Triton MTT reduction
phospholipids increase
the initial
X-100.
value
effects
were
could
the reduction
of tetrazolium
that
dissolve
in organic
at concentrations
above its
into
the detergent
fase
surrounding
into
the lower
Fig.
3. Solubilization
favor fold
the redox increase
micelles these
fase
ascorbate change
larger
than
micelles.
able
as follows. (1).
CMC, reduced and will
occurring
initial
with
Formazans
absorbance
changes
In addition,
the difference
1628
above
derived compounds X-100
preferentially in the
aqueous
was completely
extracted
in the experiment
MTT in the micelles aqueous
24 % in
of Triton
absent
chloroform
in the
of about
dissolve
the same as it
when partitioned
a 3-fold
concentrations
In the presence
MTT will
of
water-soluble
be practically
j ust
and enzyme-
agent,
X-100
are sparingly
solvents
chemical
as reducing
at Triton
salts
of reduced
that
of Triton
fase. observed
X-100
Therefore,
observed
of will
the 3-
in the presence
Triton X-100 (Fig. 1) was mainly due to the decreased concentration formazan in the aqueous fase resulting from its solubilization in detergent
of those
by the presence
and an increase
obtained
micelles,
reaction in the
were
demonstrate
be explained
from
well
the concentrations
media
influenced
With
absorbance
absorbance
CMC. These
here
of MTT are directly
and Triton in
the final its
we presented
reductions
although
in the assay
itself.
The results mediated
interactions
X-100
in
of
of the the
the time
course
Vol. 104, No. 4, 1982 of the absorbance partially
AND BIOPHYSICAL
at 570 nm in the absence
arised
formazan
BIOCHEMICAL
from
the difference
in aqueous
and organic
media
24 % higher
in 0.1
(compare
difference
the absorption
of about
MTT was completely
enhanced
in the LDH system
MTT reduction erythrocyte effect
was not
observed lipid have
experimental
results
reduction of lipid arise
since
changes
from
their salt
direct reduction
reduced
Of
3).
This
absorbance
MTT than
explains
values
change
in
the
at 570 nm
above
clearly
enzymatic in the time
for
at 570 nm was also
phospholipid
vesicles It
is
or
clear
of the enzyme since
it
that
the
reduction
chemical
to the need for assays
based
course
of the
by ascorbate. in interpreting
on tetrazolium reduction
molecules
in the incubation
mixture
influence
on the enzyme activity
of
of MTT may
reduction caution
this
was not
at 340 nm. The influence
on the enzyme-mediated
given
from
of the
or chloroform
to the assay mixture.
on NAD+ reduction
point
and lipid-like
tetrazolium
added
molecules
Our findings
X-100
in Fig.
X-100,
due to activation
the same explanation
properties
by the absorbance
when Triton
assay based
and lipid-like
shown
X-100
reduced.
were
primarily
in the
of Triton
the absorbance
% Triton
in the final
followed
membranes
since
spectra
24 % observed
once all
and presence
in the spectral
at 570 nm was about water
RESEARCH COMMUNICATIONS
salt as the amount is varied
and/or
may
the
itself.
ACKNOWLEDGEMENT: We thank Dr.Roberto D.Morero fluorescence measurements in the determination his helpful discussions and continued interest
for performing the of Triton X-100 CMC and for in these studies.
REFERENCES Wahlefeld,A.W. and Michal,G. (1974) Methods of Enzymatic 1. Mollereing,H., Analysis (Bergmeyer,H.U.ed.) Vol 1, pp 136-144. Academic Press,New York. 2. Bergmeyer,H.U. and Bernt,E. (1974) Methods of Enzymatic Analysis (Bergmeyer,H.U.ed.) Vol. 2, pp. 579-582, Academic Press, New York. 3. Fried,R. and Fried,L.W. (1974) Methods of Enzymatic Analysis (Bergmeyer, H.U.ed.) Vo1.2, pp. 644-649. Academic Press, New York. 4. Abdallah,M.A. and Biellmann,J.F. (1980) Eur.J.Biochem. 112, 331-333. 5. Tanaka,Y., Anraku,Y. and Futai,M. (1976) J.Biochem. 80, 821-830. 6. Kimura,H.and Futai,M. (1978) .J.Biol.Chem. 253, 1095-1100. 7. Fung,L.W.M., Pratt,E.A. and Ho,C. (1979) Biochemistry 18, 317-324. 8. Steck,T.L. and Kant,J.A. (1974) Methods in Enzymology. (Fleischer, S. and Packer L,eds.). Vol. xXx1, Biomembranes, part A, pp. 172-180,Academic Press, New York, San Francisco and London, 9. Batzri,S. and Korn,E.D. (1973) Biochim.Biophys.Acta 298, 1015-1019. 10. Horowitz,P. (1977) J.Colloid Interfase Sci. 61, 197-198. 11. Futai,M. (1973) Biochemistry 12, 2468-2474. 12. Helenious,A. and Soderlung,H. (1973) Biochim.Biophys.Acta 307, 287-300. 13. Kohn,L.D. and Kaback,H.R. (1973) J.Biol.Chem. 248, 7012-7017. 14. Weiner,.J.H. and Heppe1,L.A. (1972) Biochem.Biophys.Res.Commun. 47,13601365.
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