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A new kinetic assay method for quinone-reducing enzymes
ANALYTICAL
BIOCHEMISTRY
81,
328-335 (1977)
A New Kinetic Assay Method for Quinone-Reducing Enzymes E. P. TITOVETS AND G. G. PETROVSKY The Byelorussian
State Research Physiotherapy,
Institute Minsk
oj- Neurology, 22044, USSR
Neurosurgery.
and
Received December I, 1976: accepted April 4, 1977 A new kinetic method is described for the assay of quinone-reducing enzymes in various biological materials. It is based on polarographic determination of oxygen uptake in spontaneous oxidation of the diphenol formed as a result ,2 (AMOBQ) enzymic reduction. The of 4-anilino-5-methoxybenzoquinone-1 stoichiometry of the reducing equivalent transfer in the reaction sequence from NAD(P)H to oxygen has been analyzed. Data are presented on quinone-reducing activity distributions in different tissues.
The conventional method for the assay of quinone-reducing enzymes employs spectrophotometry to measure the quinone reduction rate (l-5). A number of limitations intrinsic to optical spectroscopy narrow the application range of this method. One disadvantage is due to the difficulty or even the impossibility in applying the method while working with intensely light-scattering samples, e.g., concentrated cell or mitochondrial suspensions, dense tissue homogenates, etc. In addition, this method may become quite inadequate if the specific absorption maxima overlap those of other light-absorbing components in the reaction medium. The apprearance of new synthetic substrates for quinone-reducing enzymes (6.7) with certain remarkable properties has made it possible to develop a new nonoptical assay method as reported in this paper. The new method employs polarographic monitoring of oxygen uptake, stoichiometrically with respect to the diphenol oxidation rate, the diphenol appearing as a result of enzymic reduction of 4-anilino-5-methoxybenzoquinone-1.2 (AMOBQ), a new synthetic acceptor quinone. MATERIALS
AND METHODS
Materials. The acetone preparation of NAD(P)H: (quinone acceptor) oxidoreductase [EC 1.6.99.21, also called menadione reductase, was obtained from rat liver mitochondria as described earlier (6). Tissue homogenates for quinone-reducing activity distribution experiments were prepared on l/15 M phosphate buffer, pH 7.5. Various tissues from adult domestic rabbits and white rats of both sexes were taken for Copyright All rights
IZ~ 1977 by Academic Prers. Inc. of reproductmn m any form reserved
328 ISSN 00WZhY7
NEW
QUINONE
REDUCTASE
ASSAY
329
these experiments. Rabbit brain mitochondria and other fractions of brain homogenate were obtained by a differential centrifugation technique (8). 4-Aniline-5methoxybenzoquinoneI,2 (AMOBQ) was synthesized according to Wanzlick and Jahnke (9) at the Lenin Byelorussian State University; beef liver catalase [EC 1.11.1.61 was obtained from Sigma; NAD(P)H and sodium deoxycholate were from Reanal. All of the common chemicals employed were reagent grade. Assay of quinone-reducing activity. The reaction medium for the assay of quinone-reducing enzymes consists of 1115 M KH,PO,/Na,HPO, buffer, pH 7.5, 150 FM AMOBQ, 400 PM NADPH or NADH, and 100 wg/ml of catalase, the addition of the last item being optional (see Results and Discussion). The determinations may be carried out in polarographic cells designed for oxygen consumption measurements with a Clark-type oxygen electrode and a conventional recording polarograph, e.g., Refs. (10,ll). The reaction is normally started by addition of a tissue preparation sample to the reaction medium in the polarographic cell. The ensuing oxygen depletion in the medium is stoichiometric with respect to the AMOBQ enzymic reduction rate under the given experimental conditions. Nonspecific oxygen uptake. if any, should be subtracted from the obtained values. The activity values are expressed as nanomoles of AMOBQ reduced per minute per milligram of protein, one nanomole of reduced AMOBQ (AMOBQ.H,) being equivalent to 0.5 nmol of oxygen consumed in the reaction (see Results and Discussion). Vigorous stirring of the reaction medium must be strictly maintained throughout the entire determination procedure. The experiments are carried out at 20°C. Miscellaneous methods. Oxygen concentration in the reaction medium was calculated with a reference to temperature, barometric pressure. and ionic composition of the reaction medium (12). Protein was estimated by the method of Lowry et al. (13) or by the biuret method (14). NADH oxidation was controlled at 340 nm on a Specord Uvvis automatic spectrophotometer. The kinetic study of AMOBQ.H, spontaneous oxidation was carried out at 510 nm by the stop-flow technique, using a Durrum D 110 spectrophotometer. RESULTS
AND DISCUSSION
A polarographic determination of quinone-reducing activity is demonstrated in Fig. 1. In these experiments, the reaction was initiated by adding fractional amounts of NADH (below 400 FM) to the assay medium already containing 0.3 mg/ml of the menadione reductase preparation. This was done to facilitate study of the reducing equivalent transfer stoichiometry (see below). Normally, the reaction is started by adding an enzyme sample to an otherwise complete reaction medium contain-
330
TITOVETS
66yM
AND
PETROVSKY
NADH I
I MIN
FIG. 1. Polarographic record of oxygen uptake in the assay of menadione reductase activity. The reaction medium contains 0.5 mgiml of the crude acetone fraction of menadione reductase (6). The oxygen cell volume is 1.3 ml. All additions are made as 5Oq.I volumes. Values near the curves stand for the calculated NADH/O stoichiometric ratio (for explanation. see text).
ing 400 PM NADH or NADPH, as is recommended under Materials and Methods. The linear part of the oxygen uptake trace in Fig. 1 is used for calculating the menadione reductase activity of the preparation. The oxygen uptake slows down as NADH is depleted in the reaction medium until it stops altogether. A second addition of 66 PM NADH results in a new burst of oxygen uptake. The precision of the method was calculated from 49 trial tests representing different sets of three to five replica trials. From these experiments, the standard deviation of the rate values was 0.5% of the mean value and of the range, 3.7% of it. The choice of AMOBQ as an acceptor substrate was due to the fact that it is easily reduced enzymatically and its diphenolic form is equally easily oxidized in the presence of dissolved oxygen (6,7). AMOBQ acts as a carrier of reducing equivalents to oxygen. The reaction sequence for the present method may be written as: NAD.H
+ H+ + AMOBQ
AMOBQ.H2
Enz\ NAD+
+ % 0, -
AMOBQ
+ AMOBQ.H2
[II
+ HzO,
PI
where AMOBQ. H, stands for reduced AMOBQ and Enz represents any of the quinone-reducing enzymes. An enzymatic reduction of AMOBQ takes place at Step [l]. This reaction is conducted at reasonably high concentrations of both NAD(P)H and AMOBQ and is expected to proceed at a maximal rate (6). The AMOBQ reduction rate, thus, is equal to d]AMOBQ.H,]ldr
= k,[~,],
L31
NEW
QUINONE
1.0
05 MG
OF
REDUCTASE
MENADIONE
15 REDUCTASE
331
ASSAY
20
25
PREPARATION/CELL
FIG. 2. Plot of the initial oxygen uptake rates versus the quantity of menadione reductase preparation. In these experiments. the acetone fraction of mitochondrial menadione reductase was used (6). The specific activity of the preparation is 465 nmol of AMOBQ.H,/min/mg. On the ordinate. oxygen uptake rates are expressed in arbitrary units. The assay conditions are as specified under Materials and Methods.
where Et is the total enzyme concentration, and k, is the rate constant. The reaction rate at Step [2] may be presented as: -dO/dt
= -d [AMOBQ.H,]ldt
= k [AMOBQ.H,]
[O#.
]41
Under the specified experimental conditions, the concentration of oxygen is much higher than that of AMOBQ.H, in the assay solution, and, in view of that, Eq. [4] assumes a pseudo first order in relation to the diphenol concentration: d [AMOBQ.
H,]/dt
= k’ [AMOBQ.
H,]
151
where k’ = k[O,]+. According to Eq. [5], the maxima1 possible diphenol oxidation rate may be achieved if all of the initially added AMOBQ is in a reduced form. For a correct determination of the quinone-reducing activity, the following condition should be strictly observed: k,[E,]
G k’ [AMOBQ.
H&r
[cl
where [AMOBQ. H,],, stands for all initially present AMOBQ in the reduced form. Relationship [6] ascribes the limiting reaction in the overall reaction sequence to the enzymic Step [ 11. The practical realization of this was ascertained for the experimental conditions given, and the appropriate data are reported in Fig. 2. In these experiments, a preparation containing highly active mitochondrial menadione reductase was used (6). As can be seen from Fig. 2, the initial oxygen consumption rate is linear with
332
TITOVETS
AND
PETROVSKY
respect to the amount of enzyme preparation. This points to the validity of relationship [6]. The enzyme concentrations used in these experiments are much higher than those usually found in different tissues (cf. Tables 1 and 2). It is important to evaluate the time interval within which the oxygen uptake rate in the complete system approaches a steady state or, rather, the time required to reach a pseudo steady-state which, within experimental error, may be considered to coincide with the steady-state rate. This time covers the period in which the AMOBQ. H, concentration increases from zero to [AMOBQ. HPJs.st., where the latter is the steadystate diphenol concentration. Using Eqs. [3], [4], and [5], an equation for the diphenol concentration change rate in the system may be obtained: H,]ldt
d [AMOBQ.
Integration
= k, [E,] - Ii’ [AMOBQ.
H,]
[71
of Eq. [7] gives: k’[AMOBQ.H,],.,,.Ik,[E,]
= 1 - e-“”
PI
The left hand-side of Eq. [8] is the ratio of the diphenol oxidation rate to the diphenol formation rate in an enzymatic reaction. If 95%, or a value of 0.95, is assumed for the above ratio as a reasonable practical approach to the steady-state rate, then the time necessary for this may be calculated from Eq. [8]: t = 2.99/k’
[91
The rate constant k’ from stop-flow spectrophotometric determinations was found to be 0.3 set-‘. Substituting this value for k’ in Eq. [9] gives 7.7 sec. Thus, the oxygen consumption rate, or the AMOBQ. H2 oxidation rate, becomes practically equal to the AMOBQ reduction rate within 7.7 sec. 35vM
PBO
79~“M
PBO
123vM
PBO
1
1.09 \
s D 4
l1 MIN
FIG. 3. Spectrophotometric control of NADH oxidation in the presence of menadione reductase and PBQ as acceptor substrate. The reaction medium contains 0.1 mg of the menadione reductase acetone fraction. The measurements are carried out against a blank with no enzyme preparation added. Light path = 1 cm; Specord Uvvis automatic spectrophotometer. Values at the curves stand for NADHiPBQ ratios.
NEW
QUINONE
REDUCTASE
333
ASSAY
FIG. 4. Polarographic control of oxygen uptake in spontaneous oxidation of AMOBQ. Reaction medium consists of 1115 M phosphate buffer. pH 7.5. The polarographic volume is 1.3 ml. Catalase indicates addition of 2 pg of catalase (for explanation, text).
Hz. cell see
In Eq. [2], the NAD(P)H/O ratio is assumed to be unity which, strictly speaking, should be substantiated experimentally. Theoretically, the stoichiometry is unity provided that the oxygen reduction proceeds toward H,O. On the other hand, a formation of hydrogen peroxide at Step [2], in which the auto-oxidation of diphenol occurs, can easily be envisaged and would result in lowering the overall stoichiometry. Figures 1, 3, and 4 demonstrate different experimental approaches to the study of the stoichiometry of the individual Reactions [I] and [2] as well as the complete reaction sequence. To determine the stoichiometry in Reaction [l] p -benzoquinone (PBQ) was employed because its diphenolic form is not appreciably oxidized under the experimental conditions (Fig. 3). The equilibrium in this reaction is practically TABLE DISTRIBUTION FROM
Liver Activity” N’
113.8 ? 4.7 20
OF QUINONE-REDUCING DIFFERENT TISSUES
Heart 63.6 2 4.3 19
I ACTIVITY IN HOMOGENATES OF THE WHITE RAT”
Kidney 38.5 t 2.8 18
Brain 26.4 t 3.3 15
Skeletal muscle 10.4 t 1.1 18
” The assay conditions are as given under Materials and Methods. Reducing substrate is NADH. ’ Values are expressed as nanomoles of AMOBQ. Hz per minute per milligram of protein and are given as means ? SD. Protein estimation according to Gornall et al. (14). I’ Number of trials performed on three or four pooled tissues.
334
TITOVETS
AND PETROVSKY TABLE
DISTRIBUTION
2
OF QUINONE-REDUCING BRAIN
TISSUE
ACTIVITY
IN RABBIT-
PREPARATIONS”
Homogenate
Supernatant”
Mitochondria’
Mitochondria with DOC”,”
Activity with NADH’~ N’
8.2 f 0.4 13
7.2 t 0.8 10
11.06 !I 0.4 14
30.4 i 1.0 14
Activity with NADPH’ N’
9.8 k 0.6 12
10.6 k I.2 IO
8.0 ? 0.6 12
12.3 i 0.6 I?
” The assay conditions are as given under Materials and Methods. Ir The supernatant obtained by centrifugation of the homogenate at 8000 R for 15 min. c Concentration of KCN in the reaction medium: 2 mM. ” Added to the mitochondrial suspension: I .5 mM DOC. c Values are expressed as nanomoles of AMOBQ.H, per minute per milligram of protein and are given as the means t SD. Protein estimation according to Lowry rt a/. (13). ’ Number of tests.
completely shifted toward formation of the diphenol. The experimental NADIWPBQ ratio was 1.05 ? 0.04, N = 7 (the mean -+ standard deviation and number of tests). Figure 4 illustrates polarographic experiments undertaken to determine the AMOBQ. H,/O ratio at Step [2]. In these experiments, known amounts of AMOBQ. H2 were added to the reaction medium, and oxygen consumption was measured. The stoichiometric ratio here was 0.71 5 0.04, N = 7. Addition of beef liver catalase to the reaction medium upon completion of the AMOBQ. H2 oxidation resulted in the appearance of oxygen in the cell. In the presence of catalase, the AMOBQ. HZ/O ratio was 1.03 ? 0.06, N = 6. Thus, AMOBQ. Hz spontaneous oxidation is accompanied by formation of a small quantity of hydrogen peroxide if no catalase is present in the reaction medium. The NADH/O ratio with the menadione reductase preparation (Fig. I) containing contaminating catalase activity was 1.01 2 0.05, N = 9. This points to the fact that Hz0 is the final product in the overall reaction and also confirms the validity of the initial assumption on stoichiometry in Reaction [2]. In view of the almost ubiquitous presence of catalase activity in biological materials (15). it may turn out to be unnecessary to add catalase to the reaction medium in all cases: otherwise, an addition of catalase should be made to keep the stoichiometry at unity. It should be noted that the complete reaction sequence operates as a regenerative system in respect to AMOBQ. In a special investigation, it was established that this synthetic ortho-benzoquinone does not undergo any appreciable decomposition of its chemical structure in oxidationreduction cycles (16).
NEW
QUINONE
REDUCTASE
ASSAY
335
The experimental results on quinone-reducing activity distribution in different tissues and tissue preparations studied by the new method are summarized in Tables 1 and 2. The highest activity found in rat tissues occurred in the liver, whereas the lowest activity was found to be in muscle homogenates (see Table 1). It is of interest to note an increase in quinone-reducing activity upon addition of DOC to the mitochondria (Table 2), which may be indicative of a complex enzymic quinone-reducing activity in these organelles. The problem of differentiating among the enzymes that occur together in the same material and display quinone-reducing activity is a very interesting one. The quinonereducing enzymes, e.g., menadione reductase, NADH dehydrogenase [EC 1.6.99.31, are characterized by an acceptor specificity that extends over various quinones (l-7), and. because of this, they can hardly be differentiated on the basis of acceptor specificity only. On the other hand, this problem may be quite satisfactorily solved using different donor substrates (1,2,17) and inhibitors (2,4,18), provided that a reliable and accurate assay method for the quinone-reducing activity is available. REFERENCES I. 2. 3. 4. 5. 6. 7.
8. 9. 10. Il.
12. 13. 14. 15. 16. 17. 18.
Wosilait, W. D..and Nason. A. (1954) J. Biol. Chem. 206, 255-270. Marki, F.. and Martius, C. (1960) Biochem. 2. 333, 111-135. Guiditta. A., and Strecker. H. J. (1961) Biochim. Biophys. Acta 48, 10-19. Emster. L., Danielson, L.. and Ljungren. M. (1962) Biochim. Biophys. Acta 58, 171-188. Conover, T. E., Danielson, L. and Ernster. L. (1963) Biochim. BiophgJ. Acra 67, 254-267. Titovets, E. P., and Petrovsky, G. G. (1976) Biokhimiya (USSR) 41, 1522-1530. Titovets. E. P., Lunets. E. F., and Matusevich. P. A. (1972) Proceedings of the IVth International Biophysics Congress. Abstracts of Contributed Papers, Vol. 3, pp. 195-196, Moscow. Schneider, W. C. (1948)5. Bid. Chem. 176, 259-266. Wanzlick. H.-W.. and Jahnke. U. (1968) Chem. Ber. 101, 3744-3752. Beckman. P. (1963) Anal. Chem. 35, 98. Titovets. E. P. (1973) in “Manual on the Study of Biological Oxidation by Polarographic Method” (Frank. G. M., Kondrashova. M. N.. Mokhova, E. N.. and Rotenberg. J. S.. eds.), pp. 62-64. Nauka, Moscow. Umbreit. W. W., Burris. R. H.. and Stauffer. J. F. (1957) Manometric Techniques, p. 38. Burgess, Minneapolis, Minn. Lowry. 0. H., Rosebrough. N. J.. Farr, A. L.. and Randall. R. J. (1951) J. Bid. Chem. 193, 265-275. Gornall, A. G.. Bardawall, C. J., and David, M. M. (194915. Bid. Chem. 177, 751-766. Herbert, D., and Pinsent. J. (1948) Biochem. J. 43, 203-206. Volodko, L. V.. Matusevich, P. A.. Minko. A. A., and Titovets. E. P. (1977) Biokhimiya (USSR) 42. 205-210. Hatefi, Y.. and Stempel. K. E. (1%9) .I. Biol. Chem. 244, 2350-2357. Hateti, Y.. Stempel. K. E.. and Hanstein. W. G. (1969)5. Bid. Chem. 244, ‘3.5%2365.
BIOCHEMISTRY
81,
328-335 (1977)
A New Kinetic Assay Method for Quinone-Reducing Enzymes E. P. TITOVETS AND G. G. PETROVSKY The Byelorussian
State Research Physiotherapy,
Institute Minsk
oj- Neurology, 22044, USSR
Neurosurgery.
and
Received December I, 1976: accepted April 4, 1977 A new kinetic method is described for the assay of quinone-reducing enzymes in various biological materials. It is based on polarographic determination of oxygen uptake in spontaneous oxidation of the diphenol formed as a result ,2 (AMOBQ) enzymic reduction. The of 4-anilino-5-methoxybenzoquinone-1 stoichiometry of the reducing equivalent transfer in the reaction sequence from NAD(P)H to oxygen has been analyzed. Data are presented on quinone-reducing activity distributions in different tissues.
The conventional method for the assay of quinone-reducing enzymes employs spectrophotometry to measure the quinone reduction rate (l-5). A number of limitations intrinsic to optical spectroscopy narrow the application range of this method. One disadvantage is due to the difficulty or even the impossibility in applying the method while working with intensely light-scattering samples, e.g., concentrated cell or mitochondrial suspensions, dense tissue homogenates, etc. In addition, this method may become quite inadequate if the specific absorption maxima overlap those of other light-absorbing components in the reaction medium. The apprearance of new synthetic substrates for quinone-reducing enzymes (6.7) with certain remarkable properties has made it possible to develop a new nonoptical assay method as reported in this paper. The new method employs polarographic monitoring of oxygen uptake, stoichiometrically with respect to the diphenol oxidation rate, the diphenol appearing as a result of enzymic reduction of 4-anilino-5-methoxybenzoquinone-1.2 (AMOBQ), a new synthetic acceptor quinone. MATERIALS
AND METHODS
Materials. The acetone preparation of NAD(P)H: (quinone acceptor) oxidoreductase [EC 1.6.99.21, also called menadione reductase, was obtained from rat liver mitochondria as described earlier (6). Tissue homogenates for quinone-reducing activity distribution experiments were prepared on l/15 M phosphate buffer, pH 7.5. Various tissues from adult domestic rabbits and white rats of both sexes were taken for Copyright All rights
IZ~ 1977 by Academic Prers. Inc. of reproductmn m any form reserved
328 ISSN 00WZhY7
NEW
QUINONE
REDUCTASE
ASSAY
329
these experiments. Rabbit brain mitochondria and other fractions of brain homogenate were obtained by a differential centrifugation technique (8). 4-Aniline-5methoxybenzoquinoneI,2 (AMOBQ) was synthesized according to Wanzlick and Jahnke (9) at the Lenin Byelorussian State University; beef liver catalase [EC 1.11.1.61 was obtained from Sigma; NAD(P)H and sodium deoxycholate were from Reanal. All of the common chemicals employed were reagent grade. Assay of quinone-reducing activity. The reaction medium for the assay of quinone-reducing enzymes consists of 1115 M KH,PO,/Na,HPO, buffer, pH 7.5, 150 FM AMOBQ, 400 PM NADPH or NADH, and 100 wg/ml of catalase, the addition of the last item being optional (see Results and Discussion). The determinations may be carried out in polarographic cells designed for oxygen consumption measurements with a Clark-type oxygen electrode and a conventional recording polarograph, e.g., Refs. (10,ll). The reaction is normally started by addition of a tissue preparation sample to the reaction medium in the polarographic cell. The ensuing oxygen depletion in the medium is stoichiometric with respect to the AMOBQ enzymic reduction rate under the given experimental conditions. Nonspecific oxygen uptake. if any, should be subtracted from the obtained values. The activity values are expressed as nanomoles of AMOBQ reduced per minute per milligram of protein, one nanomole of reduced AMOBQ (AMOBQ.H,) being equivalent to 0.5 nmol of oxygen consumed in the reaction (see Results and Discussion). Vigorous stirring of the reaction medium must be strictly maintained throughout the entire determination procedure. The experiments are carried out at 20°C. Miscellaneous methods. Oxygen concentration in the reaction medium was calculated with a reference to temperature, barometric pressure. and ionic composition of the reaction medium (12). Protein was estimated by the method of Lowry et al. (13) or by the biuret method (14). NADH oxidation was controlled at 340 nm on a Specord Uvvis automatic spectrophotometer. The kinetic study of AMOBQ.H, spontaneous oxidation was carried out at 510 nm by the stop-flow technique, using a Durrum D 110 spectrophotometer. RESULTS
AND DISCUSSION
A polarographic determination of quinone-reducing activity is demonstrated in Fig. 1. In these experiments, the reaction was initiated by adding fractional amounts of NADH (below 400 FM) to the assay medium already containing 0.3 mg/ml of the menadione reductase preparation. This was done to facilitate study of the reducing equivalent transfer stoichiometry (see below). Normally, the reaction is started by adding an enzyme sample to an otherwise complete reaction medium contain-
330
TITOVETS
66yM
AND
PETROVSKY
NADH I
I MIN
FIG. 1. Polarographic record of oxygen uptake in the assay of menadione reductase activity. The reaction medium contains 0.5 mgiml of the crude acetone fraction of menadione reductase (6). The oxygen cell volume is 1.3 ml. All additions are made as 5Oq.I volumes. Values near the curves stand for the calculated NADH/O stoichiometric ratio (for explanation. see text).
ing 400 PM NADH or NADPH, as is recommended under Materials and Methods. The linear part of the oxygen uptake trace in Fig. 1 is used for calculating the menadione reductase activity of the preparation. The oxygen uptake slows down as NADH is depleted in the reaction medium until it stops altogether. A second addition of 66 PM NADH results in a new burst of oxygen uptake. The precision of the method was calculated from 49 trial tests representing different sets of three to five replica trials. From these experiments, the standard deviation of the rate values was 0.5% of the mean value and of the range, 3.7% of it. The choice of AMOBQ as an acceptor substrate was due to the fact that it is easily reduced enzymatically and its diphenolic form is equally easily oxidized in the presence of dissolved oxygen (6,7). AMOBQ acts as a carrier of reducing equivalents to oxygen. The reaction sequence for the present method may be written as: NAD.H
+ H+ + AMOBQ
AMOBQ.H2
Enz\ NAD+
+ % 0, -
AMOBQ
+ AMOBQ.H2
[II
+ HzO,
PI
where AMOBQ. H, stands for reduced AMOBQ and Enz represents any of the quinone-reducing enzymes. An enzymatic reduction of AMOBQ takes place at Step [l]. This reaction is conducted at reasonably high concentrations of both NAD(P)H and AMOBQ and is expected to proceed at a maximal rate (6). The AMOBQ reduction rate, thus, is equal to d]AMOBQ.H,]ldr
= k,[~,],
L31
NEW
QUINONE
1.0
05 MG
OF
REDUCTASE
MENADIONE
15 REDUCTASE
331
ASSAY
20
25
PREPARATION/CELL
FIG. 2. Plot of the initial oxygen uptake rates versus the quantity of menadione reductase preparation. In these experiments. the acetone fraction of mitochondrial menadione reductase was used (6). The specific activity of the preparation is 465 nmol of AMOBQ.H,/min/mg. On the ordinate. oxygen uptake rates are expressed in arbitrary units. The assay conditions are as specified under Materials and Methods.
where Et is the total enzyme concentration, and k, is the rate constant. The reaction rate at Step [2] may be presented as: -dO/dt
= -d [AMOBQ.H,]ldt
= k [AMOBQ.H,]
[O#.
]41
Under the specified experimental conditions, the concentration of oxygen is much higher than that of AMOBQ.H, in the assay solution, and, in view of that, Eq. [4] assumes a pseudo first order in relation to the diphenol concentration: d [AMOBQ.
H,]/dt
= k’ [AMOBQ.
H,]
151
where k’ = k[O,]+. According to Eq. [5], the maxima1 possible diphenol oxidation rate may be achieved if all of the initially added AMOBQ is in a reduced form. For a correct determination of the quinone-reducing activity, the following condition should be strictly observed: k,[E,]
G k’ [AMOBQ.
H&r
[cl
where [AMOBQ. H,],, stands for all initially present AMOBQ in the reduced form. Relationship [6] ascribes the limiting reaction in the overall reaction sequence to the enzymic Step [ 11. The practical realization of this was ascertained for the experimental conditions given, and the appropriate data are reported in Fig. 2. In these experiments, a preparation containing highly active mitochondrial menadione reductase was used (6). As can be seen from Fig. 2, the initial oxygen consumption rate is linear with
332
TITOVETS
AND
PETROVSKY
respect to the amount of enzyme preparation. This points to the validity of relationship [6]. The enzyme concentrations used in these experiments are much higher than those usually found in different tissues (cf. Tables 1 and 2). It is important to evaluate the time interval within which the oxygen uptake rate in the complete system approaches a steady state or, rather, the time required to reach a pseudo steady-state which, within experimental error, may be considered to coincide with the steady-state rate. This time covers the period in which the AMOBQ. H, concentration increases from zero to [AMOBQ. HPJs.st., where the latter is the steadystate diphenol concentration. Using Eqs. [3], [4], and [5], an equation for the diphenol concentration change rate in the system may be obtained: H,]ldt
d [AMOBQ.
Integration
= k, [E,] - Ii’ [AMOBQ.
H,]
[71
of Eq. [7] gives: k’[AMOBQ.H,],.,,.Ik,[E,]
= 1 - e-“”
PI
The left hand-side of Eq. [8] is the ratio of the diphenol oxidation rate to the diphenol formation rate in an enzymatic reaction. If 95%, or a value of 0.95, is assumed for the above ratio as a reasonable practical approach to the steady-state rate, then the time necessary for this may be calculated from Eq. [8]: t = 2.99/k’
[91
The rate constant k’ from stop-flow spectrophotometric determinations was found to be 0.3 set-‘. Substituting this value for k’ in Eq. [9] gives 7.7 sec. Thus, the oxygen consumption rate, or the AMOBQ. H2 oxidation rate, becomes practically equal to the AMOBQ reduction rate within 7.7 sec. 35vM
PBO
79~“M
PBO
123vM
PBO
1
1.09 \
s D 4
l1 MIN
FIG. 3. Spectrophotometric control of NADH oxidation in the presence of menadione reductase and PBQ as acceptor substrate. The reaction medium contains 0.1 mg of the menadione reductase acetone fraction. The measurements are carried out against a blank with no enzyme preparation added. Light path = 1 cm; Specord Uvvis automatic spectrophotometer. Values at the curves stand for NADHiPBQ ratios.
NEW
QUINONE
REDUCTASE
333
ASSAY
FIG. 4. Polarographic control of oxygen uptake in spontaneous oxidation of AMOBQ. Reaction medium consists of 1115 M phosphate buffer. pH 7.5. The polarographic volume is 1.3 ml. Catalase indicates addition of 2 pg of catalase (for explanation, text).
Hz. cell see
In Eq. [2], the NAD(P)H/O ratio is assumed to be unity which, strictly speaking, should be substantiated experimentally. Theoretically, the stoichiometry is unity provided that the oxygen reduction proceeds toward H,O. On the other hand, a formation of hydrogen peroxide at Step [2], in which the auto-oxidation of diphenol occurs, can easily be envisaged and would result in lowering the overall stoichiometry. Figures 1, 3, and 4 demonstrate different experimental approaches to the study of the stoichiometry of the individual Reactions [I] and [2] as well as the complete reaction sequence. To determine the stoichiometry in Reaction [l] p -benzoquinone (PBQ) was employed because its diphenolic form is not appreciably oxidized under the experimental conditions (Fig. 3). The equilibrium in this reaction is practically TABLE DISTRIBUTION FROM
Liver Activity” N’
113.8 ? 4.7 20
OF QUINONE-REDUCING DIFFERENT TISSUES
Heart 63.6 2 4.3 19
I ACTIVITY IN HOMOGENATES OF THE WHITE RAT”
Kidney 38.5 t 2.8 18
Brain 26.4 t 3.3 15
Skeletal muscle 10.4 t 1.1 18
” The assay conditions are as given under Materials and Methods. Reducing substrate is NADH. ’ Values are expressed as nanomoles of AMOBQ. Hz per minute per milligram of protein and are given as means ? SD. Protein estimation according to Gornall et al. (14). I’ Number of trials performed on three or four pooled tissues.
334
TITOVETS
AND PETROVSKY TABLE
DISTRIBUTION
2
OF QUINONE-REDUCING BRAIN
TISSUE
ACTIVITY
IN RABBIT-
PREPARATIONS”
Homogenate
Supernatant”
Mitochondria’
Mitochondria with DOC”,”
Activity with NADH’~ N’
8.2 f 0.4 13
7.2 t 0.8 10
11.06 !I 0.4 14
30.4 i 1.0 14
Activity with NADPH’ N’
9.8 k 0.6 12
10.6 k I.2 IO
8.0 ? 0.6 12
12.3 i 0.6 I?
” The assay conditions are as given under Materials and Methods. Ir The supernatant obtained by centrifugation of the homogenate at 8000 R for 15 min. c Concentration of KCN in the reaction medium: 2 mM. ” Added to the mitochondrial suspension: I .5 mM DOC. c Values are expressed as nanomoles of AMOBQ.H, per minute per milligram of protein and are given as the means t SD. Protein estimation according to Lowry rt a/. (13). ’ Number of tests.
completely shifted toward formation of the diphenol. The experimental NADIWPBQ ratio was 1.05 ? 0.04, N = 7 (the mean -+ standard deviation and number of tests). Figure 4 illustrates polarographic experiments undertaken to determine the AMOBQ. H,/O ratio at Step [2]. In these experiments, known amounts of AMOBQ. H2 were added to the reaction medium, and oxygen consumption was measured. The stoichiometric ratio here was 0.71 5 0.04, N = 7. Addition of beef liver catalase to the reaction medium upon completion of the AMOBQ. H2 oxidation resulted in the appearance of oxygen in the cell. In the presence of catalase, the AMOBQ. HZ/O ratio was 1.03 ? 0.06, N = 6. Thus, AMOBQ. Hz spontaneous oxidation is accompanied by formation of a small quantity of hydrogen peroxide if no catalase is present in the reaction medium. The NADH/O ratio with the menadione reductase preparation (Fig. I) containing contaminating catalase activity was 1.01 2 0.05, N = 9. This points to the fact that Hz0 is the final product in the overall reaction and also confirms the validity of the initial assumption on stoichiometry in Reaction [2]. In view of the almost ubiquitous presence of catalase activity in biological materials (15). it may turn out to be unnecessary to add catalase to the reaction medium in all cases: otherwise, an addition of catalase should be made to keep the stoichiometry at unity. It should be noted that the complete reaction sequence operates as a regenerative system in respect to AMOBQ. In a special investigation, it was established that this synthetic ortho-benzoquinone does not undergo any appreciable decomposition of its chemical structure in oxidationreduction cycles (16).
NEW
QUINONE
REDUCTASE
ASSAY
335
The experimental results on quinone-reducing activity distribution in different tissues and tissue preparations studied by the new method are summarized in Tables 1 and 2. The highest activity found in rat tissues occurred in the liver, whereas the lowest activity was found to be in muscle homogenates (see Table 1). It is of interest to note an increase in quinone-reducing activity upon addition of DOC to the mitochondria (Table 2), which may be indicative of a complex enzymic quinone-reducing activity in these organelles. The problem of differentiating among the enzymes that occur together in the same material and display quinone-reducing activity is a very interesting one. The quinonereducing enzymes, e.g., menadione reductase, NADH dehydrogenase [EC 1.6.99.31, are characterized by an acceptor specificity that extends over various quinones (l-7), and. because of this, they can hardly be differentiated on the basis of acceptor specificity only. On the other hand, this problem may be quite satisfactorily solved using different donor substrates (1,2,17) and inhibitors (2,4,18), provided that a reliable and accurate assay method for the quinone-reducing activity is available. REFERENCES I. 2. 3. 4. 5. 6. 7.
8. 9. 10. Il.
12. 13. 14. 15. 16. 17. 18.
Wosilait, W. D..and Nason. A. (1954) J. Biol. Chem. 206, 255-270. Marki, F.. and Martius, C. (1960) Biochem. 2. 333, 111-135. Guiditta. A., and Strecker. H. J. (1961) Biochim. Biophys. Acta 48, 10-19. Emster. L., Danielson, L.. and Ljungren. M. (1962) Biochim. Biophys. Acta 58, 171-188. Conover, T. E., Danielson, L. and Ernster. L. (1963) Biochim. BiophgJ. Acra 67, 254-267. Titovets, E. P., and Petrovsky, G. G. (1976) Biokhimiya (USSR) 41, 1522-1530. Titovets. E. P., Lunets. E. F., and Matusevich. P. A. (1972) Proceedings of the IVth International Biophysics Congress. Abstracts of Contributed Papers, Vol. 3, pp. 195-196, Moscow. Schneider, W. C. (1948)5. Bid. Chem. 176, 259-266. Wanzlick. H.-W.. and Jahnke. U. (1968) Chem. Ber. 101, 3744-3752. Beckman. P. (1963) Anal. Chem. 35, 98. Titovets. E. P. (1973) in “Manual on the Study of Biological Oxidation by Polarographic Method” (Frank. G. M., Kondrashova. M. N.. Mokhova, E. N.. and Rotenberg. J. S.. eds.), pp. 62-64. Nauka, Moscow. Umbreit. W. W., Burris. R. H.. and Stauffer. J. F. (1957) Manometric Techniques, p. 38. Burgess, Minneapolis, Minn. Lowry. 0. H., Rosebrough. N. J.. Farr, A. L.. and Randall. R. J. (1951) J. Bid. Chem. 193, 265-275. Gornall, A. G.. Bardawall, C. J., and David, M. M. (194915. Bid. Chem. 177, 751-766. Herbert, D., and Pinsent. J. (1948) Biochem. J. 43, 203-206. Volodko, L. V.. Matusevich, P. A.. Minko. A. A., and Titovets. E. P. (1977) Biokhimiya (USSR) 42. 205-210. Hatefi, Y.. and Stempel. K. E. (1%9) .I. Biol. Chem. 244, 2350-2357. Hateti, Y.. Stempel. K. E.. and Hanstein. W. G. (1969)5. Bid. Chem. 244, ‘3.5%2365.