Process for the selective production of reduced oxygen species

Process for the selective production of the reduced oxygen species superoxide, hydrogen peroxide and hydroxyl radicals, wherein oxygen is reduced with NAD(P)H in the presence of an NAD(P)H-dependent, non-autoxidisable diaphorase, of an appropriate autoxidisable redox partner and optionally of an appropriate buffer system. Also, process for the determination of superoxide dismutase in which a superoxide-yielding reaction is coupled with two competing superoxide-consuming reactions, one superoxide-consuming reaction being a conventional indicator reaction and the other superoxide-consuming reaction being the superoxide dismutase reaction, wherein, as superoxide-yielding reaction, there is used the reduction of oxygen with NAD(P)H in the presence of an NAD(P)H-dependent, non-autoxidisable diaphorase, of an autoxidisable one-electron step-inducing redox partner with a one-electron redox potential in the range of from -150 mV to -500 mV and optionally of an appropriate buffer system. Also, process for the determination of NAD(P)H and of NAD(P)H-yielding reactions in which oxygen is reduced to hydrogen peroxide by NAD(P)H and the hydrogen peroxide is determined in knowm manner, wherein the reduction of the oxygen by NAD(P)H is carried out in the presence of an NAD(P)H-dependent, non-autoxidisable diaphorase, of an appropriate two-electron redox partner with a two-electron redox potential in the range of from 0 mV to 150 mV and of an appropriate buffer system. Also, reagents for carrying out these processes.

The present invention is concerned with a process for the selective 
production of reduced oxygen species and with reagents suitable for this 
purpose. 
In the case of the enzymatic reduction of the oxygen molecule, various 
products are formed, depending upon the number of transferring electrons, 
some of which products are very reactive. The following Table gives a 
survey of the possible redox reactions. Furthermore, examples are given of 
enzymes which are able to catalyse the redox reactions in question: 
TABLE 1 
______________________________________ 
Redox reactions of the oxygen molecule 
number of trans- 
No. ferring electrons 
product enzyme example 
______________________________________ 
1 1E + 2E O.sub.2.sup..- ; H.sub.2 O.sub.2 
xanthine oxidase 
2 2E H.sub.2 O.sub.2 
glucose oxidase 
3 3E OH radical non-specificity 
of xanthine 
oxidase 
4 4E H.sub.2 O cytochrome 
oxidase 
______________________________________ 
Especially the superoxide (O.sub.2.sup.-) resulting in the case of a 
one-electron step has a considerable importance in the human and animal 
organism in the defense against foreign substances. Since the discovery in 
1969 of the superoxide dismutases, i.e. enzymes which catalyse the 
dismutation of superoxide into hydrogen peroxide and oxygen, the field of 
oxygen biochemistry has achieved considerable scientific as well as 
economic interest. Questions as to which reduction products arise in the 
case of the most varied biochemical oxygen reductions, how these processes 
are influenced by the participating reaction components, which substrates 
are needed, which intermediate products arise and the like, thereby being 
in the foreground. Superoxide dismutase has already been used 
therapeutically, mainly against inflammations of the joints. A dependable 
method of determination is of economic importance for dosaging. 
In the case of this question, the problem continually arises of analysing 
the most varied oxygen reduction products, as well as of determining the 
concentration of the enzymes and substrates participating in the reduction 
processes. For this purpose, there is a series of determination processes, 
especially for superoxide, hydrogen peroxide and the hydroxyl radical, as 
well as for the superoxide dismutases. A disadvantage of these processes 
of determination has hitherto been that the known enzymatic oxygen 
reduction processes always lead to a mixture of various reduction 
products, so that definitive statements regarding the physiological action 
of the reduction products, as well as the use of the reduction products 
for the analysis of enzymes reacting them and reaction components 
participating in their formation are decisively impaired. Thus, the 
superoxide-generating system, xanthine oxidase, mainly used today for the 
analysis of superoxide or superoxide dismutase, gives not only superoxide 
but also hydrogen peroxide (cf. Table 1. No. 1) and, in part, also the 
very reactive hydroxyl radical. 
Therefore, it is an object of the present invention to provide a simple 
process with which it is possible selectively to produce in a one-, two- 
or three-electron step, superoxide, hydrogen peroxide or hydroxyl 
radicals. Surprisingly, this object can be achieved by reducing oxygen 
with NAD(P)H in the presence of an NAD(P)H-dependent, non-autoxidisable 
diaphorase and of a specially selected autoxidisable redox partner. 
Thus, according to the present invention, there is provided a process for 
the selective production of the reduced oxygen species superoxide, 
hydrogen peroxide and hydroxyl radicals, wherein oxygen is reduced with 
NAD(P)H in the presence of an NAD(P)H-dependent, non-autoxidisable 
diaphorase and of an appropriate autoxidisable redox partner. 
NAD(P)H-dependent, non-autoxidisable diaphorases are diaphorases which are 
not able to catalyse the reduction of oxygen by NAD(P)H. 
Within the meaning of the present invention, all such diaphorases can, in 
principle, be used. Especially advantageously, there is used a 
diaphorase-active NADP-ferredoxin-(Cyt c)-oxidoreductase (E.C. 1.6.99.4), 
which can be obtained, for example, from spinach or Euglena gracilis. 
Commercially available diaphorase from Clostridium kluyveri, as well as a 
diaphorase from micro-organisms which is available from Boehringer 
Mannheim GmbH can also be used. 
As autoxidisable redox partner, there can be used all substances which are 
able to transfer one or more electrons to oxygen. For the one-electron 
step, there can be used those redox partners, the one-electron redox 
potential E.sub.o of which is in the range of from -150 mV to -500 mV. In 
particular, within the scope of the present invention there have proved to 
be useful bipyridilium salts, for example 1,1'-dimethyl-4,4'-bipyridilium 
chloride (paraquat) and 1,1'-dimethylene-2,2'-bipyridilium chloride 
(diquat): triazonium salts, for example 
1-methyl-4-(4,6-dimethyl-2-sym-triazinyl)-pyridinium bromide and 
1-methyl-4-(2-sym-triazinyl)-pyridinium bromide; anthraquinone 
derivatives, for example anthraquinone-2-sulphonic acid; nitrofuran 
derivatives, for example nitrofurantoin; ferredoxins; pteridines and 
isoalloxazines, for example riboflavine. 
As redox partners for the two-electron step there can be used those 
substances, the two-electron redox potential E.sub.o of which lies in the 
range of from 0 to 150 mV. As examples thereof, there are to be mentioned, 
in particular, quinone derivatives, especially p-benzoquinone derivatives, 
such as 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 
2,3-dimethyl-5,6-methylenedioxy-p-benzoquinone. 
For the production of hydroxyl radicals, the end product of the 
three-electron step, the NAD(P)H-dependent non-autoxidisable diaphorase is 
coupled with those substances which, in the reduced state, can transfer an 
electron to hydrogen peroxide in air-saturated solution. Compounds which 
are especially preferred for this purpose include anthraquinone 
derivatives, such as anthraquinone-2-sulphonic acid; nitrofurantoin and 
ferredoxins. 
The one- and two-electron reductions of oxygen are preferably carried out 
in air- or oxygen-saturated solution. The temperature can be varied within 
relatively wide limits, it being preferable to work at 18.degree. to 
25.degree. C. or at ambient temperature. However, lower or higher 
temperatures can also be chosen, the upper temperature range being limited 
by the stability of the selected enzyme. 
An appropriate buffer can possibly be added to the reagent mixture. Within 
the scope of the present invention, all buffers can be used which are 
effective in the pH range of from 6 to 9 and which are not radical 
receptors. A phosphate buffer of pH 7 to 8 has proved to be especially 
useful. The buffer is advantageously used in a concentration of 1 to 500 
and preferably of 50 to 200 mmolar. 
For the selective production of hydroxyl radicals, hydrogen peroxide is 
added under anaerobic conditions to the reaction solution of 
NAD(P)H-dependent, non-autoxidisable diaphorase, autoxidisable redox 
partner and optionally an appropriate buffer system in an appropriate 
solvent, preferably water. It is also possible to work under aerobic 
conditions and with the addition of superoxide dismutase. The primarily 
formed superoxide is thereby rapidly dismuted to oxygen and hydrogen 
peroxide, the latter then being selectively reduced to the hydroxyl 
radical in a further one-electron step. 
The present invention also provides a reagent for carrying out the process 
according to the present invention which, possibly in separate units, 
contains in the form of a solution, of a powder mixture, of a reagent 
tablet or of a lyophilisate, NAD(P)H, an NAD(P)H-dependent, 
non-autoxidisable diaphorase and an autoxidisable redox partner, as well 
as optionally an appropriate buffer system. 
Thus, according to the process of the present invention or with the help of 
the reagent according to the present invention, it is possible selectively 
to reduce the oxygen molecule to definite reduction products. Without 
having to fear disturbing side effects, the action of the individual 
oxygen reduction products on various substances, enzymatic systems or the 
like can, therefore, be observed. In this way, questions regarding the 
differential toxicity of the individual oxygen reduction products or the 
sensitivity of various biological materials towards the oxygen reduction 
products can be selectively investigated. For this purpose, the samples to 
be investigated are mixed with a test mixture which, besides NAD(P)H, 
contains an NAD(P)H-dependent, non-autoxidisable diaphorase and an 
autoxidisable redox partner for the production of superoxide, hydrogen 
peroxide or a hydroxyl radical, as well as optionally also an appropriate 
buffer system. The effect which is brought about by the oxygen reduction 
product produced can be analysed by the usual methods known to the expert. 
In principle, in this way it is possible to measure the most varied 
reaction partners in such reactions in which the reduced oxygen species 
appears as a reaction partner or which are induced or catalysed by the 
reduced oxygen species. There is here mentioned, by way of example, the 
oxidation of hydroxylamine to nitrite ions by superoxide. Since this 
oxidation reaction proceeds stoichiometrically, the superoxide-providing 
system according to the present invention can be used for the 
determination of hydroxylamine. The nitrite ions formed by the oxidation 
of hydroxylamine are, for this purpose, determined by the conventional 
methods. 
Various antibiotics, drugs, for example adriamycin, bleomycin and 
nitrofurantoin, and xenobiotics, for example herbicides and insecticides, 
are potential autoxidisable redox partners for a one-, two- or 
three-electron reduction of the oxygen according to the principle of the 
present invention. With the help of the process according to the present 
invention, it is possible to investigate the action of a particular drug 
or of a particular antibiotic on the oxygen reduction. For this purpose, a 
test system which contains NAD(P)H and an NAD(P)H-dependent, 
non-autoxidisable diaphorase and the substance to be investigated is 
brought into contact with oxygen. The resultant oxygen reduction product 
is determined by conventional processes. Thus, for example, with the help 
of hydroxylamine, it is possible to test whether superoxide has been 
formed. Nitrite ions possibly obtained by the oxidation of hydroxylamine 
can be measured by conventional methods. 
For the detection of possibly formed hydrogen peroxide, there are available 
numerous known methods of determination. Trinder's reaction in its many 
known variants has proved to be especially suitable for this purpose. 
The test solution can be investigated for possibly formed hydroxyl radicals 
with the help of methionine, the ethylene formed in the case of this known 
test being determined gas chromatographically. The possibility of 
selectively producing superoxide in a one-electron step with an 
NAD(P)H-dependent, non-autoxidisable diaphorase and an appropriate 
autoxidisable redox partner also leads to a selective detection of 
superoxide dismutase. Superoxide dismutase (SOD) catalyses the following 
reaction: 
EQU 2O.sub.2.sup.- +2H.sup.+ .revreaction.H.sub.2 O.sub.2 +O.sub.2 
Superoxide dismutase determinations generally depend upon the following 
principle: a superoxide-yielding reaction is coupled with two 
superoxide-consuming reactions, one of the superoxide-consuming reactions 
being an indicator reaction and the other superoxide-consuming reaction 
being a superoxide dismutase reaction. Consequently, the superoxide 
dismutase reaction competes with the indicator reaction for the superoxide 
molecules present. Consequently, the more superoxide dismutase is present 
in the test solution, the more is the indicator reaction inhibited. A 
survey of the at present most common superoxide dismutase determinations 
is given in the article "Superoxide dismutase assays: A review of 
methodology" from "Superoxide and superoxide dismutases" by A. M. 
Michelson, J. M. McCord and I. Fridovich, pub. Academic Press, New York, 
1977. 
As superoxide-yielding reaction, in the case of the at present most 
frequently used superoxide dismutase test, there is employed the xanthine 
oxidase reaction. As already mentioned, the xanthine oxidase reaction 
gives superoxide non-specifically but the side reactions also form 
hydrogen peroxide and the hydroxyl radical. The superoxide dismutase 
determination is thereby considerably disturbed. As indicator reaction 
there is usually used the cytochrome reduction test, the so-called 
adrenaline test or the nitro blue tetrazolium test. These multi-component 
systems also give rise to considerable difficulties if various reduced 
oxygen species occur simultaneously. Furthermore, in the case of 
multi-component systems, there is to be reckoned with an undesired but 
always measurable additional catalysis by non-specific bound transition 
metal ions, for example iron and copper. 
If superoxide produced in the manner according to the present invention is 
used for the determination of superoxide dismutase, then a 
disturbance-free test is obtained. As indicator reaction, any known 
indicator reaction can be coupled. For the determination of superoxide 
dismutase, there has proved to be especially useful the combination of the 
superoxide-yielding reaction according to the present invention with the 
hydroxylamine test, hydroxylamine hereby being oxidised to nitrite ions by 
the resultant superoxide which, after diazotisation and subsequent azo 
coupling, can be measured photometrically. 
The present invention also provides a process for the determination of 
superoxide dismutase by coupling of a superoxide-yielding reaction with a 
superoxide-consuming indicator reaction which competes with the 
dismutation of the superoxide catalysed by the superoxide dismutase, 
whereby, as superoxide-yielding reaction, there is used the reduction of 
oxygen by NAD(P)H in the presence of an NAD(P)H-dependent, 
non-autoxidisable diaphorase and of an appropriate autoxidisable 
one-electron redox partner, as well as optionally of an appropriate buffer 
system. 
Furthermore, the present invention provides a reagent for the determination 
of superoxide dismutase which, in the form of a solution, of a powder 
mixture, of a reagent tablet or of a lyophilisate, contains NAD(P)H, an 
NAD(P)H-dependent, non-autoxidisable diaphorase, an autoxidisable 
one-electron redox partner and the components for an appropriate indicator 
reaction, as well as optionally an appropriate buffer system. 
As NAD(P)H-dependent, non-autoxidisable diaphorases, there are especially 
preferred those enzymes mentioned hereinbefore. As autoxidisable 
one-electron redox partners, there are also preferred those substances 
mentioned hereinbefore. As indicator reaction, there is preferably used 
the oxidation of hydroxylamine to nitrite ions, with the subsequent 
determination of the nitrite ions. As components of this reaction, to the 
above-mentioned reagent there is then added hydroxylamine and the 
substances necessary for the determination of the nitrite ions, for 
example sulphanilic acid and .alpha.-naphthylamine or sulphanilamide and 
naphthylethylenediamine. 
The various components are preferably contained in the reagent in such 
amount ratios that, in 2 ml. of final test solution, the following 
concentration ratios are present: 
1-10 .mu.mole NAD(P)H 
0.05-0.5 mg. diaphorase 
0.1-5 .mu.mole redox partner 
1-10 .mu.mole indicator substance 
50-200 .mu.mole buffer, pH 6-9 
Especially preferred is a test mixture which, in 2 ml. of water, contains: 
5 .mu.mole NAD(P)H 
0.5 mg. diaphorase 
1 .mu.mole redox partner 
1 .mu.mole hydroxylamine and 
60-100 .mu.mole phosphate buffer, pH 7.5-8.0. 
The selective reduction of the oxygen with NAD(P)H in a two-electron step 
to hydrogen peroxide in the presence of an NAD(P)H-dependent, 
non-autoxidisable diaphorase and of an appropriate two-electron redox 
partner, as well as optionally of an appropriate buffer system also makes 
possible a determination of NAD(P)H which is easy to carry out. For this 
purpose, the sample to be investigated is saturated with oxygen and mixed 
with a test mixture which contains an NAD(P)H-dependent, non-autoxidisable 
diaphorase, an appropriate two-electron redox partner and optionally an 
appropriate buffer system, as well as a hydrogen peroxide detection 
system. The hydrogen peroxide concentration measured with the help of the 
hydrogen peroxide detection system is a direct measure of the NAD(P)H 
present in the sample. 
The following Examples are given for the purpose of illustrating the 
present invention:

EXAMPLE 1 
Production and determination of superoxide 
2 ml. Phosphate buffer (0.2M; pH 7.8) are placed into a 10 ml. reagent 
vessel. Air is passed through the buffer solution for 30 minutes at 
ambient temperature in order to saturate it with air. Thereafter, the 
mixture is mixed with 0.1 ml. of an aqueous solution which contains 1 
.mu.mole NADH, as well as with 0.1 ml. of an aqueous solution which 
contains 0.4 .mu.mole anthraquinone-2-sulphonic acid. The solution thus 
obtained is mixed with 0.5 mg. diaphorase (Boehringer Mannheim GmbH, 
obtained from microorganisms; lyophilised) dissolved in 1 ml. distilled 
water. The resultant superoxide is detected with the help of the known 
hydroxylamine reaction. 
For this purpose, to the reaction mixture there is additionally added to 
the already mentioned substances 0.1 ml. of a hydroxylammonium chloride 
solution, prepared by dissolving 6.9 mg. hydroxylammonium chloride in 10 
ml. distilled water. The resultant nitrite ions are determined by the 
addition of a 1% (w/v) sulphanilamide solution in 25% hydrochloric acid 
and after the addition of 0.02% aqueous naphthylethylenediamine 
dihydrochloride, as well as by measurement of the azo coloured material 
formed at 540 nm. 
From the measured extinction values, there can be determined the yield of 
superoxide by comparison with a calibration curve obtained with known 
nitrite concentrations. 
Similar results are achieved when, instead of the above-mentioned 
anthraquinone-2-sulphonic acid, there are used other one-electron 
receptors, for example 1,1'-dimethyl-4,4'-bipyridilium chloride, 
1-methyl-4-(4,6-dimethyl-2-sym-triazinyl)-pyridinium bromide, 
1-methyl-4-(2-sym-triazinyl)-pyridinium bromide or nitrofurantoin. 
EXAMPLE 2 
Production of hydrogen peroxide 
2 ml. Air-saturated phosphate buffer (0.2M, pH 7.8) are prepared in a 10 
ml. reagent vessel in the manner described in Example 1. To this are added 
0.1 ml. of an aqueous solution which contains 1 .mu.mole NADH and 0.1 ml. 
of an aqueous solution which contains 0.4 .mu.mole 
2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. The so obtained solution 
is mixed with 0.5 mg. diaphorase (Boehringer Mannheim GmbH; obtained from 
micro-organisms; lyophilised), dissolved in 1 ml. of distilled water. The 
hydrogen peroxide formed is determined according to known methods, for 
example by Trinder's method. 
Comparable results are obtained when, instead of the above-mentioned redox 
partner, there are used other two-electron redox partners, for example 
2,3-dimethyl-5,6-methylenedioxy-p-benzoquinone. 
EXAMPLE 3 
Production and determination of hydroxyl radicals 
(A) 2 ml. of phosphate buffer (0.2M, pH 7.8) are placed into a 10 ml. 
reagent glass. There are successively added thereto, under anaerobic 
conditions, 0.1 ml. of an aqueous solution which contains 0.1 .mu.mole 
NADH; 0.1 ml. of an aqueous solution which contains 0.4 .mu.mole 
anthraquinone-2-sulphonic acid; 0.1 ml. of a 2% hydrogen peroxide 
solution; and 0.5 mg. diaphorase (Boehringer Mannheim GmbH; obtained from 
micro-organisms; lyophilised), dissolved in 1 ml. distilled water. 
The resultant hydroxyl radicals are detected in known manner with the help 
of methionine splitting. From the methionine, ethylene is split off, which 
can be measured gas chromatographically. 
(B) 2 ml. of phosphate buffer (0.2M; pH 7.8), which has been saturated with 
air in the manner described in Example 1, are placed in a 10 ml. reagent 
glass. There are also added 0.1 ml. of an aqueous solution which contains 
0.1 .mu.mole NADH; 0.1 ml. of an aqueous solution which contains 0.4 
.mu.mole N-(5-nitro-2-furylidene)-1-aminohydantoin(nitrofurantoin); 200 
Units (as defined in Example 4) of superoxide dismutase; and 1 mg. 
NADP-ferredoxin-(Cyt-c)-oxidoreductase, dissolved in 1 ml. distilled 
water. 
The hydroxyl radicals formed are, as described above, determined by the 
addition of methionine. 
EXAMPLE 4 
Determination of superoxide dismutase 
1. Preparation of a calibration curve 
Into a 10 ml. test tube is placed 0.5 ml. water and the following solutions 
are pipetted into it: 1.0 ml. air-saturated phosphate buffer (pH 7.8) (65 
.mu.mole), 0.1 ml. hydroxylamine hydrochloride (1.0 .mu.mole); 0.1 ml. 
anthraquinone-2-sulphonic acid (1.0 .mu.mole); and 1 ml. diaphorase (0.5 
mg. protein). 
The solution mixture is vigorously shaken and thereafter mixed with 0.1 ml. 
superoxide dismutase (increasing concentrations of 0 to 100 Units) and 0.1 
ml. NADH (5 .mu.mole). 
The solution obtained is vigorously mixed and incubated for 15 minutes at 
22.degree. C. After this reaction time, 0.5 ml. of this reaction solution 
is mixed with nitrite reagent which consists of 0.5 ml. of a 1% (w/v) 
sulphanilamide solution in 25% hydrochloric acid and 0.5 ml. of a 0.02% 
(w/v) naphthylethylenediamine dichloride solution in water. The extinction 
is followed spectrophotometrically at 540 nm. FIG. 1 of the accompanying 
drawing shows the calibration curve thus produced. 
For superoxide dismutase, one enzyme unit is given as the amount of enzyme 
which brings about a 50% inhibition of the detector reaction, i.e. in the 
present case the oxidation of hydroxylamine to nitrite ions. 
From FIG. 1, there is given for the investigated enzyme an activity of one 
unit per 0.7 .mu.g. protein according to the following equation: 
##EQU1## 
2. Determination of unknown superoxide dismutase concentrations 
In the same manner as described above, there can also be determined unknown 
superoxide dismutase concentrations in that, instead of the standard with 
known superoxide dismutase concentration, there is used the corresponding 
amount of a sample with unknown superoxide dismutase concentration, the 
measured extinction values being compared with the calibration curve, 
there thus being determined the superoxide dismutase concentration 
corresponding to the measured extinction value. 
EXAMPLE 5 
Determination of NADH 
Hydrogen peroxide is produced in the manner described in Example 2. Instead 
of the solution with a known NADH content, there is merely employed the 
sample solution with an unknown NADH content. The amount of hydrogen 
peroxide formed is, after the reaction with hydrogen peroxide formation 
from NADH has taken place with complete utilisation of the NADH, measured 
as follows: 
The following solutions are prepared: 
Solution 1: 100 mg. dichlorophenolsulphonic acid are dissolved in 4 ml. 
ethanol and diluted with 4 ml. water. 
Solution 2: 0.1 g. 4-aminoantipyrine are dissolved in 2.5 ml. water. 
Solution 3: 10 mg. peroxidase are dissolved in 10 ml. water. 
For the determination of the hydrogen peroxide, to 1 ml. of the hydrogen 
peroxide-containing solution to be tested are added 0.8 ml. phosphate 
buffer (0.1M; pH 7.0), 0.4 ml. of Solution 1, 0.1 ml. of Solution 2 and, 
for initiating the reaction, 0.1 ml. of Solution 3. After a reaction time 
of 30 minutes, the extinction is measured at 540 nm. By comparison of the 
found extinction value with an appropriate calibration curve, the NADH 
content of the investigated sample is determined. 
The calibration curve is obtained by carrying out the above determination 
process with samples which have different but known NADH concentrations. 
It will be understood that the specification and examples are illustrative 
but not limitative of the present invention and that other embodiments 
within the spirit and scope of the invention will suggest themselves to 
those skilled in the art.