JPH1075779A - Stabilization of bilirubin oxidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject enzyme useful as a clinical testing reagent for measuring bilirubin and having an improved stability in a liquid state by blending a specific heterocyclic compound containing nitrogen with a liquid containing a bilirubin oxidase. SOLUTION: This method for stabilizing a bilirubin oxidase is to blend one or two kinds or more kinds of a mercapto derivative of a heterocyclic compound containing nitrogen such as a 2-mercaptoimidazole expressed by formula I (R<1> , R<2> are both H or together form a benzene ring which may be substituted by a lower alkyl group; R<3> is H or a lower alkyl group), a 3-mercapto-1,2,4- triazole expressed by formula II (either one of R<4> , R<5> is H or a lower alkyl and the other forms a double bond between nitrogen atom bonding therewith and an adjacent carbon atom in 5th position) or a 6-mercaptopurine expressed by formula III (R<6> is H or a lower alkyl) with a liquid containing the bilirubin oxidase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for stabilizing bilirubin oxidase, and more particularly, to a method for stabilizing bilirubin oxidase for a long period of time when bilirubin oxidase is stored in a liquid state. And a bilirubin oxidase reagent to which the method is applied.

[0002]

2. Description of the Related Art Bilirubin oxidase is an enzyme that catalyzes the reaction of oxidizing bilirubin to biliverdin, and is used in clinical tests as an optical measurement method and a measurement reagent for bilirubin in a living body. Reagents for measuring bilirubin using bilirubin oxidase (herein referred to as bilirubin oxidase reagents) include a total bilirubin measurement reagent and a direct bilirubin measurement reagent, and bilirubin oxidase is a lyophilized product thereof. The powder is dissolved or dispersed in a buffer solution and used as an aqueous liquid. For the reagent for measuring total bilirubin, p
H buffer in the range of 6.0 to 9.0, for example, N- (2-acetamido) -2-aminoethanesulfonic acid (abbreviated as AC
ES) A good buffer such as a buffer or a phosphate buffer is used, and sodium cholate,
Preferably, a directing agent such as sodium lauryl sulfate (abbreviated as SDS) is added to the buffer.

[0003] Reagents for measuring bilirubin directly have a pH of 3.
Buffers in the range of 0 to 4.5, for example, lactate-sodium lactate buffer, citrate buffer and the like are used. Examples of bilirubin oxidase include, for example, myrocesium-derived bilirubin oxidase, shrimp mushroom (Trachydermatsum
odae) is known, and the amount of bilirubin oxidase to be used is appropriately determined within a range showing the desired enzyme activity.
0 units / ml, preferably 0.1 for total bilirubin determination.
08-4 units / ml, 0.4 for direct bilirubin measurement
It is used in the range of ８8 units / ml. However, bilirubin oxidase is liquid and very unstable, and is only stable for 96 hours in a 0.1M phosphate buffer at pH 9.2-9.7 even at low temperatures [Agricultural and
Biological Chemistry (Agric. Biol. Chem.), Vol. 46, No. 8, 2031-2034, 1982],
Even when the enzyme is stored at a stable pH at room temperature (25 ° C.), only 14% of the remaining activity is shown after 14 days.

[0004] In general, bilirubin oxidase reagents are commercially available as powders such as freeze-dried products.
At the time of use, the required amount must be prepared in a liquid form, which is troublesome, and once prepared, must be used within a short period of time. On the other hand, if the bilirubin oxidase reagent can be obtained in a liquid state, the operation of measuring bilirubin can be easily performed. For that purpose, it is necessary that the bilirubin oxidase be in a liquid state and stable for a long time. Conventionally, as a method for stabilizing bilirubin oxidase in a liquid form, p
H8 or more, preferably an alkaline buffer having a pH of 8 to 10, for example, sodium diethyl barbiturate / hydrochloric acid buffer, trishydroxyaminomethane / hydrochloric acid buffer, or the like, or lactose alanine, glycine, bovine albumin, Stabilizers such as cyclodextrin,
Method of storing in a solution containing sodium nitride and so on
(JP-A-60-151561), a phosphate buffer having a pH of about 7 to 9,
Tris / hydrochloride buffer, dimethyl glutaric acid-NaOH
Method of storing in buffer solution (JP-A-61-209587), and pH
A method of preserving a 5 to 10.5 0.1M phosphate buffer (Japanese Patent Publication No. 62-33880) and the like are known. However, it is difficult to maintain bilirubin oxidase activity for a long period of time even by using these methods.

As another method for stabilizing the activity of bilirubin oxidase, a method of adding aspartic acid and / or tryptophan (JP-A-6-28488).
6), among these stabilizers, tryptophan has a stabilizing effect, but when tryptophan is added to a bilirubin oxidase-containing test solution, it gradually turns brown during storage, and the change in absorbance is measured. It is not suitable for a reagent for measuring bilirubin. Recently, as a method for maintaining the activity of bilirubin oxidase stably for a long period of time, a method for stabilizing bilirubin oxidase with a halide has been proposed.
(JP-A-7-203962) and N, N-bis (2-
A method for stabilizing bilirubin oxidase using a buffer such as (hydroxyethyl) glycine and a carboxylic acid such as sodium potassium tartrate (JP-A-8-66196) has been disclosed. If these stabilization methods are used, refrigeration (7 ° C)
Can maintain about 50% of the residual activity even after storage for one year, but further long-term stabilization has been desired.

[0006]

[Problems to be solved by the present invention] In view of such circumstances,
An object of the present invention is to provide a method for stably storing an unstable bilirubin oxidase in an aqueous liquid for a long period of time, and a stable bilirubin oxidase reagent to which the method is applied.

[0007]

Means for Solving the Problems As a result of extensive studies, the present inventors have surprisingly found that the general formula (1):

Embedded image [Wherein, R ¹ and R ² are both hydrogen atoms or together form a benzene ring which may be substituted with a lower alkyl group. R ³ is a hydrogen atom or a lower alkyl group];

Embedded image [Wherein one of R ⁴ and R ⁵ is a hydrogen atom or a lower alkyl group, and the other forms a double bond between the nitrogen atom to which it is bonded and the adjacent carbon atom at the 5-position] Or the general formula (3):

Embedded image [Wherein R ⁶ is a hydrogen atom or a lower alkyl group]
By adding one or more of mercapto derivatives of a nitrogen-containing heterocyclic compound represented by the formula to a liquid bilirubin oxidase reagent, it has been found that the stability of bilirubin oxidase is dramatically improved, and to complete the present invention. Reached.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, one or two or more mercapto derivatives of a nitrogen-containing heterocyclic compound represented by the above general formula (1), (2) or (3) are mixed with a bilirubin oxidase-containing solution. A method for stabilizing bilirubin oxidase, and a stable bilirubin oxidase reagent prepared as described above. In a preferred embodiment of the present invention, a buffer is added to the bilirubin oxidase-containing solution so as to have a buffering capacity between pH 7.0 and 12.0.
Provided is a method for stabilizing bilirubin oxidase, which comprises mixing one or more of mercapto derivatives of a nitrogen-containing heterocyclic compound represented by (3), and a stable bilirubin oxidase reagent prepared as described above. Things. Specific examples of the mercapto derivative of the nitrogen-containing heterocyclic compound used in the present invention include 2-mercapto-
1-methylimidazole, 3-mercapto-1,2,4
-Triazole, 6-mercaptopurine, 3-mercapto-4-methyl-4H-1,2,4-triazole, 2-mercaptobenzimidazole and the like, preferably 2-mercapto-1-methylimidazole and 3 -Mercapto-1,2,4-triazole.

The amount of the mercapto derivative of the nitrogen-containing heterocyclic compound is not particularly limited as long as a sufficient stabilizing effect can be obtained. Generally, the final concentration of the bilirubin oxidase aqueous solution at the time of use is 5 mM or more. , A desired stabilizing effect can be obtained, and usually a final concentration of 5 mM to 7
It is 50 mM, preferably 10 mM to 500 mM, more preferably 15 mM to 250 mM. These mercapto derivatives of the nitrogen-containing heterocyclic compound may be used in the form of an alkali metal salt or a hydrochloride thereof, if necessary. The above-mentioned buffer used in the present invention is not limited to a buffer having a buffering ability in a stable pH range of bilirubin oxidase (pH 7.0 to 12.0). These can be used alone or in combination of two or more. Preferred buffers are 2-morpholinoethanesulfonic acid monohydrate (abbreviated MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (abbreviated Bi).
s-Tris), N- (2-acetamido) iminodiacetic acid (abbreviation ADA), piperazine-1,4-bis (2-ethanesulfonic acid) (abbreviation PIPES), N- (2-acetamido) -2-amino Ethanesulfonic acid (abbreviation ACE)
S), 2-hydroxy-3-morpholinopropanesulfonic acid (abbreviation MOPSO), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (abbreviation BE)
S), 3-morpholinopropanesulfonic acid (abbreviation MOP)
S), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (abbreviation TES), 2- [4- (2-
Hydroxyethyl) -1-piperazine] ethanesulfonic acid (abbreviation HEPES), piperazine-1,4-bis (2-
(Hydroxy-3-propanesulfonic acid) dihydrate (PI
PSO), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (abbreviation DIPSO), 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid
(Abbreviation TAPSO), 2-hydroxy-3- [4- (2-
Hydroxyethyl) -1-piperazine] propanesulfonic acid monohydrate (abbreviation HEPPSO), 3- [4- (2-hydroxyethyl) -1-piperazine] propanesulfonic acid (abbreviation EPPS), N- [tris (hydroxy Methyl)
Methyl] glycine (abbreviation Tricine), N, N-bis (2-hydroxyethyl) glycine (abbreviation Bicin)
e), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (abbreviated TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (abbreviated CHE)
S), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (abbreviation CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (abbreviation CAP)
S), 2- (ethylamino) ethanol (abbreviation EAE),
Diethanolamine (abbreviation DEA), tris (hydroxymethyl) aminomethane (abbreviation Tris), tris (hydroxymethyl) aminomethane maleate (abbreviation Tris)
-Maleic acid), glycinamide, glycylglycine,
Glycine, borate buffer [boric acid (H ₃ BO ₄ ) and sodium tetraborate (Na ₂ B ₄ O ₇ )], or phosphate buffer
[Dihydrogen phosphate (Na ₂ HPO ₄ ) and sodium dihydrogen phosphate (NaH ₂ PO ₄ )], and more preferably N, N-bis (2-hydroxyethyl) glycine (B
icine), N-tris (hydroxymethyl) methyl-
3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N
-Cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-3-
Aminopropanesulfonic acid (CAPS), 2- (ethylamino) ethanol (EAE), diethanolamine (DE
A) or a borate buffer, particularly preferably N, N-bis (2-hydroxyethyl) glycine (Bic
ine) and borate buffer. The amount of the buffer added is not particularly limited as long as a sufficient stabilizing effect can be obtained. Generally, if the final concentration in the bilirubin oxidase-containing solution at the time of use is 10 mM or more, the desired stabilizing effect is obtained. Is usually obtained at a final concentration of 10 mM to 1
M, preferably 10 mM to 100 mM.

Of the combinations of one or more of these mercapto derivatives of nitrogen-containing heterocyclic compounds with one or more of buffering agents, the preferred combination is 2-
Mercapto-1-methylimidazole, 3-mercapto-1,2,4-triazole, 6-mercaptopurine,
One or more selected from 3-mercapto-4-methyl-4H-1,2,4-triazole and 2-mercaptobenzimidazole and N, N-bis (2-hydroxyethyl) glycine (Bicine); N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), 2- (ethylamino) ethanol (E
AE), diethanolamine (DEA), and a combination of two or more selected from borate buffers, and more preferably 2-mercapto-1-methylimidazole, 3-mercapto-1,2,4-triazole , 6-mercaptopurine, 3-mercapto-4-methyl-4H-1,2,4-triazole and 2-mercaptobenzimidazole, and N, N-bis (2-hydroxyethyl )glycine
(Bicine) and a combination of two or more selected from borate buffers.
One or two selected from mercapto-1-methylimidazole and 3-mercapto-1,2,4-triazole and N, N-bis (2-hydroxyethyl) glycine
(Bicine) and a borate buffer.

The stabilizing effect of bilirubin oxidase by combining these mercapto derivatives of nitrogen-containing heterocyclic compounds with a buffer is remarkably improved when one or more carboxylic acids are combined. As the carboxylic acid to be combined with the mercapto derivative of the nitrogen-containing heterocyclic compound and the buffer, tartaric acid, sodium tartrate, potassium tartrate, potassium hydrogen tartrate, sodium potassium tartrate, and sodium citrate are preferable, and sodium potassium tartrate is particularly preferable. As the bilirubin oxidase, any known enzyme is used.For example, myrothecium (Myrothecium) genus bacteria, for example, myrothecium velcaria
ucaria) -derived bilirubin oxidase, shrimp mushroom (Trachy
dermatsumodae) produced bilirubin oxidase,
Penicillium janthinell
um) -derived bilirubin oxidase, Bacillus licheniformis-derived bilirubin oxidase, Agaricus bisporus (Agaricus bisporus)
us) Bilirubin oxidase derived from fungi and the like.
The amount of bilirubin oxidase to be used is appropriately used within a range showing the desired enzyme activity, but usually 0.04 in the reaction solution.
-10 to 10 units / ml, preferably 0.08 to 4 units / ml for total bilirubin determination, and 0.0 to 4 units / ml for direct bilirubin measurement.
It is used in the range of 4 to 8 units / ml.

To stabilize the bilirubin oxidase according to the present invention, the pH value of a bilirubin oxidase-containing liquid containing a buffer prepared as described above and one or more mercapto derivatives of a nitrogen-containing heterocyclic compound is blended. Is usually pH 7.0 to 12.0, preferably pH 8.0 to 1
1.0, more preferably pH 9.0-11.0. If necessary, other components such as ordinary buffers and preservatives may be added to the bilirubin oxidase reagent. Further, when stabilizing bilirubin oxidase using a mercapto derivative of a nitrogen-containing heterocyclic compound, a compound having a mercapto group such as dithiothreitol, L-cysteine, N-acetyl-L-cysteine, methionine, and mercaptoethanol; If a reducing agent such as histidine is added together, the stabilizing effect of bilirubin oxidase is further improved.

According to the present invention, in order to stabilize bilirubin oxidase, a bilirubin oxidase-containing solution is combined with one or more of the above-mentioned buffers and one or more of mercapto derivatives of a nitrogen-containing heterocyclic compound. The stability can be further improved by adding one or more carboxylic acids to this combination. In practice, a buffer is used in a conventional manner to prepare a normal pH adjuster, for example, an inorganic or organic acid such as hydrochloric acid, sulfuric acid, lactic acid, or acetic acid, or an inorganic base such as sodium hydroxide, potassium hydroxide, or ammonium hydroxide. Alternatively, by preparing a buffer having an appropriate pH range using an organic base, dispersing or dissolving bilirubin oxidase in this, and adding one or more of mercapto derivatives of a nitrogen-containing heterocyclic compound before and after the buffer. Done.

The bilirubin oxidase reagent of the present invention comprises
It may be a liquid or a solid such as a powder.
The preparation of the liquid reagent is performed at a pH of 7.0 to 1 as described above.
It is carried out by blending one or more of 5 mM or more mercapto derivatives of a nitrogen-containing heterocyclic compound with a bilirubin oxidase-containing solution having a buffering capacity between 2.0 and 2.0. The preparation of the solid reagent may be lyophilized from a bilirubin oxidase-containing liquid containing one or more of the above buffering agents and one or more of the mercapto derivatives of the nitrogen-containing heterocyclic compound, In powdered bilirubin oxidase, the concentration of one or more of the buffering agents and one or more of the mercapto derivatives of the nitrogen-containing heterocyclic compound is 10 mM or more and 5 mM or more, respectively, when made into a liquid state. It is performed by blending as follows.

The bilirubin oxidase reagent of the present invention may be in the form of a kit. For example, a solid mixture of powdered bilirubin oxidase, one or more mercapto derivatives of a nitrogen-containing heterocyclic compound, and a buffering agent One or two of a solid mixture of powdered bilirubin oxidase and a buffer or a freeze-dried product and a mercapto derivative of a nitrogen-containing heterocyclic compound for dissolving the same. There is a combination with a buffer solution containing the above, and the like, and they are used by mixing at the time of use. The method and the reagent of the present invention can store bilirubin oxidase in a liquid state very stably, and can be effectively used in a measurement method using bilirubin oxidase, for example, total bilirubin measurement or direct bilirubin measurement.

[0016]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples. The residual activity of bilirubin oxidase in the examples was measured as follows. Reagent sample for activity measurement : Bilirubin oxidase (derived from the genus Myrocesium) First reagent solution: 50 mM phosphate buffer pH 7.2 1% sodium cholate Second reagent solution: Bilirubin control serum 5 mg / dl [commercial control serum (Nescor (Japan) to Shoji Co., Ltd.)), a bilirubin oxidase solution 2μl prepared as a the Biriru bottle (ICN Co.) as was added in an amount of 5 mg / dl] or less activity assay 0.02 units / ml After adding 270 μl of the first test solution and incubating at 37 ° C. for 5 minutes, the second test solution 9
0 μl was added thereto, and the change in absorbance at 450 nm from 3 minutes to 5 minutes was measured, and the activity was indicated by the amount of change in absorbance per minute. One unit (U) of the enzyme is obtained under the following conditions.
It is defined as the amount of enzyme required to oxidize 1 μmole of albumin-bound bilirubin per minute.

Example 1 Bilyl with 2-mercapto-1-methylimidazole
Long-term stabilizing effect of vinoxidase As a basic prescription, a prescription prepared by adding 25 U of sodium potassium tartrate to 25 mM bicine buffer and adding 4 U / ml of bilirubin oxidase to adjust the pH to 10.0 was used. An enzyme solution (pH 10.0) prepared so as to contain 25 mM 2-mercapto-1-methylimidazole (hereinafter referred to as MMI) in this basic formulation was stored at 7 ° C., and the residual activity after 8 months and 11 months Was measured by the above measurement method and compared with the basic prescription. Table 1 shows the results.

[Table 1] From Table 1, it can be seen that the basic formulation using the conventional technique leaves only 42.3% residual activity after storage at 7 ° C for 11 months. However, when 2-mercapto-1-methylimidazole is added, it is stored at 7 ° C. It can be seen that the remaining activity of 84.8% is maintained even after months.

Example 2 Birring of various nitrogen-containing heterocyclic compounds with mercapto derivatives
Stabilizing effect of rubin oxidase Bilirubin oxidase 2U was added to 100 mM borate buffer.
/ Ml and an enzyme solution (pH 10.0) prepared to contain 25 mM of a substance shown in Table 2 below under severe conditions (3.
(0 ° C.), and the residual activity after 6 days was measured by the above-mentioned measuring method and compared with the basic formulation. Table 2 shows the results.

[Table 2] As shown in Table 2, 2-mercapto-1-methylimidazole, 3-mercapto-1,2,4-triazole,
6-mercaptopurine, 3-mercapto-4-methyl-
It was recognized that 4H-1,2,4-triazole and 2-mercaptobenzimidazole had a large stabilizing effect. In particular, the effect of 2-mercapto-1-methylimidazole was remarkable.

Example 3 Concentration of 2-mercapto-1-methylimidazole and
Relationship with stabilizing effect Bilirubin oxidase 2U was added to 100 mM borate buffer.
/ Ml of an enzyme solution (pH 10.
To 0), various concentrations of 2-mercapto-1-methylimidazole were added and stored at 30 ° C., and the residual activity after 5 days was measured and compared by the above measurement method. Table 3 shows the results.

[Table 3] From Table 3, it was found that 2-mercapto-1-methylimidazole exhibits a stabilizing effect between 5 mM and 750 mM. It was also found that the addition of 750 mM or more weakened the stabilizing effect.

Example 4 Formulation with 2-mercapto-1-methylimidazole added
Comparison with added formulation Reagents at the time of preparation in which a formulation containing 2-mercapto-1-methylimidazole and a formulation without 2-mercapto-1-methylimidazole were applied to a reagent for measuring total bilirubin and a reagent for directly measuring bilirubin. The correlation was examined in. FIG. 1 shows the results when applied to a total bilirubin measurement reagent, and FIG. 2 shows the results when applied directly to a bilirubin measurement reagent. VLT-BIL and VLD-BIL (manufactured by Nippon Shoji Co., Ltd.) were used as a reagent for measuring total bilirubin and a reagent for directly measuring bilirubin. FIG.
From FIG. 2 and FIG. 2, it was found that the prescription containing 2-mercapto-1-methylimidazole exhibited the same performance as that without the addition, and the addition of these stabilizers did not affect the bilirubin measurement performance.

Example 5 Comparison of the reagent stored at 7 ° C. for one year with the reagent at the time of preparation 2-mercapto-1-methylimidazole was added,
Using the reagent stored at 1 ° C. for one year, the correlation with the reagent at the time of preparation was examined. The result is shown in FIG. According to FIG.
It was found that the performance was the same as that at the time of preparation even after 1 year at ℃.

[0022]

According to the present invention, the pH is 7.0 to 12.0.
The addition of one or more mercapto derivatives of a nitrogen-containing heterocyclic compound represented by the above general formula (1), (2) or (3) to a bilirubin oxidase-containing solution having a buffering capacity between the two. A simple, economical method can stabilize bilirubin oxidase in a liquid state for a long period of time, eliminating the need to prepare a new bilirubin oxidase reagent every time bilirubin is measured. In addition, since the obtained bilirubin oxidase reagent solution can be used for measurement as it is, desired total bilirubin and direct bilirubin can be measured very simply and easily.

[Brief description of the drawings]

FIG. 1. M when applied to a reagent for measuring total bilirubin
It is a graph which shows the correlation of MI prescription and non-prescription.

FIG. 2 is a graph showing a correlation between an MMI-added prescription and a non-added prescription when applied to a direct bilirubin measurement reagent.

FIG. 3 shows M when applied to a reagent for measuring total bilirubin.
4 is a graph showing the correlation between the time of preparation and the time after storage at 7 ° C. for one year in the MI-added formulation.

Claims (4)

[Claims] 1. A bilirubin oxidase-containing solution containing a compound of the general formula (1): [Wherein, R ¹ and R ² are both hydrogen atoms or together form a benzene ring which may be substituted with a lower alkyl group. R ³ is a hydrogen atom or a lower alkyl group]; Formula (2) [Wherein one of R ⁴ and R ⁵ is a hydrogen atom or a lower alkyl group, and the other forms a double bond between the nitrogen atom to which it is bonded and the adjacent carbon atom at the 5-position] Or the general formula (3): [Wherein R ⁶ is a hydrogen atom or a lower alkyl group]
A method for stabilizing bilirubin oxidase, which comprises mixing one or more of mercapto derivatives of a nitrogen-containing heterocyclic compound represented by the formula (1). 2. The mercapto derivative of a nitrogen-containing heterocyclic compound is 2-mercapto-1-methylimidazole, 3-mercapto-1,2,4-triazole, 6-mercaptopurine, 3-mercapto-4-methyl-4H- 1,2,4
The method for stabilizing bilirubin oxidase according to claim 1, which is -triazole and 2-mercaptobenzimidazole. 3. The method for stabilizing bilirubin oxidase according to claim 1, wherein a buffer is added to the bilirubin oxidase-containing solution and stored under a buffering capacity between pH 7.0 and 12.0. . 4. The bilirubin according to claim 3, wherein N, N-bis (2-hydroxyethyl) glycine, 2- (ethylamino) ethanol, diethanolamine, or borate buffer is used as a buffer. A method for stabilizing oxidase.