WO2009107966A2 - Method of measuring nadph-cytochrome p450 reductase activity in a sample using 5-cyano-2,3-ditolyl tetrazolium chloride as substrate, method of screening a material modulating nadph-cytochrome p450 reductase activity using the method, and kit for measuring nadph-cytochrome p450 reductase activity - Google Patents

Method of measuring nadph-cytochrome p450 reductase activity in a sample using 5-cyano-2,3-ditolyl tetrazolium chloride as substrate, method of screening a material modulating nadph-cytochrome p450 reductase activity using the method, and kit for measuring nadph-cytochrome p450 reductase activity Download PDF

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WO2009107966A2
WO2009107966A2 PCT/KR2009/000877 KR2009000877W WO2009107966A2 WO 2009107966 A2 WO2009107966 A2 WO 2009107966A2 KR 2009000877 W KR2009000877 W KR 2009000877W WO 2009107966 A2 WO2009107966 A2 WO 2009107966A2
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ctc
cpr
activity
measuring
nadph
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WO2009107966A3 (en
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Chul Ho Yun
Sung Kun Yim
Dong Hyun Kim
Keon Hee Kim
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Industry Foundation Of Chonnam National University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90209—Oxidoreductases (1.) acting on NADH or NADPH (1.6), e.g. those with a heme protein as acceptor (1.6.2) (general), Cytochrome-b5 reductase (1.6.2.2) or NADPH-cytochrome P450 reductase (1.6.2.4)

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  • the present invention relates to a method of measuring NADPH-cytochrome P450 reductase (CPR) activity in a sample using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) as a substrate, a method of screening a material modulating CPR activity by using the method, and a kit for measuring CPR activity in the sample.
  • CPR NADPH-cytochrome P450 reductase
  • a microsome NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) mediates electron transport from NADPH to cytochrome P450 (P450 or CYP), other microsome proteins, and cytochrome c .
  • the CPR also catalyzes reduction of various drugs and exogenous compounds, such as potassium ferricyanide, 2,6-dichloroindophenol, 1,1-diphenyl-2-picrylhydrazyl (DPPH) [Yim et al, 2004], 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [Yim et al, 2005], or mitomycine C [Sevrioukova and Peterson, 1995].
  • drugs and exogenous compounds such as potassium ferricyanide, 2,6-dichloroindophenol, 1,1-diphenyl-2-picrylhydrazyl (DPPH) [Yim et al, 2004], 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [Yim et al, 2005], or mitomycine C [Sevrioukova and Peterson
  • CPR deficiency causes a newly found adrenal and gonadal steroid generation disease [Miller, 2004].
  • Patients, in general, show evidence of adrenal dysfunction (increased ACTH), and evidence of partially deficient 17 ⁇ -hydroxylase deficiency (increased progesterone) and partially deficient 21- hydroxylase activity (increased 17OH-progesterone).
  • Many patients also are born from mothers who become virilized during pregnancy, which reflects partially deficient aromatase activity. All of these activities are catalyzed by a cytoplasm type of P450 that requires electron donation by CPR.
  • Most patients having CPR deficiency also have an Antley-Bixler skeletal malformation syndrome.
  • CPR mutation results in a milder enzymatic consequence.
  • additional clinical tests and assay need to be performed on CPR sequences.
  • a tetrazolium salt including MTT 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), p-Nitroblue tetrazolium chloride (NBT), indo nitro tetrazolium violet chloride (INT) or nitrotetrazolium violet (NTV), a corresponding formazan is precipitated in an aqueous solution.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt
  • XTT 2,3-bis(2-methoxy-4-nitro-5-sul
  • This reaction is widely used as an indicator of cell survival, metabolic activity, and an oxidation reaction (generation of oxidized product).
  • This reaction is also used to screen a reduction system in cells, tissues, and cytokine assays.
  • 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) that is a monotetrazolium redox dye is very useful because CTC-formazan is fluorescent. Accordingly, accurately screening a small amount of CTC-formazan may be easier than screening non-fluorescent, colored precipitations.
  • the highest sensitivity and resolution for screening CTC-formazan in a viable cell medium can be provided by a fluorescent confocal microscope.
  • CTC is used only as a cellular redox indicator of respiration (that is, electron delivery) activity in cellular chemical experiments [Bernas and Dobrucki, 1999 and references included therein].
  • the present invention provides a method of easily, quickly, and highly-sensitively measuring NADPH-cytochrome P450 reductase (CPR) activity in a sample using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) as a substrate.
  • CPR NADPH-cytochrome P450 reductase
  • the present invention also provides a method of screening a material modulating CPR activity by using the method of measuring CPR activity described above.
  • the present invention also provides a kit for measuring CPR activity in a sample, wherein the kit includes CTC and NADPH.
  • a method of measuring CPR activity in a sample comprising:
  • CPR NADPH-cytochrome P450 reductase
  • CTC 5-cyano-2,3-ditolyl tetrazolium chloride
  • a method of screening a material for modulating CPR activity comprising:
  • kits for measuring CPR activity in a sample comprising CTC and NADPH.
  • a method of measuring CPR activity in a sample includes: reacting a sample including an enzyme having an NADPH-cytochrome P450 reductase (CPR) activity with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) in the presence of NADPH, thereby converting CTC into CTC-formazan; and measuring the concentration of the CTC-formazan by using a spectrophotometric method or a spectrofluorometric method.
  • CPR NADPH-cytochrome P450 reductase
  • CTC 5-cyano-2,3-ditolyl tetrazolium chloride
  • the method according to the present invention includes reacting a sample including an enzyme having CPR activity with CTC in the presence of NADPH, thereby converting CTC into CTC-formazan.
  • the CPR may have an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQIDNOS: 1 to 3.
  • CPRs having the amino acid sequences set forth in SEQIDNOS: 1 to 3 are NADPH-cytochrome P450 reductase of humans, rats, and rabbits, respectively.
  • the sample may be a purified CPR or a biological sample including CPR.
  • the biological sample includes a sample directly or indirectly derived from organisms. Examples of the biological sample include blood, saliva, urine, tissues, biopsies, cell or tissue culture, clinical samples, or items extracted after surgery.
  • the biological sample can also be other materials.
  • the concentration of CTC may be in a range of 1 to 500 ⁇ M but is not limited thereto
  • the concentration of NADPH may be in a range of 1 to 500 ⁇ M but is not limited thereto
  • the concentration of CPR may be in a range of 0.1 to 100 nM but is not limited thereto.
  • the method according to the present invention also includes measuring the concentration of the CTC-formazan by using a spectrophotometric method or a spectrofluorometric method.
  • the spectrophotometric method may include measuring absorption at a wavelength in a range of about 400 nm to about 600 nm, specifically about 400 nm to about 500 nm, and more specifically about 450 nm.
  • the spectrofluorometric method may include irradiating an excitation light having a wavelength in a range of about 400 nm to about 500 nm, for example about 488 nm and measuring fluorescence at a wavelength in a range of about 600 nm to about 700 nm, for example about 620 nm.
  • the concentration of the CTC-formazan may be measured by irradiating an excitation light having a wavelength in a range of about 400 nm to about 500 nm and measuring fluorescence at a wavelength in a range of about 600 nm to about 700 nm with the naked eye. When exposed to the excitation light, a red fluorescent light is emitted and identified with the naked eye.
  • the method according to the present invention may further include identifying CPR activity by measuring a conversion rate from CTC to CTC-formazan using the concentration of the CTC-formazan.
  • a method of screening a material for modulating CPR activity including: measuring CPR activity in a sample in the presence of a test material by using the method of measuring CPR activity described above; comparing the CPR activity with control CPR activity that is measured in the sample in the absence of the test material by using the method of measuring CPR activity described above.
  • the method of screening a material for modulating CPR activity according to the present invention includes measuring CPR activity in a sample in the presence of a test material by using the method of measuring CPR activity described above.
  • the test material is a material that is used to identify an activity for modulating CPR activity.
  • This method of measuring the activity for modulating CPR activity is the same as the method of measuring CPR activity in the sample except that CPR activity is measured in the presence of the test material.
  • the method of screening a material for modulating CPR activity according to the present invention also includes comparing the CPR activity with control CPR activity that is measured in the sample in the absence of the test material by using the method of measuring CPR activity described above. If CRP activity is enhanced in the presence of the test material, it is determined that the test material has an activity for increasing CPR activity. If CRP activity does not change in the presence of the test material, it is determined that the test material does not affect CPR activity. If CRP activity is reduced in the presence of the test material, it is determined that the test material has an activity for decreasing CPR activity.
  • kits for measuring CPR activity in a sample wherein the kit includes CTC and NADPH.
  • the kit may include a manual for using CTC and NADPH according to the method of measuring CPR activity in the sample.
  • CPR activity can be easily, quickly, and highly-sensitively measured using CTC as a substrate for CPR due to the following advantages of CTC.
  • a CTC stock solution is stable for several days at room temperature.
  • CTC can be dissolved in water until the concentration of CTC is 50 mM.
  • the color of CTC solution changes from no color to red. Such color change is recognizable with the naked eye and is suitable for a high-capacity colorimetric high throughput assay.
  • CTC Since the turnover number of CTC is similar to those of ferricyanide and 1,1-diphenyl-2-picryl hydrazyl (DPPH), CTC can be used to perform a continuous spectrophotometric assay of CPR. Finally, CTC-formazan that is a fluorescent product can be formed by CPR. This spectrofluorometric assay has a higher degree of sensitivity than the spectrophotometric assay, when used to measure CPR activity.
  • the method according to the present invention may use a commercially available substrate. In addition, when the method is used, CPR activity can be easily measured and thus, an assay time period is short.
  • CTC-formazan is fluorescent and thus, even in a small amount, can be easily and precisely screened compared to a colored precipitation that is not fluorescent.
  • the highest sensitivity and resolution for screening CTC-formazan in a viable cell medium is provided by a fluorescent confocal microscope.
  • CTC can be used to detect CPR in a culture system.
  • the material for modulating CPR activity can be easily, quickly, and highly-sensitively measured.
  • a material for modulating CPR activity can be easily, quickly, and highly-sensitively measured.
  • FIG. 1 is a diagram for explaining a reduction process whereby 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is catalyzed by NADPH-cytochrome P450 reductase (CPR) and reduced to CTC-formazan in the presence of NADPH;
  • CTC 5-cyano-2,3-ditolyl tetrazolium chloride
  • CPR NADPH-cytochrome P450 reductase
  • FIG. 2A is a graph illustrating results of concentration-dependent reduction of CTC catalyzed by CPR
  • FIG. 2B is a graph of a CTC reduction kinetic parameter measured by using a method described in the Material and Method
  • FIG. 2C is a graph illustrating continuous spectrophotometric spectra of CTC reduced by CPR
  • FIG. 3A is a graph illustrating results of concentration-dependent reduction of CTC catalyzed by CPR.
  • FIG. 3B is a graph of a CTC reduction kinetic parameter measured by using a method described in the Material and Method.
  • CTC CTC
  • ⁇ -NADPH potassium phosphate monobasic
  • Sigma-Aldrich Chemical Co St. Louis, USA. All the other chemical materials used were assay-level materials.
  • a rat CPR was recombinantly expressed in E. coli and purified as described in Hanna et al ., 1998.
  • absorption spectra were measured by using a Shimadzu UV-1601 spectrophotometer (Tokyo, Japan).
  • a spectrometric test in a wavelength range of 290 to 700 nm was performed by using a standard 1 cm disposable cuvette. All the experiments were performed at a temperature of 23 o C.
  • Fluorescence experiments were performed at 30°C, and before these tests, a sample (sample volume was 2.0 ml) was maintained in a circulating water tank for 5 minutes. Fluorescence emission spectra were measured using a Shimadzu RF-5301 PC spectrofluorometer equipped with a thermostated cuvette compartment using a quartz cuvette. Emission fluorescence of CTC-formazan was measured at a wavelength of 620 nm after the CTC-formazan was excited at a wavelength of 488 nm. For all the fluorescence experiments, experimental results were compensated with respect to an inner filter effect caused by light scattering and absorption, as described in Subbarao and MacDonald, 1993.
  • a reaction to measure spectrofluorometric spectra of CTC reductase activity was performed under the following conditions: 500 ⁇ M CTC and 0.5 nM CPR in 100 mM potassium phosphate buffer (pH 7.4). An increase in fluorescence at a wavelength of 620 nm was determined after 100 ⁇ M NADPH was added thereto and the resultant solution was exposed to excitation light having a wavelength of 488 nm. The reduction rate of CTC was measured by using relative fluorescence of a standard compound and a reduced CTC (CTC-formazan) (Bernas and Dobrucki, 1999).
  • a reaction mixture included CPR (10 nM for spectrophotometric assay and 0.5 nM for spectrofluorometric assay) in 100 mM potassium phosphate buffer, and 2 ml of a substrate having various concentrations (1 to 500 ⁇ M CTC).
  • a reaction was initiated by adding 100 ⁇ M NADPH.
  • Kinetic parameters K m and k cat ) were calculated in nonlinear regression by using Graph-Pad Prism software (San Diego, CA).
  • 2 cuvettes were filled with 100 mM potassium phosphate buffer (pH 7.4) and a base line of equivalent light absorption of the buffer in a dual-beam spectrophotometer was measured at a wavelength in a range of 700 nm to 350 nm and was recorded.
  • the buffer in the sample cuvettes was replaced with the same buffer containing 100 ⁇ M MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and then spectrophotometric spectra were monitored until no change occurred. Then, 20 nM CPR was added thereto and then corresponding spectrophotometric spectra were recorded. A fresh NADPH solution (100 ⁇ M) was added thereto and then corresponding spectrophotometric spectra were recorded every minute.
  • Example 1 Spectrophotometric Evaluation of CPR activity in a sample
  • a CTC (20 mM) stock solution was prepared in 100 mM potassium phosphate buffer (pH 7.4).
  • the CTC stock solution was prepared every day, stored in an amber vial, and diluted to obtain various concentrations as illustrated in FIG. 2A.
  • CPR-catalyzed reduction of CTC was measured while the concentration of CTC was increased at a wavelength of 450 nm (see FIGS. 2A and 2B).
  • An increase of A 450 during the initial 30 seconds was used to determine a reduction rate of CTC at a designated concentration.
  • FIG. 1 is a diagram for explaining a reduction process whereby CTC is catalyzed by CPR and reduced to CTC-formazan in the presence of NADPH.
  • FIG. 2A is a graph illustrating a concentration-dependent reduction of CTC catalyzed by CPR. Reduction of CTC was spectrophotometrically measured in the presence of NADPH (100 ⁇ M) for 30 seconds at various concentrations in a range of 0.1-1000 ⁇ M at a wavelength of 450 nm.
  • FIG. 2B is a graph of a CTC reduction kinetic parameter using a method described in the Material and Method. A nonlinear regression gradient was calculated in nonlinear regression by using Graph-Pad Prism software (San Diego, US).
  • the kinetic parameter assay was performed by using CTC as an electron receptor. The results are shown in Table 1.
  • Table 1 also shows kinetic parameters of known substrates (ferricyanide, DPPH, and MTT) for CPR to compare with CTC.
  • the k cat value of CTC was 2520 min -1 which is similar to those of potassium ferricyanide (1,860 min -1 ), DPPH (1,690 min -1 ), and MTT (1,910 min -1 ).
  • the K m value of CTC was 50 ⁇ M which is higher than those of ferricyanide (9.4 ⁇ M), DPPH (28 ⁇ M), and MTT (20 ⁇ M). Such results show that CTC can be used as a substrate for CPR assay at a wavelength of 450 nm.
  • extinction coefficient (mM -1 cm -1 ) was used to evaluate a reduction rate of the substrate: ferricyanide, 1.02 at 420 nm (Schellenberg and Hellerman, 1958); DPPH, 4.09 at 520 nm (Yim et al ., 2004); MTT, 11.3 at 610 nm (Yim et al ., 2005); and CTC, 0.016 at 450 nm (Smith and McFeters, 1996).
  • a kinetic parameter was determined according to the Material and Method. Average ⁇ standard deviations of three independent experiments are shown.
  • FIG. 2C is a graph illustrating continuous spectrophotometric spectra of CTC reduced by CPR.
  • CTC-formazan is fluorescent
  • a fluorescent substrate has many advantages compared to when a colorimetric assay is performed.
  • a spectrofluorometric assay has a higher degree of sensitivity than a spectrophotometric assay.
  • the spectrofluorometric assay needs much lower concentration of an enzyme than the spectrophotometric assay.
  • the fluorescent product was formed in the presence of CPR and NADPH.
  • the CPR-catalyzed reduction product of CTC was excited at a wavelength of 488 nm and then assayed at 620 nm, while the concentration of CTC was increased (see FIGS. 3A and 3B).
  • An increase of F 620 during the initial 30 seconds was used to determine a reduction rate at a designated concentration of CTC.
  • the spectrofluorometric assay was sufficiently performed only using 0.50 nM CPR, 1/40 of the concentration of CPR needed for the spectrophotometric assay.
  • the kinetic parameter of CTC for the spectrofluorometric assay is very similar to that for the spectrophotometric assay (see Table 1, and FIGS. 2 and 3). Such results show that the spectrofluorometric assay is very reliable.
  • FIG. 3A is a graph illustrating results of concentration-dependent reduction of CTC catalyzed by CPR.
  • CTC at a varying concentration in a range of 0.1 to 500 ⁇ M was reacted with CPR in the presence of NADPH (100 ⁇ M) for 30 seconds to reduce CTC, and the reduction product was excited at a wavelength of 488 nm, and then spectrofluorometrically measured at a wavelength of 620 nm.
  • FIG. 3B is a graph of a CTC reduction kinetic parameter measured by using the method described in the Material and Method. A nonlinear regression gradient was calculated in nonlinear regression by using Graph-Pad Prism software (San Diego, US).
  • CPR activity can be easily, quickly measured using CTC as a substrate.
  • the method according to the present invention is characterized in that reduction of CTC can be directly assayed in a reaction medium by continuous spectrophotometric and spectrofluorometric assays.
  • NADPH emits electrons by CPR and the emitted electrons are transported to CTC, and CTC reduction may be spectrophotometrically and spectrofluorometrically analyzed since CTC reduction cause the formation of a light red fluorescent formazan.
  • the method according to the present invention requires a short assay time, enables continuous monitoring of CPR activity, and uses a commercially available substrate.

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Abstract

Provided are a method of measuring CPR activity in a sample, the method including: reacting a sample including an enzyme having an NADPH-cytochrome P450 reductase (CPR) activity with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) in the presence of NADPH, thereby converting CTC into CTC-formazan; and measuring the concentration of the CTC-formazan by using a spectrophotometric method or a spectrofluorometric method, a method of screening a material for modulating CPR activity by using the method of measuring CPR activity, and a kit for measuring CPR activity in a sample.

Description

METHOD OF MEASURING NADPH-CYTOCHROME P450 REDUCTASE ACTIVITY IN A SAMPLE USING 5-CYANO-2,3-DITOLYL TETRAZOLIUM CHLORIDE AS SUBSTRATE, METHOD OF SCREENING A MATERIAL MODULATING NADPH-CYTOCHROME P450 REDUCTASE ACTIVITY USING THE METHOD, AND KIT FOR MEASURING NADPH-CYTOCHROME P450 REDUCTASE ACTIVITY Technical Field
This application claims the benefit of Korean Patent Application No. 10-2008-0018534, filed on February 28, 2008, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
The present invention relates to a method of measuring NADPH-cytochrome P450 reductase (CPR) activity in a sample using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) as a substrate, a method of screening a material modulating CPR activity by using the method, and a kit for measuring CPR activity in the sample.
Background Art
A microsome NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) mediates electron transport from NADPH to cytochrome P450 (P450 or CYP), other microsome proteins, and cytochrome c. The CPR also catalyzes reduction of various drugs and exogenous compounds, such as potassium ferricyanide, 2,6-dichloroindophenol, 1,1-diphenyl-2-picrylhydrazyl (DPPH) [Yim et al, 2004], 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [Yim et al, 2005], or mitomycine C [Sevrioukova and Peterson, 1995]. Meanwhile, there are many important proteins that receive electrons from CPR to perform physiological functions. Examples of such proteins include cytochrome b 5, heme oxygenase and squalene epoxidase. CPR also initiates lipid peroxidation by one-electron reduction of molecular oxygen.
CPR deficiency causes a newly found adrenal and gonadal steroid generation disease [Miller, 2004]. Patients, in general, show evidence of adrenal dysfunction (increased ACTH), and evidence of partially deficient 17α-hydroxylase deficiency (increased progesterone) and partially deficient 21- hydroxylase activity (increased 17OH-progesterone). Many patients also are born from mothers who become virilized during pregnancy, which reflects partially deficient aromatase activity. All of these activities are catalyzed by a cytoplasm type of P450 that requires electron donation by CPR. Most patients having CPR deficiency also have an Antley-Bixler skeletal malformation syndrome. According to an in-vitro assay about CPR activity, for these cases, CPR mutation results in a milder enzymatic consequence. To verify whether such milder type of CPR deficiency is a major or rare cause of PCOS, amenorrhea or infertility, additional clinical tests and assay need to be performed on CPR sequences.
As a result of cell-mediated reduction of a tetrazolium salt including MTT, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), p-Nitroblue tetrazolium chloride (NBT), indo nitro tetrazolium violet chloride (INT) or nitrotetrazolium violet (NTV), a corresponding formazan is precipitated in an aqueous solution. This reaction is widely used as an indicator of cell survival, metabolic activity, and an oxidation reaction (generation of oxidized product). This reaction is also used to screen a reduction system in cells, tissues, and cytokine assays. In these aspects, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) that is a monotetrazolium redox dye is very useful because CTC-formazan is fluorescent. Accordingly, accurately screening a small amount of CTC-formazan may be easier than screening non-fluorescent, colored precipitations. The highest sensitivity and resolution for screening CTC-formazan in a viable cell medium can be provided by a fluorescent confocal microscope. CTC is used only as a cellular redox indicator of respiration (that is, electron delivery) activity in cellular chemical experiments [Bernas and Dobrucki, 1999 and references included therein].
Accordingly, even with the conventional techniques described above, there is still a need to develop a method of easily, quickly, and highly-sensitively measuring CPR activity.
 Technical Problem
The present invention provides a method of easily, quickly, and highly-sensitively measuring NADPH-cytochrome P450 reductase (CPR) activity in a sample using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) as a substrate.
The present invention also provides a method of screening a material modulating CPR activity by using the method of measuring CPR activity described above.
The present invention also provides a kit for measuring CPR activity in a sample, wherein the kit includes CTC and NADPH.
According to an aspect of the present invention, there is provided a method of measuring CPR activity in a sample, the method comprising:
reacting a sample comprising an enzyme having an NADPH-cytochrome P450 reductase (CPR) activity with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) in the presence of NADPH, thereby converting CTC into CTC-formazan; and
measuring the concentration of the CTC-formazan by using a spectrophotometric method or a spectrofluorometric method.
According to an aspect of the present invention, there is provided a method of screening a material for modulating CPR activity, the method comprising:
measuring CPR activity in a sample in the presence of a test material by using the method of the present invention; and
comparing the CPR activity with control CPR activity that is measured in the sample in the absence of the test material by using the method of the present invention.
According to an aspect of the present invention, there is provided a kit for measuring CPR activity in a sample, comprising CTC and NADPH.
 Technical Solution
According to an aspect of the present invention, there is provided a method of measuring CPR activity in a sample, wherein the method includes: reacting a sample including an enzyme having an NADPH-cytochrome P450 reductase (CPR) activity with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) in the presence of NADPH, thereby converting CTC into CTC-formazan; and measuring the concentration of the CTC-formazan by using a spectrophotometric method or a spectrofluorometric method.
The method according to the present invention includes reacting a sample including an enzyme having CPR activity with CTC in the presence of NADPH, thereby converting CTC into CTC-formazan.
For example, the CPR may have an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQIDNOS: 1 to 3. CPRs having the amino acid sequences set forth in SEQIDNOS: 1 to 3 are NADPH-cytochrome P450 reductase of humans, rats, and rabbits, respectively.
The sample may be a purified CPR or a biological sample including CPR. The biological sample includes a sample directly or indirectly derived from organisms. Examples of the biological sample include blood, saliva, urine, tissues, biopsies, cell or tissue culture, clinical samples, or items extracted after surgery. The biological sample can also be other materials.
In the reacting, the concentration of CTC may be in a range of 1 to 500 μM but is not limited thereto, the concentration of NADPH may be in a range of 1 to 500 μM but is not limited thereto, and the concentration of CPR may be in a range of 0.1 to 100 nM but is not limited thereto.
The method according to the present invention also includes measuring the concentration of the CTC-formazan by using a spectrophotometric method or a spectrofluorometric method.
The spectrophotometric method may include measuring absorption at a wavelength in a range of about 400 nm to about 600 nm, specifically about 400 nm to about 500 nm, and more specifically about 450 nm.
The spectrofluorometric method may include irradiating an excitation light having a wavelength in a range of about 400 nm to about 500 nm, for example about 488 nm and measuring fluorescence at a wavelength in a range of about 600 nm to about 700 nm, for example about 620 nm.
The concentration of the CTC-formazan may be measured by irradiating an excitation light having a wavelength in a range of about 400 nm to about 500 nm and measuring fluorescence at a wavelength in a range of about 600 nm to about 700 nm with the naked eye. When exposed to the excitation light, a red fluorescent light is emitted and identified with the naked eye.
The method according to the present invention may further include identifying CPR activity by measuring a conversion rate from CTC to CTC-formazan using the concentration of the CTC-formazan.
According to another aspect of the present invention, there is provided a method of screening a material for modulating CPR activity, the method including: measuring CPR activity in a sample in the presence of a test material by using the method of measuring CPR activity described above; comparing the CPR activity with control CPR activity that is measured in the sample in the absence of the test material by using the method of measuring CPR activity described above.
The method of screening a material for modulating CPR activity according to the present invention includes measuring CPR activity in a sample in the presence of a test material by using the method of measuring CPR activity described above. The test material is a material that is used to identify an activity for modulating CPR activity. This method of measuring the activity for modulating CPR activity is the same as the method of measuring CPR activity in the sample except that CPR activity is measured in the presence of the test material.
The method of screening a material for modulating CPR activity according to the present invention also includes comparing the CPR activity with control CPR activity that is measured in the sample in the absence of the test material by using the method of measuring CPR activity described above. If CRP activity is enhanced in the presence of the test material, it is determined that the test material has an activity for increasing CPR activity. If CRP activity does not change in the presence of the test material, it is determined that the test material does not affect CPR activity. If CRP activity is reduced in the presence of the test material, it is determined that the test material has an activity for decreasing CPR activity.
According to another aspect of the present invention, there is provided a kit for measuring CPR activity in a sample, wherein the kit includes CTC and NADPH.
The kit may include a manual for using CTC and NADPH according to the method of measuring CPR activity in the sample.
 Advantageous Effects
According to a method of measuring CPR activity in a sample, according to the present invention, CPR activity can be easily, quickly, and highly-sensitively measured using CTC as a substrate for CPR due to the following advantages of CTC. First, a CTC stock solution is stable for several days at room temperature. Second, CTC can be dissolved in water until the concentration of CTC is 50 mM. Third, when CTC is reduced by CPR, the color of CTC solution changes from no color to red. Such color change is recognizable with the naked eye and is suitable for a high-capacity colorimetric high throughput assay. Since the turnover number of CTC is similar to those of ferricyanide and 1,1-diphenyl-2-picryl hydrazyl (DPPH), CTC can be used to perform a continuous spectrophotometric assay of CPR. Finally, CTC-formazan that is a fluorescent product can be formed by CPR. This spectrofluorometric assay has a higher degree of sensitivity than the spectrophotometric assay, when used to measure CPR activity. The method according to the present invention may use a commercially available substrate. In addition, when the method is used, CPR activity can be easily measured and thus, an assay time period is short. CTC-formazan is fluorescent and thus, even in a small amount, can be easily and precisely screened compared to a colored precipitation that is not fluorescent. The highest sensitivity and resolution for screening CTC-formazan in a viable cell medium is provided by a fluorescent confocal microscope. CTC can be used to detect CPR in a culture system.
According to a method of screening a material for modulating CPR activity, the material for modulating CPR activity can be easily, quickly, and highly-sensitively measured.
According to a kit for measuring CPR activity in a sample, a material for modulating CPR activity can be easily, quickly, and highly-sensitively measured.
 Description of Drawings
The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:
FIG. 1 is a diagram for explaining a reduction process whereby 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is catalyzed by NADPH-cytochrome P450 reductase (CPR) and reduced to CTC-formazan in the presence of NADPH;
FIG. 2A is a graph illustrating results of concentration-dependent reduction of CTC catalyzed by CPR;
FIG. 2B is a graph of a CTC reduction kinetic parameter measured by using a method described in the Material and Method;
FIG. 2C is a graph illustrating continuous spectrophotometric spectra of CTC reduced by CPR;
FIG. 3A is a graph illustrating results of concentration-dependent reduction of CTC catalyzed by CPR; and
FIG. 3B is a graph of a CTC reduction kinetic parameter measured by using a method described in the Material and Method.
 Best Mode
Examples:
The present invention will be described in further detail with reference to following examples. These examples are for illustrative purpose only and are not intended to limit the scope of the present invention.
1. Material and Method
Material: CTC, β-NADPH, and potassium phosphate monobasic were purchased from Sigma-Aldrich Chemical Co (St. Louis, USA). All the other chemical materials used were assay-level materials. A rat CPR was recombinantly expressed in E. coli and purified as described in Hanna et al., 1998.
Apparatus: absorption spectra were measured by using a Shimadzu UV-1601 spectrophotometer (Tokyo, Japan). A spectrometric test in a wavelength range of 290 to 700 nm was performed by using a standard 1 cm disposable cuvette. All the experiments were performed at a temperature of 23oC.
Fluorescence experiments were performed at 30℃, and before these tests, a sample (sample volume was 2.0 ml) was maintained in a circulating water tank for 5 minutes. Fluorescence emission spectra were measured using a Shimadzu RF-5301 PC spectrofluorometer equipped with a thermostated cuvette compartment using a quartz cuvette. Emission fluorescence of CTC-formazan was measured at a wavelength of 620 nm after the CTC-formazan was excited at a wavelength of 488 nm. For all the fluorescence experiments, experimental results were compensated with respect to an inner filter effect caused by light scattering and absorption, as described in Subbarao and MacDonald, 1993.
Experimental Example 1: NADPH-cytochrome P450 reductase (CPR) activity.
All the continuous spectrophotometric spectra with respect to CPR activity were obtained using a standard 1 cm disposable cuvette having an entire reaction volume of 1 ml.
A reaction to measure spectrophotometric spectra of CTC reductase activity was performed under the following conditions: 500μM CTC and 10 nM CPR in 100 mM potassium phosphate buffer (pH 7.4). An increase in absorption at a wavelength of 450 nm was determined after 100μM NADPH was added. A reduction rate of CTC was measured by using e450 = 0.016 mM-1cm-1 (Smith and McFeters, 1996) with respect to CTC that is reduced.
A reaction to measure spectrofluorometric spectra of CTC reductase activity was performed under the following conditions: 500μM CTC and 0.5 nM CPR in 100 mM potassium phosphate buffer (pH 7.4). An increase in fluorescence at a wavelength of 620 nm was determined after 100 μM NADPH was added thereto and the resultant solution was exposed to excitation light having a wavelength of 488 nm. The reduction rate of CTC was measured by using relative fluorescence of a standard compound and a reduced CTC (CTC-formazan) (Bernas and Dobrucki, 1999).
Experimental Example 2: Calculation of Kinetic Parameter
A reaction mixture included CPR (10 nM for spectrophotometric assay and 0.5 nM for spectrofluorometric assay) in 100 mM potassium phosphate buffer, and 2 ml of a substrate having various concentrations (1 to 500 μM CTC). A reaction was initiated by adding 100μM NADPH. Kinetic parameters (K m and k cat ) were calculated in nonlinear regression by using Graph-Pad Prism software (San Diego, CA).
Experimental Example 3: CTC absorption spectra assay
2 cuvettes were filled with 100 mM potassium phosphate buffer (pH 7.4) and a base line of equivalent light absorption of the buffer in a dual-beam spectrophotometer was measured at a wavelength in a range of 700 nm to 350 nm and was recorded. The buffer in the sample cuvettes was replaced with the same buffer containing 100 μM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and then spectrophotometric spectra were monitored until no change occurred. Then, 20 nM CPR was added thereto and then corresponding spectrophotometric spectra were recorded. A fresh NADPH solution (100 μM) was added thereto and then corresponding spectrophotometric spectra were recorded every minute.
Example 1: Spectrophotometric Evaluation of CPR activity in a sample
A CTC (20 mM) stock solution was prepared in 100 mM potassium phosphate buffer (pH 7.4). The CTC stock solution was prepared every day, stored in an amber vial, and diluted to obtain various concentrations as illustrated in FIG. 2A. CPR-catalyzed reduction of CTC was measured while the concentration of CTC was increased at a wavelength of 450 nm (see FIGS. 2A and 2B). An increase of A450 during the initial 30 seconds was used to determine a reduction rate of CTC at a designated concentration.
FIG. 1 is a diagram for explaining a reduction process whereby CTC is catalyzed by CPR and reduced to CTC-formazan in the presence of NADPH.
FIG. 2A is a graph illustrating a concentration-dependent reduction of CTC catalyzed by CPR. Reduction of CTC was spectrophotometrically measured in the presence of NADPH (100μM) for 30 seconds at various concentrations in a range of 0.1-1000 μM at a wavelength of 450 nm.
FIG. 2B is a graph of a CTC reduction kinetic parameter using a method described in the Material and Method. A nonlinear regression gradient was calculated in nonlinear regression by using Graph-Pad Prism software (San Diego, US).
The kinetic parameter assay was performed by using CTC as an electron receptor. The results are shown in Table 1.
Table 1. CPR-catalyzed Reduction of Compound Substrate
Figure PCTKR2009000877-appb-I000007
Table 1 also shows kinetic parameters of known substrates (ferricyanide, DPPH, and MTT) for CPR to compare with CTC. With respect to CPR, the k cat value of CTC was 2520 min-1 which is similar to those of potassium ferricyanide (1,860 min-1), DPPH (1,690 min-1), and MTT (1,910 min-1). With respect to CPR, the K m value of CTC was 50 μM which is higher than those of ferricyanide (9.4 μM), DPPH (28 μM), and MTT (20 μM). Such results show that CTC can be used as a substrate for CPR assay at a wavelength of 450 nm. In Table 1, extinction coefficient (mM-1cm-1) was used to evaluate a reduction rate of the substrate: ferricyanide, 1.02 at 420 nm (Schellenberg and Hellerman, 1958); DPPH, 4.09 at 520 nm (Yim et al., 2004); MTT, 11.3 at 610 nm (Yim et al., 2005); and CTC, 0.016 at 450 nm (Smith and McFeters, 1996). a: kinetic parameter was determined according to the Material and Method. Average ± standard deviations of three independent experiments are shown.
Interestingly, all the substrates shown in Table 1 have similar kinetic parameters. Respective substrates may be exposed to light having different wavelengths according to assay conditions.
Visible absorption spectra were measured to analyze a reduction process of CTC catalyzed by CPR in the presence of NAPDH. FIG. 2C is a graph illustrating continuous spectrophotometric spectra of CTC reduced by CPR. A typical spectrum of CTC-formazan that is a reduction product of CTC appeared as a reaction product was generated (λmax of the reaction product appears at a wavelength of 485 nm between 400 nm and 600 nm). An isosbestic point appeared at 380 nm.
Example 2: Spectrofluorometric Evaluation of CPR activity in a sample
Since the fact that CTC-formazan is fluorescent is well known, this experiment was performed to identify whether CTC can be a substrate for CPR. There are no conventional fluorescent substrates for CPR. In general, a fluorescent substrate has many advantages compared to when a colorimetric assay is performed. When a reaction product is detected, a spectrofluorometric assay has a higher degree of sensitivity than a spectrophotometric assay. The spectrofluorometric assay needs much lower concentration of an enzyme than the spectrophotometric assay.
For a fluorescent product of CTC formed by CPR, the fluorescent product was formed in the presence of CPR and NADPH. The CPR-catalyzed reduction product of CTC was excited at a wavelength of 488 nm and then assayed at 620 nm, while the concentration of CTC was increased (see FIGS. 3A and 3B). An increase of F620 during the initial 30 seconds was used to determine a reduction rate at a designated concentration of CTC. The spectrofluorometric assay was sufficiently performed only using 0.50 nM CPR, 1/40 of the concentration of CPR needed for the spectrophotometric assay. With respect to CPR, the kinetic parameter of CTC for the spectrofluorometric assay is very similar to that for the spectrophotometric assay (see Table 1, and FIGS. 2 and 3). Such results show that the spectrofluorometric assay is very reliable.
FIG. 3A is a graph illustrating results of concentration-dependent reduction of CTC catalyzed by CPR. CTC at a varying concentration in a range of 0.1 to 500μM was reacted with CPR in the presence of NADPH (100μM) for 30 seconds to reduce CTC, and the reduction product was excited at a wavelength of 488 nm, and then spectrofluorometrically measured at a wavelength of 620 nm.
FIG. 3B is a graph of a CTC reduction kinetic parameter measured by using the method described in the Material and Method. A nonlinear regression gradient was calculated in nonlinear regression by using Graph-Pad Prism software (San Diego, US).
As described above, when the method according to the present invention is used, CPR activity can be easily, quickly measured using CTC as a substrate. At a wavelength of 450 nm, the extinction coefficient with respect to CTC (ε450 = 0.016 mM-1cm-1) was very small compared to MTT (ε610 = 11.3 mM-1cm-1), DPPH (ε520 = 4.09 mM-1cm-1) and ferricyanide (ε420 = 1.02 mM-1cm-1). The method according to the present invention is characterized in that reduction of CTC can be directly assayed in a reaction medium by continuous spectrophotometric and spectrofluorometric assays. NADPH emits electrons by CPR and the emitted electrons are transported to CTC, and CTC reduction may be spectrophotometrically and spectrofluorometrically analyzed since CTC reduction cause the formation of a light red fluorescent formazan. The CTC reduction follows a typical Michaelis-Menten kinetic theory (Km = 50 μM, k cat = 2520 min-1) (refer to Table 1). The method according to the present invention requires a short assay time, enables continuous monitoring of CPR activity, and uses a commercially available substrate.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
References:
1. Bernas T, Dobrucki J. (1999) Biochim Biophys Acta. 1451(1):73-81.
2. Hanna IH, Teiber JF, Kokones KL, Hollenberg PF. (1998). Arch Biochem Biophys. 350(2):324-332.
3. Miller WL. (2004), Trends Endocrinol Metab. 15(7):311-315.
4. Sevrioukova IF, Peterson JA. (1995) Biochimie. 77(7-8):562-572.
5. Schellenberg KA, Hellerman L. (1958) J Biol Chem. 231(1):547-556.
6. Smith JJ, McFeters GA. (1996) J Appl Bacteriol. 80(2):209-215.
7. Subbarao NK, MacDonald RC (1993) Analyst, 118, 913-916.
8. Yim SK, Yun CH, Ahn T, Jung HC, Pan JG. (2005) J Biochem Mol Biol.38(3):366-369.
9. Yim SK, Yun SJ, Yun CH. (2004) J Biochem Mol Biol. 37(5):629-633.

Claims (10)

  1. A method of measuring CPR activity in a sample, the method comprising:
    reacting a sample comprising an enzyme having an NADPH-cytochrome P450 reductase (CPR) activity with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) in the presence of NADPH, thereby converting CTC into CTC-formazan; and
    measuring the concentration of the CTC-formazan by using a spectrophotometric method or a spectrofluorometric method.
  2. The method of claim 1, wherein the CPR has an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQIDNOS: 1 to 3.
  3. The method of claim 1, wherein the sample comprises a purified CPR or a biological sample.
  4. The method of claim 1, wherein, in the reacting, the concentration of CTC is in a range of about 1 to about 500 μM, and the concentration of NADPH is in a range of about 1 to about 500 μM.
  5. The method of claim 1, wherein the spectrophotometric method comprises measuring absorption at a wavelength in a range of about 400 nm to about 600 nm.
  6. The method of claim 1, wherein the spectrofluorometric method comprises irradiating an excitation light having a wavelength in a range of about 400 nm to about 500 nm and measuring fluorescence at a wavelength in a range of about 600 nm to about 700 nm.
  7. The method of claim 1, wherein the concentration of the CTC-formazan is measured by irradiating an excitation light having a wavelength in a range of about 400 nm to about 500 nm and measuring fluorescence at a wavelength in a range of about 600 nm to about 700 nm with naked eyes.
  8. The method of claim 1, further comprising identifying CPR activity by measuring a conversion rate from CTC to CTC-formazan using the concentration of the CTC-formazan.
  9. A method of screening a material for modulating CPR activity, the method comprising:
    measuring CPR activity in a sample in the presence of a test material by using the method of any one of claims 1-8; and
    comparing the CPR activity with control CPR activity that is measured in the sample in the absence of the test material by using the method of any one of claims 1-8.
  10. A kit for measuring CPR activity in a sample, comprising CTC and NADPH.
PCT/KR2009/000877 2008-02-28 2009-02-24 Method of measuring nadph-cytochrome p450 reductase activity in a sample using 5-cyano-2,3-ditolyl tetrazolium chloride as substrate, method of screening a material modulating nadph-cytochrome p450 reductase activity using the method, and kit for measuring nadph-cytochrome p450 reductase activity WO2009107966A2 (en)

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CN113218928A (en) * 2021-05-21 2021-08-06 宁德师范学院 Colorimetric method for rapidly determining antibacterial activity based on fluorescent probe

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113218928A (en) * 2021-05-21 2021-08-06 宁德师范学院 Colorimetric method for rapidly determining antibacterial activity based on fluorescent probe

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