CN103376247A - Detection method for enzyme activity of succinic acid-Q reductase and reagent - Google Patents
Detection method for enzyme activity of succinic acid-Q reductase and reagent Download PDFInfo
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Abstract
The invention provides a detection method for enzyme activity of succinic acid-Q reductase (EC1.3.5.1) (mitochondrial respiratory chain compound II), and meanwhile further provides a reagent for determining the enzyme activity of succinic acid-Q reductase. The method and reagent can be used for analyzing and determining the enzyme activity of succinic acid-Q reductase in a sample to be detected, so that the method and reagent can be applied to analysis, detection and diagnosis of clinical diseases. The principle of the invention is as follows: succinic acid-Q reductase reducing oxidized fluorochrome can generate reductive fluorochrome molecules which can emit fluorescent light under special exciting light. The reaction rate is in direct proportion to the fluorescence intensity of generating the fluorochrome molecules. The enzyme activity of succinic acid-Q reductase in the sample to be detected can be calculated by detecting change of fluorescence values.
Description
Technical field
The invention belongs to the biological medicine technology field, belong to again the clinical diagnosis detection field of Inherited Metabolic Disorders simultaneously.Particularly the invention provides a kind of mensuration succinic acid-Q reductase (succinate:ubiquinone reductase; Succinate dehydrogenase; EC 1.3.5.1) method of enzyme activity, simultaneously the present invention also provides as succinic acid-Q reductase (succinate:ubiquinone reductase; Succinate dehydrogenase; EC 1.3.5.1) enzyme activity assay and the reagent of detection.
Background technology
Mitochondria is a kind of organelle in the eukaryotic, and coated by two membranes, adventitia is level and smooth, and inner membrance inwardly is folded to form ridge, and the chamber is arranged between the two membranes, is called intermembrane space, and mitochondria central authorities are matrix.Have respiratory chain enzyme system and ATP combined enzyme agent on the mitochondrial inner membrane.Mitochondria is Cellular Oxidation phosphorylation and the main place that forms ATP, and the title of cell " power factory " (power plant) is arranged.In addition, mitochondria has DNA and the genetic system of self, but the gene dosage of mitochondrial genomes is limited, and therefore, mitochondria is a kind of semi-autonomous organelle.
Mitochondriopathy (Mitochondrial disorders) is because gene cause one group of multisystem disease that the defective of Metabolism of Mitochondria enzyme occurs unusually, is also referred to as mitochondrial cytopathies.
Mitochondriopathy can be caused by karyogene (nDNA) defective or chondriogen (mtDNA) defective, so this class disease can have multiple hereditary pattern, and such as matrilinear inheritance, Mendelian inheritance, perhaps both all have.
Almost the DIRECT ENERGY of all cells source all is mitochondria in the human body, and therefore, mitochondriopathy can be described as a kind of disease that causes the multisystem disorder, and it can damage more than a kind of body cell, tissue or organ.Since normal directly not identical with the mitochondrial composition of pathology in the mitochondriopathy patient body, so its symptom that causes also varies with each individual.
At first, the mitochondriopathy phenotype is various and can be overlapped.Same mtDNA sudden change can produce different phenotypes, and different mtDNA sudden changes can produce similar phenotype.
The second, except the minority mitochondriopathy only affects the one organ, the overwhelming majority can affect many tracts.Mitochondrial respiratory chain be aerobic metabolism essential with final path, thereby often those depend on the tissue of aerobic metabolism and organ after one's own heart, brain and muscle gets involved at first, and symptom is outstanding.
The 3rd, mitochondriopathy all can occur in any age.In general, fallen ill in the infancy by nDNA caused by abnormal person, then fallen ill childhood and after growing up by mtDNA caused by abnormal person.
Because myocyte and neurocyte are large especially to the demand of energy, so the problem of neuromuscular aspect---such as muscle weakness, dyskinesia, hearing disability, balance ability go down, faint from fear and learn can not---all be the principal character of mitochondriopathy.Other general complication of this disease comprise cataract, amyocardia, diabetes and depauperation etc.Generally speaking, the mitochondriopathy patient have above two to a plurality of illnesss, wherein some often occur simultaneously, to such an extent as to we classify as certain syndrome to them.Can cause the mitochondriopathy of obvious muscle problem to be called mitochondrial myopathy (mitochondrial myopathy), that causes simultaneously obvious muscle and nervous symptoms then is called mitochondrial encephalomyopathy (mitochondrial encephalomyopathy).
Because mitochondria is the main place of carrying out aerobic respiration in the cell, so the enzyme system that carries out aerobic respiration in the mitochondria is called mitochondrial respiratory chain.Mitochondrial respiratory chain is comprised of one group of compound, comprises I, II, III, IV, V.The effect of composite I, II, III, IV is to backhaul transmission of electricity son at assembly line, so we are referred to as electron transport chain, and the effect of V complex is " synthesize " ATP, so is called again the ATP synzyme.
Any or several enzyme hypofunction or defective all can cause the generation of above-mentioned mitochondriopathy in the mitochondrial respiratory chain cpd.
Mitochondrial succinate-Q reductase referred to as Complex II (Complex II), is a kind of transmembrane protein on the mitochondrial inner membrane.The function of Complex II is that the succinic acid in the catalysis tricarboxylic acid cycle generates fumaric acid, and electronics is passed to ubiquinone.The encoding gene of Complex II is karyogene, wherein the dysfunction of SDHA gene coded protein and MT-C2D(mitochondrial complex II deficiency), LS (Leigh syndrome), CMD1GG(cardiomyopathy dilated type 1GG), PGL5(paragangliomas type 5) etc. relevant; The dysfunction of SDHB gene coded protein and PCC(pheochromocytoma), PGL4(paragangliomas type 4), PGGSS(paraganglioma and gastric stromal sarcoma), CWDLS(Cowden-like syndrome) etc. relevant.
Foreign study witness line plastochondria Respiratory Chain Complex I I functional defect the line is busy 2% of plastochondria respiratory chain disease.Yet at present for the research means of mitochondriopathy substantially take clinical for basic, but because this disease is multisystem disease, relate to organ or the systems such as heredity, nerve, muscle, blood, bone, eyes, Symptoms is complicated, and to multiple common disease similar clinical manifestation is arranged, therefore the doctor is difficult to accumulate diagnostic experiences from Symptoms, therefore fail to pinpoint a disease in diagnosis, mistaken diagnosis and delay diagnosis be ubiquitous problem.The laboratory detection means is also mostly to be indirect modes, as detecting lactic acid/pyruvic acid ratio, serum CK level etc.There is the Individual testwas chamber can do genetic test and muscle pathology detection, but the existence because of the mononucleotide sequence polymorphism, possible some sudden change is exactly normal, can be not diseases induced, therefore bring uncertainty to analysis, and mitochondrial gene mutation also shows as and just can fall ill after reaching certain certain value, so genetic test equally can not be as the reliable basis of diagnosis.And the muscle pathology detection can cause certain wound.
But the direct factor that causes mitochondriopathy to produce be exactly in the patient body related enzyme activity reduce or complete disappearance, and coincident with severity degree of condition is often closely related with the residual ratio of enzymatic activity, therefore judge whether the activity of patient's mitochondria relevant enzyme is normal, in conjunction with patient's clinical symptoms performance, just can make diagnosis to patient's disease rapidly and accurately simultaneously.Therefore the mitochondria relevant enzyme especially the diagnosis basis that detects as mitochondriopathy of mitochondrial respiratory chain cpd enzymatic activity have the advantage that additive method can not be compared, carrying out related enzyme activity by the patient to doubtful mitochondriopathy clinically detects, can realize early diagnosis, early treatment, to the mitochondriopathy prognosis, it is significant to family and social impact and the hidden danger of bringing to reduce disease.The proof of clinical practice both at home and abroad detection line plastochondria related enzyme activity is auxiliary this sick best means of making a definite diagnosis.But the detection method that adopts at present mitochondrial respiratory chain cpd enzymatic activity all is colourimetry, and namely by the enzymatic activity of spectrophotometer detection line plastochondria respiratory chain complex, the shortcoming of this method is that sensitivity is low, and poor accuracy is large to the demand of sample.Compare with spectrocolorimetry, it is few that the work of Fluorometric assay enzyme has an albumen consumption, and detection sensitivity is high, and specificity is high, the advantage that accuracy is high.
The present invention just is based on the current demand of these technical backgrounds and clinical diagnosis, characteristics according to the enzymatic activity of succinic acid-Q reductase (mitochondrial respiratory chain Complex II), utilization contains the oxidized form dyestuff of fluorophor as the substrate of enzymic catalytic reaction, the succinic acid in can the mensuration sample to be tested of rapid sensitive-Q reductase enzyme activity.Succinic acid in the sample to be tested-Q reductase can generate the reduced form fluorescence molecule after containing the oxidized form reducing dyes of fluorophor, fluorescence can occur in the reduced form fluorescence molecule under specific exciting light, and the speed of succinic acid-Q reductase catalysis succinic acid (salt) and oxidized fluorescence dyestuff reaction is directly proportional with the fluorescence intensity of release fluorescence molecule, so just can calculate succinic acid in the sample to be tested-Q reductase enzyme activity.Adopt the mensuration reagent of this method preparation to have convenient, fast and highly sensitive characteristics, easy to utilize.
Summary of the invention
The technical matters that the present invention solves is: provide a kind of fast, succinic acid-Q reductase (succinate:ubiquinone reductase accurately and reliably; Succinate dehydrogenase; EC 1.3.5.1) fluorescence detection of enzyme activity, simultaneously the present invention also is provided for the detection reagent of fluorescence spectrometry succinic acid-Q reductase activity, adopt the method and reagent to use at fluorescence detection devices such as semi-automatic or full automatic fluorophotometer or microplate reader, and detection sensitivity is high, specificity is high, easy and simple to handle, thereby can obtain practical promoting the use of.
Be the technical solution problem, technical scheme provided by the invention is as follows:
Succinic acid of the present invention-Q reductase activity determination method principle is as follows:
Succinic acid-Q reductase can be transferred to the electronics in the succinic acid oxidized coenzyme Q(CoQ under given conditions) generate reduced coenzyme Q, reduced form CoQ further is transferred to electronics the R that electron accepter R generates reduced form; The R of reduced form can launch the fluorescence of special wavelength under specific excitation wavelength, therefore can detect under given conditions the fluorescent value of reduced form R, because the speed of succinic acid-Q reductase catalytic substrate reaction is directly proportional with the turnout of reduced form R, therefore detect the variation of the fluorescent value of the reduced form R under specific wavelength, just can calculate the enzyme activity of succinic acid-Q reductase.
Above-mentioned reaction principle also can simply represent with following reaction equation:
Succinic acid+CoQ fumaric acid+CoQ(reduced form)
The CoQ(reduced form)+and the R(oxidized form) CoQ+ R(reduced form).
Electron accepter R provided by the invention, it is characterized in that: it be a kind of in the physics and chemistry field general received any luminescent dye molecule that can in redox reaction, be reduced, oxidized form R produces the fluorescence molecule R of reduced form after by the reduction of succinic acid-Q reductase, reduced form fluorescence molecule R can be under particular excitation light emitting fluorescence.Its common representative molecule is resazurin (Resazurin), and its structural formula is:
Resazurin generates resorufin (Resorufin) after reduction, resorufin is the reduced form luminescent dye molecule, can be in the 530-580nm excitation wavelength with general fluorescence detection device, detect special fluorescence under the emission wavelength of 560-620nm (the suitableeest emission wavelength is 590nm).The structural formula of resorufin is:
It should be noted that above-mentioned resazurin (Resazurin) is applied to succinic acid provided by the invention-Q reductase (succinate:ubiquinone reductase; Succinate dehydrogenase; EC 1.3.5.1) detection method of enzyme activity and reagent are a part of the present invention, it also is a Typical Representative in the general received any luminescent dye molecule that can in redox reaction, be reduced in physics and chemistry field, although this also is the part of claims of the present invention, but it does not represent the present invention all contain content, this is not the intention restriction content that contains of the present invention for example.
A kind of succinic acid provided by the invention-Q reductase activity detects reagent, and it is characterized in that: it develops preparation according to claims 1 described principle and method, and it is mainly used in analysis and the detection of the enzymatic activity of succinic acid-Q reductase.
Succinic acid provided by the invention-Q reductase activity detects reagent, it is characterized in that: its principal ingredient comprises the described electron accepter R such as claims 1-3, the maximum characteristics of this electron accepter R be it be a kind of in the physics and chemistry field general received any luminescent dye molecule that can in redox reaction, be reduced, oxidized form R produces the fluorescence molecule R of reduced form after by the reduction of succinic acid-Q reductase, reduced form fluorescence molecule R can be under particular excitation light emitting fluorescence.Aforesaid resazurin (Resazurin) is exactly modal one and represents molecule, but the invention is not restricted to this a kind of luminescent dye molecule of resazurin (Resazurin).
Succinic acid provided by the invention-Q reductase activity detects reagent, and it is characterized in that: its principal ingredient can also comprise electron donor succinic acid or succinate, and it includes but not limited to following instance: succinic acid, sodium succinate, Potassium Suceinate; But also can comprise as claimed in claim 1 ubiquinone (CoQ), i.e. ubiquinone, it can be the parent molecule of ubiquinone, also can be various derivants or the trim of CoQ parent molecule.The structural formula of ubiquinone parent molecule is:
Hydrocarbon chain is connected on 6 C of parent molecule, because of various derivants or the trims of the different CoQ of formation of the group parent molecule that connects, such as CoQ1, CoQ2, CoQ10, Decylubiquinone(DB) etc. quinones, its structural formula is as follows:
Reagent with Fluorometric assay succinic acid-Q reductase activity provided by the invention is characterized in that: it is based on exploitation preparation on the basis of the described principle of claims 1-8, method and reagent, and its principal ingredient comprises:
Damping fluid 10-1000mmol/L
PH scope 6.0-9.0
Stabilizing agent 0.01%-10%
CoQ 0.005—2mmol/L
Succinic acid (salt) 0.5-50mmol/L
Inhibitor 0-20mmol/L
Oxidized form substrate R 0.002-10mmol/L
This detection reagent is mainly used in enzyme activity assay and the detection of succinic acid-Q reductase.
Experiment shows, considers from the accuracy of measurement result and economy two aspects of preparation cost, no matter is two agent or three doses, and it is comparatively desirable that the succinic acid of the present invention of following composition relation-Q reductase detects reagent:
Damping fluid 25-100mmol/L
PH scope 7.2-8.5
Stabilizing agent 0.1-0.5%
CoQ 0.05—0.5mmol/L
Succinic acid (salt) 5-20mmol/L
Inhibitor 0.05-10mmol/L
Substrate R 0.005-0.02mmol/L
Succinic acid of the present invention-Q reductase enzyme activity detects reagent can be made into following pair of agent reagent: reagent 1
Damping fluid, stabilizing agent, CoQ, succinic acid (salt), R
Reagent 2
Inhibitor
Reagent can be to make dry powder, uses after the dissolving, or is made into liquid reagent, can directly use.
Also above-mentioned pair of agent reagent can be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, CoQ, succinic acid (salt)
Reagent 2
Damping fluid, stabilizing agent, R
Reagent 3
Inhibitor
Reagent can be to make dry powder, uses after the dissolving, or is made into liquid reagent, can directly use.
A kind of succinic acid provided by the invention-Q reductase activity detects reagent, it is characterized in that its principal ingredient comprises inhibitor, and this inhibitor is general received various reagent or the molecules that can suppress succinic acid-Q reductase enzymatic activity on zymetology.The specific enzymes activity of succinic acid-Q reductase can be by calculating succinic acid-Q reductase the difference of gross activity (without inhibitor) and the non-specific activity (adding certain density inhibitor) of succinic acid-Q reductase obtain.This detection reagent is mainly used in analysis and the detection of the enzymatic activity of succinic acid-Q reductase.It includes but not limited to following instance: malonic acid, oxaloacetic acid, thenoyltrifluoroacetone.
Succinic acid provided by the invention-Q reductase activity detects reagent, described stabilizing agent mainly comprises the various surfactants (detergent) of generally being accepted on physics, chemistry and the zymetology and using, and it is mainly used in analysis and the detection of the enzymatic activity of succinic acid-Q reductase.It comprises following instance but is not limited to these examples: Triton x-100, Tween20, NP40, Brij-35
Succinic acid provided by the invention-Q reductase enzymatic activity detects buffer reagent in the damping fluid described in the reagent by the generally accepted reagent that the pH value of system can be cushioned or stablize in 6.0-9.0 the scope on physics, chemistry and the zymetology, and it is mainly used in analysis and the detection of the enzymatic activity of succinic acid-Q reductase.It comprises following instance but is not limited to these examples:
Citric acid/sodium citrate: pH 3.0-6.6
Hydrophosphate/citric acid (salt): pH 2.2-8.0
Phosphate: pH 4.9-8.2
Citric acid/NaOH/hydrochloric acid: pH 2.2-6.5
Tris-hydrochloric acid: pH 7.1-8.9
Boric acid-borate buffer solution: pH7.4-9.0
Succinic acid provided by the invention-Q reductase method for detecting enzymatic activity and reagent, can be for the enzyme activity of succinic acid-Q reductase in the various body fluid of enzyme activity, particularly human body, tissue or the cell sample of the succinic acid of analyzing and detect various samples to be tested-Q reductase.These detected samples include but not limited to brain tissue, hepatic tissue, muscular tissue, leucocyte, fibroblast, the succinic acid of the mitochondria of extraction, the various purity of extraction-Q reductase sample etc.
Intact cell, the enzyme activity of the succinic acid of smudge cells, complete line plastochondria and broken mitochondria-Q reductase can be used for be analyzed and be detected to succinic acid provided by the invention-Q reductase method for detecting enzymatic activity and reagent.
Succinic acid provided by the invention-Q reductase activity detection method and reagent, the various diseases that can be used for analyze, the enzyme activity of the succinic acid of diagnosis and detection sample to be tested-Q reductase causes unusually, the various heredity and the metabolic disease that are particularly unusually caused by the enzyme activity of succinic acid-Q reductase.Thereby can be widely applied to clinical detection or the diagnosis disease relevant with the enzyme activity of succinic acid-Q reductase.
Description of drawings:
Fig. 1: mitochondrial respiratory chain Complex II gross activity and non-specific active testing result.1. expression mitochondrial respiratory chain Complex II gross activity detects: do not add malonic acid in the detection system, the curve linear equation is: y=61.245x+259.05 (R
2=0.9968) (n=3).2. the non-specific active detection of expression mitochondrial respiratory chain Complex II: add malonic acid in the detection system, concentration is 0.1mmol/L.The curve linear equation is: y=21.836x+225.27 (R
2=0.9939) (n=3).
Embodiment
For comprehend and application the present invention, hereinafter describe the present invention in detail with reference to embodiment, described embodiment only is intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.Scope of the present invention is specifically limited by claims of the present invention.
Embodiment one
It is two agent reagent that the succinic acid of present embodiment-Q reductase detects reagent, comprises
Reagent 1
Phosphate buffer (pH7.5) 50mmol/L
Tween 20 0.1%
CoQ 0.2mmol/L
Succinic acid (salt) 10mmol/L
Resazurin sodium 0.01mmol/L
Reagent 2
Malonic acid 0.1mmol/L
The human leukocytes Mitochondria of the sample of tested succinic acid-Q reductase for separating, after break process, the total protein consumption is 0.06ug/ul.This detection method adopts enzyme kinetics assay method detection line plastochondria Respiratory Chain Complex I I enzymatic activity.
Gross activity is measured: place in advance 30 ℃ of environment temperature to bathe 3 minutes reagent 1, then reagent 1 is joined in the microplate reader, be 530nm in excitation wavelength, emission wavelength is 590nm, carries out baseline scan 1 minute under the 30 degree conditions, adds protein sample and starts reaction, the variation of fluorescent value under the scanning 590nm, be 10 minutes sweep time, and recording curve changes, and calculates the slope of reaction.
Non-specific determination of activity: reagent 1 and reagent 2 mixed in advance be placed in 30 ℃ of environment temperature and bathed 3 minutes, then reagent mixed liquor is joined in the microplate reader, be 530nm in excitation wavelength, emission wavelength is 590nm, carries out baseline scan 1 minute under the 30 degree conditions, adds protein sample and starts reaction, the variation of fluorescent value under the scanning 590nm, be 10 minutes sweep time, and recording curve changes, and calculates the slope of reaction.
Succinic acid-Q reductase activity specific=gross activity-non-specific activity
Typical curve according to resorufin calculates succinic acid-Q reductase activity specific.
The reagent 1 blank fluorescence value of reading average out to 281, add the leucocyte mitochondria startup reaction after the normal person separates fragmentation, detect 10 minutes and measure the Complex II gross activity, the gross activity slope be 61(as shown in Figure 1), reagent 1 and the reagent 2 mixed blank fluorescence value of reading average out to 237, add the leucocyte mitochondria that the normal person separates after the fragmentation and start reaction, detects the non-specific activity of 10 minutes mensuration Complex IIs, the slope of non-specific activity be 21(as shown in Figure 1).
The specific activity slope of Complex II=gross activity slope-non-specific active slope=40.By the fluorescence standard curve, calculate the enzyme activity of succinic acid-Q reductase.
Experimental result shows, the method for detection succinic acid of the present invention-Q reductase enzyme activity and reagent can effectively detect succinic acid in the sample-Q reductase enzyme activity, can be applicable to the unusual relevant disease of clinical detection and succinic acid-Q reductase enzyme activity.
Embodiment two
It is two agent reagent that the succinic acid of present embodiment-Q reductase detects reagent, comprises
Reagent 1
Phosphate buffer (pH7.5) 50mmol/L
Tween 20 0.1%
CoQ 0.2mmol/L
Succinic acid (salt) 10mmol/L
Resazurin sodium 0.01mmol/L
Reagent 2
Malonic acid 0.1mmol/L
The human leukocytes Mitochondria of the sample of tested succinic acid-Q reductase for separating, after break process, the total protein consumption is 0.06ug/ul.This detection method adopts end-point method detection line plastochondria Respiratory Chain Complex I I enzymatic activity.
Gross activity is measured: reagent 1 is mixed being placed on warm the bath 10 minutes in 30 ℃ of environment with mitochondrial protein, then with reaction terminating, the mixed liquor of getting after the termination joins in the microplate reader, is 530nm in excitation wavelength, emission wavelength is the fluorescent value that detects product under the 590nm condition, is designated as F
Always
Non-specific determination of activity: reagent 1 and reagent 2, mitochondrial protein mixed be placed in 30 ℃ of environment temperature and bathed 10 minutes, then with reaction terminating, the mixed liquor of getting after the termination joins in the microplate reader, be 530nm in excitation wavelength, emission wavelength is the fluorescent value that detects product under the 590nm condition, is designated as F
Non-
Succinic acid-Q reductase activity specific=gross activity-non-specific activity
Calculate the reaction rate of succinic acid-Q reductase according to typical curve.
The reagent 1 blank fluorescence value of reading average out to 281, add the leucocyte mitochondria startup reaction after the normal person separates fragmentation, detect 10 minutes and measure the Complex II gross activity, gross activity fluorescence value of reading (deduction reagent blank) is 605, reagent 1 and the reagent 2 mixed blank fluorescence value of reading average out to 237, add the leucocyte mitochondria startup reaction after the normal person separates fragmentation, detect the non-specific activity of measuring Complex II in 10 minutes, fluorescence value of reading of non-specific activity (deduction reagent blank) is shown in Table 1 for 214().
Specific activity=the gross activity of Complex II-non-specific activity.By the fluorescence standard curve, calculate the enzyme activity of succinic acid-Q reductase.
Table 1 mitochondrial respiratory chain Complex II gross activity and non-specific active testing result
Experimental result shows, the method of detection succinic acid of the present invention-Q reductase enzyme activity and reagent can effectively detect succinic acid in the sample-Q reductase enzyme activity and measure stable (n=3, CV＜3%), can be applicable to clinical detection and the unusual relevant disease of succinic acid-Q reductase enzyme activity.
In a word, facts have proved, adopt method and the reagent of detection succinic acid of the present invention-Q reductase enzyme activity can draw required measurement result by general fluorescence detection device detection fully, and highly sensitive, degree of accuracy is good, specificity is high, is not subjected to pollution and the interference of interior allogenic material, and is easy to utilize in the unusual relevant disease of clinical detection and succinic acid-Q reductase enzyme activity.
List of references
Birch-Machin MA, Turbull DM. Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues.
Methods Cell Biol, 2001,65:97-117.
Burgeois M, Goutieres F, Chretien D, et al. Deficiency in complex II of the respiratory chain, presenting as a leukodystrophy in two sisters with Leigh syndrome.
Brain Dev, 1992, 14(6):404-408.
Cameron JM, Levandovskiy V, Mackay N, et al. Respiratory chain analysis of skin fibroblasts in mitochondrial disease.
Mitochondrion, 2004, 4(5-6):387-394.
Chretien D, Rustin P, Bourgeron T, et al. Reference charts for respiratory chain activities in human tissues.
Clin Chim Acta, 1994, 228(1):53-70.
Ghezzi D, Goffrini P, Uziel G, et al. SDHAF1, encoding a LYP complex II specific assembly factor, is mutated in SDH-defective infantile leukoencephalophathy.
Nat Genet, 2009, 41(6):654-656.
Thorburn DR, Chow CW, Kirby DM. Respirtory chain enzyme analysis in muscle and liver.
Mitochondrion, 2004,4(5-6):363-375.
Taylor RW, Birch-Machin MA, Schaefer J, et al. Deficiency of complex II of the mitochondrial respiratory chain in late-onset atrophy and ataxia.
Ann Neurol, 1996, 39(2):224-232。
Claims (14)
1. succinic acid-Q reductase activity assay method is characterized in that its principle is as follows:
Succinic acid-Q reductase can be transferred to the electronics in the succinic acid oxidized coenzyme Q(CoQ under given conditions) generate reduced form CoQ, reduced form CoQ further is transferred to electronics the R that electron accepter R generates reduced form; The R of reduced form can launch the fluorescence of special wavelength under specific excitation wavelength, therefore can detect under given conditions the fluorescent value of reduced form R, because the speed of succinic acid-Q reductase catalytic substrate reaction is directly proportional with the turnout of reduced form R, therefore detect the variation of the fluorescent value of the reduced form R under specific wavelength, just can calculate the enzyme activity of succinic acid-Q reductase.
2. electron accepter R as claimed in claim 1, it is characterized in that: it be a kind of in the physics and chemistry field general received any luminescent dye molecule that can in redox reaction, be reduced, oxidized form R produces the fluorescence molecule R of reduced form after by the reduction of succinic acid-Q reductase, reduced form fluorescence molecule R can be under particular excitation light emitting fluorescence.
3. such as the described electron accepter R of claims 1-2, it is characterized in that its common representative molecule is resazurin (Resazurin), its structural formula is:
A kind of succinic acid-Q reductase activity detects reagent, and it is characterized in that: it develops preparation according to claims 1 described principle and method, and it is mainly used in analysis and the detection of the enzymatic activity of succinic acid-Q reductase.
4. succinic acid-Q reductase activity detects reagent as claimed in claim 4, it is characterized in that: its principal ingredient comprises the described electron accepter R such as claims 1-3, and this detection reagent is mainly used in analysis and the detection of the enzymatic activity of succinic acid-Q reductase.
5. detect reagent such as the described succinic acid of claims 4-5-Q reductase activity, it is characterized in that: its principal ingredient can also comprise the electron donor succinic acid, and it also can be the various salt compounds of succinic acid; But also can comprise as claimed in claim 1 ubiquinone (CoQ), be ubiquinone, it can be the parent molecule of ubiquinone, also can be various derivants or the trim of CoQ parent molecule, such as quinoness such as CoQ1, CoQ2, CoQ10, Decylubiquinone.
6. detect reagent such as the described succinic acid of claims 4-6-Q reductase activity, it is characterized in that: can also comprise inhibitor in its composition, this inhibitor is general received various reagent or the molecules that can suppress succinic acid-Q reductase enzyme activity on zymetology.
7. detect reagent such as the described succinic acid of claims 4-6-Q reductase activity, it is characterized in that its composition can also comprise damping fluid and stabilizing agent, described stabilizing agent is by the various surfactants of generally accepting on physics, chemistry and the zymetology and using; Buffer reagent in the described damping fluid can or be stablized reagent in 6.0-9.0 the scope with the pH value of system buffering by generally accepted on physics, chemistry and the zymetology, and this detection reagent is mainly used in enzyme activity assay and the detection of succinic acid-Q reductase.
8. a succinic acid-Q reductase activity detects reagent, it is characterized in that: it is based on exploitation preparation on the basis of the described principle of claims 1-8, method and reagent, and its principal ingredient comprises:
Damping fluid 10-1000mmol/L as claimed in claim 8
PH scope 6.0-9.0
Stabilizing agent 0.01%-10% as claimed in claim 8
CoQ 0.005-2mmol/L as claimed in claim 6
Succinic acid (salt) 0.5-50mmol/L as claimed in claim 6
Inhibitor 0-20mmol/L as claimed in claim 7
Such as the described electron accepter R of claims 1-3 0.002-10mmol/L
This detection reagent is mainly used in enzyme activity assay and the detection of succinic acid-Q reductase.
9. described succinic acid-Q reductase activity detects reagent according to claim 9, it is characterized in that: form two agent reagent by damping fluid, stabilizing agent, CoQ, succinic acid (salt), inhibitor, R; Reagent 1 is comprised of damping fluid, stabilizing agent, CoQ, succinic acid (salt), R; Reagent 2 is comprised of inhibitor.
10. described succinic acid-Q reductase activity detects reagent according to claim 9, it is characterized in that: form three doses of reagent by damping fluid, stabilizing agent, CoQ, succinic acid (salt), inhibitor, R; Reagent 1 is comprised of damping fluid, stabilizing agent, CoQ, succinic acid (salt); Reagent 2 is comprised of damping fluid, stabilizing agent, R; Reagent 3 is comprised of inhibitor.
11. the reagent such as the described succinic acid of claims 1-11-Q reductase activity method for measuring and enzymatic activity detection is characterized in that, this detection method and reagent can be used for analysis and detect the enzyme activity of the succinic acid of various samples to be tested-Q reductase.
12. the reagent such as the described succinic acid of claims 1-11-Q reductase activity method for measuring and enzymatic activity detection, it is characterized in that this detection method and reagent can be used for analyze and the enzyme activity of the succinic acid of the various body fluid of human body, tissue and cell sample-Q reductase.
13. the reagent such as the described succinic acid of claims 1-11-Q reductase activity method for measuring and enzymatic activity detection, it is characterized in that intact cell, the enzyme activity of the succinic acid of smudge cells, complete line plastochondria and broken mitochondria-Q reductase can be used for be analyzed and be detected to this detection method and reagent.
14. the reagent such as the described succinic acid of claims 1-14-Q reductase activity method for measuring and enzymatic activity detection, it is characterized in that, this detection method and reagent can be used for the various diseases that the enzyme activity of analysis, diagnosis and detection and succinic acid-Q reductase causes unusually, the various heredity and the metabolic disease that are particularly unusually caused by the enzyme activity of succinic acid-Q reductase.
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| US3384555A (en) * | 1965-11-30 | 1968-05-21 | Army Usa | Direct fluorometric method for measuring dehydrogenase activity |
| CN102282465A (en) * | 2008-11-14 | 2011-12-14 | 梅拉尼.M.赫尔 | Apparatus and method for detecting glycol |
| CN103374597A (en) * | 2012-04-26 | 2013-10-30 | 北京和信非凡生物技术有限公司 | NADH-Q (Nicotinamide Adenine Dinucleotide) reductase enzymatic activity detection method and reagent |
| CN103424384A (en) * | 2012-05-23 | 2013-12-04 | 北京和信非凡生物技术有限公司 | Enzyme activity detection method for mitochondrial respiratory chain compound I and reagents |
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| US3384555A (en) * | 1965-11-30 | 1968-05-21 | Army Usa | Direct fluorometric method for measuring dehydrogenase activity |
| CN102282465A (en) * | 2008-11-14 | 2011-12-14 | 梅拉尼.M.赫尔 | Apparatus and method for detecting glycol |
| CN103374597A (en) * | 2012-04-26 | 2013-10-30 | 北京和信非凡生物技术有限公司 | NADH-Q (Nicotinamide Adenine Dinucleotide) reductase enzymatic activity detection method and reagent |
| CN103424384A (en) * | 2012-05-23 | 2013-12-04 | 北京和信非凡生物技术有限公司 | Enzyme activity detection method for mitochondrial respiratory chain compound I and reagents |
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