CN106290351A - A kind of phospholipase A2activity determination method - Google Patents
A kind of phospholipase A2activity determination method Download PDFInfo
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Abstract
The present invention relates to enzyme assay, specifically provide a kind of phospholipase A2Activity determination method.Described method utilizes the phospholipase A adding gradient dilution in the buffer solution of pH8.02Standard solution and testing sample, it is then respectively adding substrate 4-nitro-3-octanoyloxy benzoic acid and carries out water-bath, terminator detects absorbance after terminating reaction, utilize absorbance to draw standard curve with concentration, calculate phospholipase A in testing sample by the absorbance of standard curve and testing sample2Concentration.Present invention also offers a kind of for detecting phospholipase A in factor X activator stock solution2The method of activity.The method of the invention is wider compared to prior art detection range, is 20～180U/mL；Linear preferable, R2>0.990；And there is good veracity and precision, experimental cost is low, can carry out phospholipase A more easily2Determination of activity.
Description
Technical field
The present invention relates to enzyme assay, specifically, relate to a kind of phospholipase A2Activity survey
Determine method.
Background technology
Phospholipase A2(EC 3.1.1.4, phospholipase A2, PLA2) it is a kind of permissible
Sn-2 position ester bond in hydrolytic phosphatide molecule, obtains the enzyme (Wang little Qiang of lysophosphatide and fatty acid
Deng. agkistrodon halyx pallas venom acid phosphorus lipase A2Crystal structure research [J] of high calcium-containing amount. biochemistry and life
Thing Acta Physica Sinica, 1997,29 (2): 142-149).Phospholipase A2It it is the widely distributed enzyme of a class
Family, be present in the viruses such as various animal tissue, especially snake venom, scorpion venom, Venenum apis and
In the pancreas juice of mammal (Hao Cai etc. Guangdong cobra venom phospholipase A2Separation pure
Change and enzymatic properties [J]. Products in China magazine, 2011,24 (5): 575-578).Ginseng
Degraded with membrane phospholipid and metabolism, signal conduction, host defense, blood coagulation, cell membrane weight
Build, docosane acid precursors generation etc. (Yang Wu etc. secreting type phospholipase A2Subfamily: structure
With function [J]. Chinese biological chemistry and molecular biosciences journal, 2013,29 (10): 932-940).
Wherein phospholipase A in snake venom2Also have some important toxic action (as neurotoxicity,
Hemorrhage toxicity, musclar toxicity, cardiac toxicity etc.) and multiple other biological function (as sterilized,
Inducing cell apoptosis, prevention HIV enter host cell, cause edema etc.).Just because of phosphorus
Lipase A2Multiple toxicity and biological function, examine and determine its activity extremely important.On the one hand,
We need by its activity its biological function that may bring of deduction, on the other hand, due to
There is toxic action, phospholipase A2Calibrating as the impurity in sample is the most particularly important.
Phospholipase A2The more commonly used method of determination of activity is to utilize automatic potentiometric titration, with
Fresh egg yolk is substrate, adds required calcium ion and sodium ion and detergent such as deoxycholic acid
Sodium, Triton X 100 etc., tune about pH8.0, the fatty acid discharged by titration,
Obtain PLA2Vigor.This method is simple to operate, and substrate is to PLA2The most sensitive, but by
In egg yolk, there is impurity, requiring the highest occasion so being used in more.Also have survey live time need by
PH electrode immerses in substrate, and this just requires that survey false bottom thing is no less than 5ml every time, and pH can not
The highest or too low.
Also having research with phenol red as pH indicator, surveyed by colorimetry and live, this method is fitted
For a large amount of mensuration to sample, the most rapidly, required substrate is the most less, but because of indicator pair
PLA2Impact and PLA2Vigor by pH restriction etc., accuracy the highest (Zhang Xiang etc.
Snake venom phospholipase A2: the assessment [J] of different measuring method for activity. the chemistry of life, 1994,
14(2):41-42)。
With radiolabeled synthetic substrate (Dole V P and Meinertz H,
Microdetermination of Long-chain Fatty Acids in Plasma and Tissues[J].
J Biol Chem, 1960,235:2595), can be greatly improved and survey the sensitivity lived.Because connecing
Contact isotope operation and above have inconvenience more.
Nearest by (Matthew Holzer and Stephen P. such as MATTHEW HOLZER
Mackessy.An aqueous endpoint assay of snake venom phospholipase
A2 [J] .Toxicon, 1996,34 (10): 1149-55) with 4-nitro-3-octanoyloxy benzene first
Acid is substrate, utilizes phospholipase A2In the presence of calcium ion, energy catalytic substrate 4-nitro-3-is pungent
Acetoxybenzoic hydrolyzes, the product 4-nitro-3-hydroxy benzoic acid containing chromophoric group of generation,
By measuring its amount evaluation response speed that product generates within the unit interval, thus reflect phospholipid
Enzyme A2Activity.The method does not relies on specific apparatus, such as automatical potentiometric titrimeter, coordination
Element detecting instrument；Operation safety, does not have radioactivity；Simple to operate；The detection cycle is short.
Phospholipase A2Catalytic substrate hydrolysis equation, as follows:
But, find in actual tests, the method for MATTHEW HOLZER exist with
Lower defect: detection limit is higher, can only achieve 100U/mL；Test difficulty is big, is difficult to obtain good
Good linear；Recovery of standard addition can only achieve about 50%；Sample and standard substance needed for method system
Volume is relatively big, increases experimentation cost.
Summary of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of phospholipid
Enzyme A2Activity determination method.
In order to realize the object of the invention, technical scheme is as follows:
A kind of phospholipase A2Activity determination method, described method comprises the steps:
1) to the phospholipase A of gradient dilution2Standard substance and testing sample are separately added into pH8.0
Buffer solution mixing；It is then respectively adding substrate 4-nitro-3-octanoyloxy benzoic acid and carries out water
Bath reaction, terminator detects absorbance after terminating reaction, utilizes absorbance to draw with concentration
Standard curve；
2) absorbance by standard curve and testing sample calculates the phospholipase of testing sample
A2Activity；
Wherein, described buffer solution is containing Tris-HCl, CaCl2With the buffer solution of NaCl,
Tris-HCl:CaCl2: the molar concentration scope ratio of NaCl is 6.0～16.7:1:5.0～13.9.
The activity test method that the present invention provides, compared with prior art, buffer solution therein
Molar concentration is relatively big, and buffer capacity strengthens, so that reaction system pH during Activity determination
The most stable so that the method linearly more preferably, detection range wider.
Further, described buffer solution is containing 180～300mM Tris-HCl, 18～30mM
CaCl2, 150～the buffer solution of pH8.0 of 250mM NaCl.
As preferably, described buffer solution is containing 220mM Tris-HCl, 18mM CaCl2、
The buffer solution of the pH8.0 of 200mM NaCl.
Further, the temperature of described water-bath is 40～50 DEG C, and the response time is
30～40min；It is further preferred that bath temperature is 43 DEG C in described method, the response time
For 30min.
Inventor is found by lot of experiments research, phospholipid in the activity determination method of the present invention
Enzyme A2It is not its optimum temperature at conventional enzyme reaction temperature 37 DEG C, reacts 20 minutes the most not
It it is optimum reacting time.In test, catalytic reaction is very slow, through the most just dashing forward when starting
So rise, i.e. there is period of delay.When controlling in the response time at 30-40 minute, make mensuration
Time depart from catalytic reaction period of delay so that being linearly improved of the inventive method.Send out simultaneously
Existing, when reaction temperature being controlled at 40～50 DEG C, avoid the optimal reactive temperature of other enzymes,
And inhibiting the activity of other enzymes, the accuracy of the present invention is also improved.
Further, described terminator is water soluble and the organic solvent not making buffer salt separate out
Or Cr solion；As preferably, described organic solvent is selected from ethanol, acetonitrile or methanol；More
For preferably, described organic solvent is ethanol.
Use water soluble of the present invention and do not make buffer salt separate out organic solvent as terminator,
Reaction can be terminated quickly and effectively, make the result recording experiment will not produce relatively large deviation.Simultaneously
Next-step operation can also be carried out in time, save the operating time.
Further, described method add buffer solution and phospholipase A2Standard substance and treat test sample
The volume ratio of product is 1:1；
Further, the substrate of addition is 1:5 with the volume ratio of buffer solution；
Further, the terminator of addition is 1:1 with the volume ratio of buffer solution.
Further, described concentration of substrate is 7.5-15mM；It is preferably 9mM.
The substrate total amount that the inventive method adds is few, it is possible to decrease testing cost.At preferred substrate
In concentration range, during mensuration, concentration of substrate is sufficiently large, and in the unit interval, enzyme's reaction speeding is relative
Stable, improve linear and accuracy.
In an embodiment of the present invention, further, described testing sample arranges comparison, comparison
The buffer solution of middle addition is except without CaCl2Outward, remaining composition, concentration and volume with treat test sample
Added by product, buffer solution is consistent.
Further, described method can be used for detecting the phospholipid in factor X activator stock solution
Enzyme A2Activity.
Further, described method is survey absorbance under 425nm.
The beneficial effects of the present invention is:
The invention provides a kind of new mensuration phospholipase A2The method of activity, compared to original side
The topmost technique effect of method is embodied in: detection range width, for 20-180U/mL；Good linearity,
Linear value R2 ＞0.990, accuracy is all significantly improved with precision, and concrete improvement is embodied in
Following several respects:
1, in the present invention, reaction system buffer capacity strengthens, mainly due to the buffer added
Middle Tris-HCl molar concentration increases and the special ratios of three kinds of material molar concentrations, improves
The buffer capacity of reaction system, has buffered and has produced a large amount of octanoic acid in course of reaction, made reaction system
PH is the most stable so that the method good linearity, linear value R2> 0.990, much larger than prior art
Linear value R2=0.986, detection range width 20-180U/mL, lowest detectable limit 20U/mL,
Detection minimum 100U/mL far below prior art.
2, the method for the present invention has groped phospholipase A2When bath temperature during reaction and reaction
Between, find to control reaction temperature 40～50 DEG C, the response time control at 30-40 minute time,
The period of delay of catalytic reaction is departed from so that linearly being carried of the inventive method when determination of activity
High.Find, when reaction temperature being controlled at 40～50 DEG C, compared with prior art originally simultaneously
The recovery of standard addition of invention can bring up to 80～110% from about 50%, and method accuracy is higher.
3, the inventive method uses water soluble and the organic solvent conduct not making buffer salt separate out
Terminator, can terminate reaction completely, and in 30 minutes, measurement result is stable, and RSD is less than 2%,
The result recording experiment will not produce relatively large deviation；Operating process does not produce bubble simultaneously, just
In operation.
4, improving concentration of substrate, when making mensuration, concentration of substrate is sufficiently large, enzymatic in the unit interval
Reaction rate is the most stable, improves linear and accuracy.
5, the volume of buffer system and the volumetric usage of substrate reduce, it is simple to operation, the end simultaneously
The overall consumption of thing reduces, and saves experimentation cost.
Embodiments of the invention are also provided with sample controls, the thick poison of minimizing and stock solution contain
Have the impact of some protein of slight hydrolysis to substrate, testing result is more accurate, it is to avoid
The phenomenon that testing result is the most higher relative to actual value.The method can be used for detecting Stuart factor
Phospholipase A in activator stock solution2Activity, thus see whether stock solution meets medicinal requirements.
Accompanying drawing explanation
Fig. 1 is the canonical plotting described in the embodiment of the present invention 1；
Fig. 2 is the canonical plotting of the inventive method described in embodiment 2 step 3；
Fig. 3 be the absorbance of MATTHEW HOLZER method described in embodiment 2 step 3 with
Reaction density graph of a relation；
Fig. 4 is the standard curve of MATTHEW HOLZER method described in embodiment 2 step 3
Figure；
When Fig. 5 is the different test of MATTHEW HOLZER method described in embodiment 2 step 4
Between OD variation diagram；
Fig. 6 is the canonical plotting described in embodiment 3；
Fig. 7 is the canonical plotting described in embodiment 4.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
Main agents:
4-nitro-3-octanoyloxy benzoic acid: (producer: Enzo Life Sciences, article No.:
BML-ST506-0250)
Phospholipase A2Standard solution: buy from Sigma, in Sigma official website, inquiry is originally
The examining report of batch standard substance, therefrom checks in corresponding standard substance activity.Add ultra-pure water to dissolve,
Make its activity for 200U/ml.0.3ml/ props up subpackage, is saved in-80 DEG C, 6 months effect duration.
The present invention is with documents MATTHEW HOLZER method as contrast test.
Embodiment 1 phospholipase A2Determination of activity
1, prepared by standard substance and testing sample
1.1 each dilution factor standard substance preparations
Phospholipase A2Standard solution adds ultra-pure water and dissolves so that its activity is 200U/ml.
0.3ml/ props up subpackage, is saved in-80 DEG C.
Water and phospholipase A is added with reference to following table2Standard solution, prepares each dilution factor standard substance and makes
Each standard substance activity is respectively 0,20,60,100,140,180U/mL, correspond to respectively
A0-A5。
Prepared by 1.2 testing samples
Thick poison: the round speckle adder of purchase after slightly poison water is formulated as the solution of 10mg/ml, dilutes
60,70 and 100 times of high, normal, basic 3 concentration.
Factor X activator (RVV-X) stock solution: according to " Guangxi circle speckle viper venom blood coagulation
The research of the isolated and purified and anastalsis of tenase agent " described in method prepare blood coagulation
Tenase agent stock solution.Utilize 3K super filter tube ultrafiltration displacement sample solvent for water (4000g from
Heart 60min, add water supplementing solvent, is repeated 3 times), adjustment protein concentration is about 1mg/mL.
2, testing sample activity is measured
2.1 have added to every respectively containing 100 μ L 220mM Tris-HCl, 18mM CaCl2,
The EP pipe of the pH8.0 buffer solution of 200mM NaCl adds the above-mentioned each dilution factor prepared
Standard substance and each dilution factor testing sample, each standard substance or sample in triplicate, after mixing from
The heart, then it is added thereto to 20 μ L 9mM substrate 4-nitro-3-octanoyloxy benzoic acid, vortex mixes
Even 5s is centrifuged, and places 43 DEG C of water-bath 30min.Reaction tube is placed on rapidly on mixture of ice and water
Cooling rapidly, mixing is centrifugal.Rapidly join 100 μ L ice cold ethanol, vortex mixing 5s, terminate
Reaction.
2.2 take 200 μ L from each EP pipe adds in 96 orifice plates, surveys rapidly under 425nm
Examination absorbance, draws standard curve.By the absorbance meter of standard curve and testing sample
Calculate the phospholipase A of testing sample2Activity.
3, testing sample comparison
While above-mentioned steps 3 measures testing sample, the detection of each testing sample pipe arranges to be measured
Sample controls pipe, adds in testing sample control tube containing 100 μ L 220mM Tris-HCl,
The pH8.0 buffer solution of 200mM NaCl, is then separately added into the 100 high, normal, basic concentration of μ L thick
Poison sample and bulk samples.
4, testing result
According to the method described above, standard substance measure under 425nm and obtain following result:
Table 1 standard substance and sample absorbance under 425nm
Its OD is deducted as blank in A0 hole, obtains each point OD on standard curve, by absorbance
Value and concentration do linear regression, obtain standard curve as it is shown in figure 1, its R2=0.998.From mark
Directrix curve understands, the present embodiment activity test method linear in the range of 20～180U/ml.
Sample cell and sample controls pipe OD value are as follows:
Table 2 testing sample pipe and testing sample control tube OD value result
Sample cell test result substitutes in standard curve, calculates phospholipase A2Activity, is tied
Fruit is as follows:
Table 3 does not sets the sample activity result of testing sample matched group
As seen from the above table, the low concentration of mensuration slightly poison, the phospholipase A of stock solution2Activity is respectively
137.960,94.029U/mL, senior middle school's concentration slightly poison measurement result is more than 180U/ml.For relatively
High concentration testing sample need to dilute again, makes enzymatic activity result in 20～180U/ml detection ranges
In.
Testing sample pipe test result deducts the testing sample results of comparison of correspondence, then brings standard into
In curve, calculate phospholipase A2Activity, obtains following result:
Table 4 sets the sample determination result of testing sample matched group
From result, the phospholipase A of the high, normal, basic concentration of mensuration slightly poison2Activity is respectively
168.68、123.806、63.858U/mL.Stock solution measurement result is not less than 20U/mL standard substance
Point, i.e. activity is less than 20U/mL.
In the present embodiment, it is not provided with testing sample comparison and compares standard bent with arranging testing sample
The drafting of line does not affect, and all can detect that the activity of testing sample.
Phospholipase A2It is a kind of calcium ion dependent form enzyme, does not has in the solution lacking calcium ion
Hydrolysing activity, and factor X activator is without Ca2+In the case of just have proteoclastic
Activity (Takeya, H..Coagulation factor Xactivating enzyme from Russell ' s
viper venom(RVV-X).A novel metalloproteinase with disintegrin(platelet
aggregation inhibitor)-like and C-typelectin-like
domains.J.Biol.Chem.267(1992)14109-14117).Additionally, we are also by experiment
Show, lack the solution of calcium ion along with factor X activator content increase in solution, survey
The OD value of examination rises, it was demonstrated that factor X activator has slight hydrolysis to substrate.
Because there is factor X activator, factor X activator pair in thick poison and stock solution
Substrate has slight hydrolysis so that the end value being not provided with testing sample matched group is bigger than normal.This
Embodiment arranges testing sample matched group and eliminates RVV-X in testing sample
The impact that the end value that causes the hydrolysis of substrate is higher, result is more accurate.Meanwhile, if
Put testing sample matched group and can be additionally used in phospholipase A in detection factor X activator stock solution2
Activity.
Embodiment 2 method validation
1, accuracy
Be 50 to activity by three analysis personnel simultaneously, 90, three testing samples of 130U/ml
Carry out detecting (according to arranging matched group method), everyone each sample detection 3 days.Totally 9 weights
Multiple experimental calculation response rate evaluation methodology accuracy.
Result investigated by table 5 accuracy (recovery of standard addition)
As seen from the results in Table 5, the inventive method test result when low middle and high concentration all has preferably
Accuracy, recovery of standard addition is between 83-108%.
The recovery of standard addition of contrast test MATTHEW HOLZER method is about 50%.
2, precision
2.1 it is repeated
One laboratory technician measures low middle and high concentration (50,90,130U/ml) testing sample and (presses
According to arranging matched group method), the experimental result being repeated 6 times altogether, calculate RSD, be used for the side of evaluation
The repeatability of method.
Table 6 repeatability investigates result
As seen from the results in Table 6, method has good repeatability, and RSD is less than 10%.
2.2 Intermediate precision
Three laboratory technicians measure low middle and high concentration (50,90,130U/ml) at not same date and treat
Test sample product (according to arranging matched group method), everyone is repeated 3 times, totally 9 experimental results, meter
Calculate RSD, for the Intermediate precision of evaluation methodology.
Table 7 Intermediate precision investigates result
As seen from the results in Table 7, method Intermediate precision is good, and RSD is less than 10%.
To sum up result, the low middle and high concentration testing sample RSD of the present invention is respectively less than 10%.With " raw
Tetramune Quality Control Analysis method validation technology rule " in the essence of method for detecting enzymatic activity
Density requirements is compared less than 20%, and the present invention has good precision.
3, linearity and range
From Fig. 2 result, this method phospholipase A2Activity between 20-180U/mL time linear
Well, R2=0.998 > 0.990.
The absorbance of contrast test MATTHEW HOLZER method and reaction density relation
Figure (Fig. 3) and standard curve (Fig. 4) display thereof, contrast test detection line is higher, is more than
100U/mL, after rejecting 250U/mL point, linear coefficient R2=0.986 < 0.99.
4, latter 30 minutes build-in test results of reaction are terminated
(testing sample comparison is set), after reaction terminating, different time is operated according to embodiment 1
Testing result such as table 8:
Table 8 terminates latter 30 minutes build-in test phospholipase As of reaction2Activity Results
From result, the detection method of the present invention is at reaction terminating 30min build-in test, different
Result RSD of time test is less than 2%.
Contrast test MATTHEW HOLZER method measures suction within the different testing times
Shading value, the standard substance test absorbance of each concentration is rising (Fig. 5) always.To having a competition
The stop buffer Triton X-100 tested terminates reaction the most completely, even has in document and mentions decontamination
Agent its vigor is had facilitation (Wang Zhi is clear. cobra venom acid phosphorus lipase A is produced in Guangxi2's
Isolated and purified and character research [J]. Serpentis will .1999,11 (4): 12-17).The method water-bath 20min is also
Without departing from period of delay.And the membership that adds of surfactant Triton X-100 makes system produce greatly
Amount aeration experimental implementation.
Embodiment 3: change the phospholipase A of buffer solution2Determination of activity
Operate with the step arranging testing sample matched group in embodiment 1 step 1-3, at data
Reason method processes with the method in embodiment 1 step 4 testing result, and difference is:
Described standard substance are 180mM Tris-HCl, 30mM with buffer solution in testing sample pipe
CaCl2, the buffer solution of 250mM NaCl, pH8.0, slow in described testing sample control tube
Dissolved liquid is the buffer solution of 180mM Tris-HCl, 250mM NaCl, pH8.0, substrate
Concentration is 7.5mM, and bath temperature is 40 degrees Celsius, and the response time is 30 minutes, described in have
Machine solvent is acetonitrile.
Obtain standard curve as shown in Figure 6, its R2=0.997.Knowable to standard curve, this reality
Execute the linear in the range of 20～180U/ml of example activity test method.
OD value in table 9 is the testing sample comparison that testing sample pipe test result deducts correspondence
Meansigma methods after result, brings standard curve into, calculates phospholipase A2Activity, obtains detection knot
Fruit is as follows:
Table 9 arranges the sample determination result after matched group
From result, the phospholipase A of the high, normal, basic concentration of mensuration slightly poison2Activity is respectively
195.00、148.65、90.53U/mL.Stock solution measurement result not less than 20U/mL standard substance point,
I.e. activity is less than 20U/mL.
In like manner, the method in embodiment 2 carrying out Method validation, mode of operation is with reference to implementing
Example 2:
1) accuracy: the recovery of standard addition recorded, between 80-110%, illustrates in embodiment 3
Method have preferable accuracy；
2) precision: the RSD that repeatability and precision measure is respectively less than 10%, illustrates to implement
Method repeatability and precision in example 3 are good.
Embodiment 4 changes the phospholipase A of buffer solution and reaction condition2Determination of activity
Operate with the step arranging testing sample matched group in embodiment 1 step 1-3, at data
Reason method processes with the method in embodiment 1 step 4 testing result, and difference is:
Described standard substance are 300mM Tris-HCl, 18mM with buffer solution in testing sample pipe
CaCl2, the buffer solution of 150mM NaCl, pH8.0；Described testing sample control tube is delayed
Dissolved liquid is the buffer solution of 300mM Tris-HCl, 150mM NaCl, pH8.0, substrate
Concentration is 15mM, and bath temperature is 50 degrees Celsius, and the response time is 40 minutes, described in have
Machine solvent is Cr solion.
Obtain standard curve as it is shown in fig. 7, its R2=0.995.Knowable to standard curve, this reality
Execute the linear in the range of 20～180U/ml of example activity test method.
OD value in table 10 is the testing sample pair that testing sample pipe test result deducts correspondence
According to the meansigma methods after result, bring standard curve into, calculate phospholipase A2Activity, is detected
Result is as follows:
Table 10 arranges the sample determination result after matched group
From result, the phospholipase A of the high, normal, basic concentration of mensuration slightly poison2Activity is respectively
165.62、121.814、65.522U/mL.Stock solution measurement result is not less than 20U/mL standard substance
Point, i.e. activity is less than 20U/mL.
In like manner, the method in embodiment 2 carrying out Method validation, mode of operation is with reference to implementing
Example 2:
1) accuracy: the recovery of standard addition recorded, between 80-110%, illustrates in embodiment 4
Method have preferable accuracy；
2) precision: the RSD that repeatability and precision measure is respectively less than 10%, illustrates to implement
Method repeatability and precision in example 4 are good.
Although, the most with a general description of the specific embodiments the present invention has been made in detail
Most description, but on the basis of the present invention, it can be made some modifications or improvements, this is to this
It is apparent from for skilled person.Therefore, on the basis without departing from spirit of the present invention
Upper these modifications or improvements, belong to the scope of protection of present invention.
Claims (10)
1. a phospholipase A2Activity determination method, it is characterised in that described method includes
Following steps:
1) to the phospholipase A of gradient dilution2Standard substance and testing sample are separately added into pH8.0
Buffer solution mixing；It is then respectively adding substrate 4-nitro-3-octanoyloxy benzoic acid and carries out water
Bath reaction, terminator detects absorbance after terminating reaction, utilizes absorbance to draw with concentration
Standard curve；
2) absorbance by standard curve and testing sample calculates the phospholipase of testing sample
A2Activity；
Wherein, described buffer solution is containing Tris-HCl, CaCl2With the buffer solution of NaCl,
Tris-HCl:CaCl2: the molar concentration scope ratio of NaCl is 6.0～16.7:1:5.0～13.9.
Assay method the most according to claim 1, it is characterised in that described buffering is molten
Liquid is containing 180～300mM Tris-HCl, 18～30mM CaCl2, 150～250mM NaCl
The buffer solution of pH8.0.
Assay method the most according to claim 2, it is characterised in that described buffering is molten
Liquid is containing 220mM Tris-HCl, 18mM CaCl2, pH8.0 slow of 200mM NaCl
Dissolved liquid.
4. according to the assay method described in any one of claim 1-3, it is characterised in that institute
The temperature stating water-bath is 40～50 DEG C, and the response time is 30～40min；It is further preferred that
In described method, temperature is 43 DEG C, and the response time is 30min.
5. according to the assay method described in claim 1-4, it is characterised in that described terminator
For water soluble and do not make organic solvent or the Cr solion that buffer salt separates out；Further preferably
Ground, described organic solvent is selected from ethanol, acetonitrile or methanol；It is further preferred that it is described organic
Solvent is ethanol.
6. according to the assay method described in claim 1-5, it is characterised in that described method adds
The buffer solution entered and phospholipase A2The volume ratio of standard substance and testing sample is 1:1；Enter one
Preferably, the substrate that described method adds is 1:5 with the volume ratio of buffer solution to step；The most excellent
Choosing, the terminator that described method adds is 1:1 with the volume ratio of buffer solution.
7. according to the assay method described in claim 1-6, it is characterised in that described substrate is dense
Degree is 7.5-15mM；It is further preferred that described concentration of substrate is 9mM.
8. according to the assay method described in claim 1-7, it is characterised in that described in treat test sample
Product arrange comparison, and the buffer solution added in comparison is except without CaCl2Outward, remaining composition, dense
Spend with volume and testing sample added by buffer solution consistent.
Assay method the most according to claim 8, it is characterised in that described method can be used
Phospholipase A in detection factor X activator stock solution2Activity.
10. according to the arbitrary described assay method of claim 1-9, it is characterised in that described
Method is survey absorbance under 425nm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020198260A1 (en) * | 2001-03-23 | 2002-12-26 | Brian Keller | Compounds and methods for inhibition of phospholipase a2 and cyclooxygenase - 2 |
| CN102388311A (en) * | 2009-02-10 | 2012-03-21 | 阿特罗瓦西公司 | Enzymatic assay for the quantitative determination of phospholipase A1 or A2 activity in a sample |
| CN102798632A (en) * | 2011-05-25 | 2012-11-28 | 兆科药业（合肥）有限公司 | Method for detecting activity of batroxobin |
-
2015
- 2015-06-03 CN CN201510307217.1A patent/CN106290351A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020198260A1 (en) * | 2001-03-23 | 2002-12-26 | Brian Keller | Compounds and methods for inhibition of phospholipase a2 and cyclooxygenase - 2 |
| CN102388311A (en) * | 2009-02-10 | 2012-03-21 | 阿特罗瓦西公司 | Enzymatic assay for the quantitative determination of phospholipase A1 or A2 activity in a sample |
| CN102798632A (en) * | 2011-05-25 | 2012-11-28 | 兆科药业（合肥）有限公司 | Method for detecting activity of batroxobin |
Non-Patent Citations (7)
| Title |
|---|
| ESHEL FARAGGI: "《Protein Structure》", 20 April 2012 * |
| MATTHEW HOLZER ET AL.: "AN AQUEOUS ENDPOINT ASSAY OF SNAKE VENOM PHOSPHOLIPASE A2", 《TOXICON》 * |
| MAURICIO AURELIO GOMES HELENO ET AL.: "Biochemical Characterization and Pharmacological Properties of New Basic PLA2 BrTX-I Isolated from Bothrops roedingeri (Roedinger′s Lancehead) Mertens, 1942, Snake Venom", 《BIOMED RES INT.》 * |
| SALOMÓN HUANCAHUIRE-VEGA ET AL.: "PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry", 《TOXINS》 * |
| 居乃琥: "《酶工程手册》", 31 August 2011, 中国轻工业出版社 * |
| 杨艳杰: "《医用化学》", 31 August 2006, 第四军医大学出版社 * |
| 郝彩 等: "广东眼镜蛇毒磷脂酶A2的分离纯化及其酶活性质", 《中国生物制品学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114047150A (en) * | 2021-11-11 | 2022-02-15 | 浙江伊利康生物技术有限公司 | Detection reagent for lipoprotein-associated phospholipase A2, preparation method and application thereof |
| CN114047150B (en) * | 2021-11-11 | 2022-07-26 | 浙江伊利康生物技术有限公司 | Detection reagent for lipoprotein-associated phospholipase A2, preparation method and application thereof |
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