CN106442484A - Method for detecting hydrolase activity - Google Patents

Method for detecting hydrolase activity Download PDF

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Publication number
CN106442484A
CN106442484A CN201611025014.4A CN201611025014A CN106442484A CN 106442484 A CN106442484 A CN 106442484A CN 201611025014 A CN201611025014 A CN 201611025014A CN 106442484 A CN106442484 A CN 106442484A
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detection
substrate
hydrolase
acid
added
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孔金明
胡琼
何玟辉
梅亚琦
张学记
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Nanjing University of Science and Technology
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Nanjing University of Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry

Abstract

The invention discloses a method for detecting hydrolase activity. The method comprises the following steps: hatching detection liquid containing substrate, Cu<2+> and bicinchoninic acid with to-be-detected hydrolase solution, carrying out reaction on the substrate and hydrolase to generate a reducing product, reducing Cu<2+> into Cu<+> by the reducing product, and bonding Cu<+> with bicinchoninic acid to form a purple Cu<2+>-bicinchoninic acid compound, wherein the purple Cu<2+>-bicinchoninic acid compound has a strong characteristic absorption peak at 562nm. The higher the activity of hydrolase in a sample is, the deeper the colour of the detection liquid is, so that detection on hydrolase activity is realized. The method disclosed by the invention has high detection sensitivity on the hydrolase activity, good selectivity, strong antijamming capability and good reproducibility, is easy in operation and short in detection time and can be used for rapid and high sensitivity detection on the hydrolase activity in a clinical sample, and detection cost is greatly lowered.

Description

A kind of method of detection hydrolytic enzyme activities
Technical field
The invention belongs to bioassay technique field, it is related to a kind of method of detection hydrolytic enzyme activities and in particular to one kind The method that hydrolytic enzyme activities are detected based on colorimetric analysis.
Background technology
Hydrolase is the general name of the class of enzymes of catalytic hydrolysis reaction, is distributed widely in human body, to human metabolism with And the vital movement such as bioelectric detecting plays an important role.High-level or low-level hydrolytic enzyme activities all can be different degrees of lead to The disorder of function of human body, causes a series of relevant diseases.The detection of therefore hydrolytic enzyme activities is in the sieving and diagnosis of disease and prognosis Etc. aspect all have very important significance.
Because enzyme has highly single-minded catalysis activity, thus can by measure its corresponding substrate or production concentration change into And realize the detection to enzymatic activity.Detection for hydrolytic enzyme activities adopts XRF and colorimetric analysis both at home and abroad at present Method, wherein colorimetric method have that testing equipment is simple, easy and simple to handle, analyze speed is fast, accuracy is high, reproducibility is good and suitable big The good characteristics such as batch sample.
, the colorimetric methods currently used for its determination of activity have two categories below taking alkaline phosphatase as a example：(1) with to nitre Base benzenephosphonic acid disodium is chromogenic substrate, because substrate hydrolyzes the end product generating yellow under the catalytic action of alkaline phosphatase Thing, and have maximum absorption band at～405nm, thus realizing the Quantitative colorimetric analysis to alkaline phosphatase activities, but should There is poor selectivity in method, sensitivity is low, the shortcomings of detection range is narrow and antijamming capability is weak；And the stability of substrate is poor, Easily decompose under illumination condition and lead to " false positive " result (Farley J R, et al. (1981) .Improved method for quantitative determination in serum of alkaline phosphatase of skeletal origin[J].Clinical Chemistry,27(12):2002-2007.)；(2) with nm of gold as probe, based on nm of gold Accumulation shape changes and causes the change of solution colour, realizes the Quantitative colorimetric analysis to alkaline phosphatase activities, but should Method has that probe preparation process is complicated, and detection cycle is long, high cost and defect (Choi Y, the et al. such as antijamming capability is weak (2007).Sensing phosphatase activity by using gold nanoparticles[J].Angewandte Chemie International Edition,46(5):707-709.).
Bicinchoninic acid energy and Cu+High specific combines, and forms purple compound, and this compound is often used as colorimetric probe Quantitative determination is carried out to protein concentration.At present, not yet have been reported that as colorimetric probe, hydrolytic enzyme activities are entered with this compound Row colorimetric detection.
Content of the invention
It is an object of the invention to provide a kind of method of detection hydrolytic enzyme activities, the method is based on colorimetric analysis detection water Solution enzymatic activity, has easy and simple to handle, and sensitivity is high, high specificity, and detection time is short, with low cost, strong antijamming capability, can weigh The advantages of now property is good, can be used for the clinical detection of hydrolytic enzyme activities.
For achieving the above object, the present invention adopts following technical solution：
A kind of method of detection hydrolytic enzyme activities, comprises the following steps：Will be containing substrate, Cu2+Inspection with bicinchoninic acid Survey liquid and hydrolysis enzyme solutions incubation to be measured, generate Cu+- bicinchoninic acid compound, measures light absorbs at 562nm for the reactant liquor Intensity, according to the calibration curve relation of optical absorption intensity and hydrolytic enzyme activities, is calculated hydrolase in hydrolysis enzyme solutions to be measured Activity；Described substrate is irreducibility substrate, generates reproducibility product with hydrolysis enzyme reaction to be measured.
Described hydrolase is selected from alkaline phosphatase, Soil neutral phosphatase, acid phosphatase, alpha-galactosidase, β-gala Glycosidase, alpha-glucosidase, beta-glucosidase, alpha-Mannosidase or beta-Mannosidase.
When described hydrolase is alkaline phosphatase, substrate can be ascorbic acid -2- phosphoric acid.
When described hydrolase is Soil neutral phosphatase, substrate can be ascorbic acid -2- phosphoric acid.
When described hydrolase is acid phosphatase, substrate can be ascorbic acid -2- phosphoric acid.
When described hydrolase is alpha-galactosidase, substrate can be p-amino phenyl- α-D- galactopyranoside.
When described hydrolase is beta galactosidase, substrate can be p-amino phenyl- β-D- galactopyranoside.
When described hydrolase is alpha-glucosidase, substrate can be p-amino phenyl- α-D- glucopyranoside.
When described hydrolase is beta-glucosidase, substrate can be p-amino phenyl- β-D- glucopyranoside.
When described hydrolase is alpha-Mannosidase, substrate can be p-amino phenyl- α-D- mannopyranose glycosides.
When described hydrolase is beta-Mannosidase, substrate can be p-amino phenyl- β-D- mannopyranose glycosides.
The method of the detection hydrolytic enzyme activities of the present invention, substrate does not have reproducibility it is impossible to by Cu2+It is reduced into Cu+, substrate Reproducibility product can be generated in the presence of hydrolase, the reproducibility product of generation can be by Cu2+It is reduced into Cu+, Cu+With two quinolines Quinoline formic acid combines to form Cu+- bicinchoninic acid compound.Containing substrate, Cu2+Detection liquid and water to be measured with bicinchoninic acid During solution enzyme solutions incubation, substrate generates reproducibility product with hydrolysis enzyme reaction, and reproducibility product is by Cu2+It is reduced into Cu+, Cu+With bicinchoninic acid with 1:2 proportioning combines to form stable Cu+- bicinchoninic acid compound, Cu+- bicinchoninic acid is combined Thing has very strong characteristic absorption peak at 562nm.The present invention passes through to measure the absorption intensity in 562nm for the reactant liquor, final basis Optical absorption intensity and the calibration curve relation of hydrolytic enzyme activities, obtain the activity of hydrolase in hydrolysis enzyme solutions to be measured.
The present invention only needs the single stepping i.e. achievable detection to hydrolytic enzyme activities, has sensitivity height, selectively good, resists Interference performance is strong, and reproducibility is good, easy and simple to handle, the advantages of detection time is short and with low cost, can be used for water in clinical sample Quick, the highly sensitive detection of solution enzymatic activity, significantly reduces testing cost.
Brief description
Fig. 1 is that wherein, a is all examinations containing the UV-visible absorption spectrum in the case of different component in embodiment 1 Agent all adds, and b is to be not added with ascorbic acid -2- phosphoric acid, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+And bicinchoninic acid, e is not Plus Cu2+, f is to be not added with alkaline phosphatase.
Fig. 2 is the graph of relation in embodiment 2 between detection signal and alkaline phosphatase activities.
Fig. 3 is the comparison diagram of the detection liquid absorption value at 562nm containing different proteins sample in embodiment 3, wherein, A is alkaline phosphatase, and b is trypsase, and c is acid phosphatase, and d is fibrin ferment, and e is human serum albumins, and f is blood red egg In vain, g is blank.
Fig. 4 is the graph of relation in embodiment 4 between detection signal and different volumes serum sample.
Fig. 5 is that wherein, a is all examinations containing the UV-visible absorption spectrum in the case of different component in embodiment 5 Agent all adds, and b is to be not added with ascorbic acid -2- phosphoric acid, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+And bicinchoninic acid, e is not Plus Cu2+, f is to be not added with Soil neutral phosphatase.
Fig. 6 is that wherein, a is all examinations containing the UV-visible absorption spectrum in the case of different component in embodiment 6 Agent all adds, and b is to be not added with ascorbic acid -2- phosphoric acid, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+And bicinchoninic acid, e is not Plus Cu2+, f is to be not added with acid phosphatase.
Fig. 7 is that wherein, a is all examinations containing the UV-visible absorption spectrum in the case of different component in embodiment 7 Agent all adds, and b is to be not added with p-amino phenyl- α-D- galactopyranoside, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+With two quinolines Quinoline formic acid, e is to be not added with Cu2+, f is to be not added with alpha-galactosidase.
Fig. 8 is that wherein, a is all examinations containing the UV-visible absorption spectrum in the case of different component in embodiment 8 Agent all adds, and b is to be not added with p-amino phenyl- β-D- galactopyranoside, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+With two quinolines Quinoline formic acid, e is to be not added with Cu2+, f is to be not added with beta galactosidase.
Fig. 9 is that wherein, a is all examinations containing the UV-visible absorption spectrum in the case of different component in embodiment 9 Agent all adds, and b is to be not added with p-amino phenyl- α-D- glucopyranoside, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+With two quinolines Quinoline formic acid, e is to be not added with Cu2+, f is to be not added with alpha-glucosidase.
Figure 10 is that wherein, a is all containing the UV-visible absorption spectrum in the case of different component in embodiment 10 Reagent all adds, and b is to be not added with p-amino phenyl- β-D- glucopyranoside, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+With two Quinolinecarboxylic acid, e is to be not added with Cu2+, f is to be not added with beta-glucosidase.
Figure 11 is that wherein, a is all containing the UV-visible absorption spectrum in the case of different component in embodiment 11 Reagent all adds, and b is to be not added with p-amino phenyl- α-D- mannopyranose glycosides, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+With two Quinolinecarboxylic acid, e is to be not added with Cu2+, f is to be not added with alpha-Mannosidase.
Figure 12 is that wherein, a is all containing the UV-visible absorption spectrum in the case of different component in embodiment 12 Reagent all adds, and b is to be not added with p-amino phenyl- β-D- mannopyranose glycosides, and c is to be not added with bicinchoninic acid, and d is to be not added with Cu2+With two Quinolinecarboxylic acid, e is to be not added with Cu2+, f is to be not added with beta-Mannosidase.
Specific embodiment
With reference to embodiment and accompanying drawing, the invention will be further described.
Embodiment 1
The inventive method is used for the feasibility analysis experiment of detection of alkaline phosphatase activity.
By 20 μ L 0.5mg mL-1Alkaline phosphatase enzyme solutions are added to 600 μ L and contain 10mM ascorbic acid -2- phosphoric acid, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record its ultraviolet-visible Absorption spectrum.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.It will be seen from figure 1 that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid Ascorbic acid -2- phosphoric acid (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+(e) or alkaline phosphatase During enzyme (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for the inspection of alkaline phosphatase activities Survey.
Embodiment 2
The inventive method is used for the relation between the detection signal of detection of alkaline phosphatase activity and alkaline phosphatase activities.
By 0, the 20,40,60,80,100,120,140,160,180,200 of 20 μ L and 220mU mL-1Hydrolysis enzyme solutions It is added separately to 600 μ L and contain 10mM ascorbic acid -2- phosphoric acid, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, gained After solution is incubated 15min at 25 DEG C, record its uv-visible absorption spectra.
Figure it is seen that with the increase of sample activity change of Alkaline phosphatase, detect absorption at～562nm for the liquid Value is corresponding to be increased, and absorption intensity has good quantitative relationship within the specific limits with alkaline phosphatase activities.As can be seen here, originally Inventive method can be used for the quantitative determination of alkaline phosphatase activities.
Embodiment 3
The inventive method is used for the selectivity analysis of detection of alkaline phosphatase activity.
By 20 μ L 0.5mg ml-1Protein solution be added to 600 μ L and contain 10mM ascorbic acid -2- phosphoric acid, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after gained mixed liquor is incubated 15min at 25 DEG C, records it and inhale at～562nm Receipts value.
From figure 3, it can be seen that in the case of adding alkaline phosphatase (a) in detection liquid, detection liquid has at～562nm Very strong absworption peak；And be trypsase (b) when add in detection liquid, acid phosphatase (c), fibrin ferment (d), the white egg of serum In vain (e), hemoglobin (f) or when being not added with albumen (g), it no substantially absorbs at～562nm.As can be seen here, the one of the present invention The method planting detection of alkaline phosphatase activity has goodish selectivity, strong antijamming capability in complicated sample environment.
Embodiment 4
The inventive method is used for detection performance during detection of alkaline phosphatase activity in blood serum sample.
4,8,12,16,20,24,28 μ L blood serum samples are added separately to containing 10mM ascorbic acid -2- phosphoric acid, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid (final volume is 600 μ L), after resulting solution is incubated 15min at 25 DEG C, record Its uv-visible absorption spectra.
From fig. 4, it can be seen that with the increase of blood serum sample volume, detect that liquid accordingly increases in the absorption value at～562nm Greatly, and the volume of absorption intensity and blood serum sample has good quantitative relationship.As can be seen here, the inventive method can be used for serum The direct quantitative detection of sample activity change of Alkaline phosphatase, has good practicality.
Embodiment 5
The inventive method is used for detecting the feasibility analysis experiment of Soil neutral phosphatase activity.
By 20 μ L 0.5mg mL-1Soil neutral phosphatase solution is added to 600 μ L and contains 10mM ascorbic acid -2- phosphoric acid, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record its ultraviolet-visible Absorption spectrum.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.From fig. 5, it can be seen that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid Ascorbic acid -2- phosphoric acid (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+(e) or neutral phosphoric acid During enzyme (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for the inspection of Soil neutral phosphatase activity Survey.
Embodiment 6
The inventive method is used for detecting the feasibility analysis experiment of activity of acid phosphatase.
By 20 μ L 0.5mg mL-1Acid phosphatase enzyme solutions are added to 600 μ L and contain 10mM ascorbic acid -2- phosphoric acid, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record its ultraviolet-visible Absorption spectrum.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.From fig. 6, it can be seen that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid Ascorbic acid -2- phosphoric acid (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+(e) or acid phosphatase During enzyme (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for the inspection of activity of acid phosphatase Survey.
Embodiment 7
The inventive method is used for detecting the feasibility analysis experiment of alpha-galactosidase activity.
By 20 μ L 0.01mg mL-1Alpha-galactoside enzyme solutions are added to 600 μ L and contain 10mM p-amino phenyl- α-D- pyrrole Mutter galactoside, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record Its uv-visible absorption spectra.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.From figure 7 it can be seen that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid P-amino phenyl- α-D- galactopyranoside (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+ E, when () or alpha-galactosidase (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for α-half The detection of gal activity.
Embodiment 8
The inventive method is used for the feasibility analysis experiment of detectionofβ-galactosidaseactivity.
By 20 μ L 0.01mg mL-1Beta galactosidase solution is added to 600 μ L and contains 10mM p-amino phenyl- β-D- pyrrole Mutter galactoside, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record Its uv-visible absorption spectra.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.From figure 8, it is seen that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid P-amino phenyl- β-D- galactopyranoside (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+ E, when () or beta galactosidase (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for β-half The detection of gal activity.
Embodiment 9
The inventive method is used for detecting the feasibility analysis experiment of alpha-glucosidase activity.
By 20 μ L 0.05mg mL-1Alpha-glucosaccharase enzyme solutions are added to 600 μ L and contain 10mM p-amino phenyl- α-D- pyrrole Glucopyranoside glycosides, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record Its uv-visible absorption spectra.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.From fig. 9, it can be seen that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid P-amino phenyl- α-D- glucopyranoside (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+ E, when () or alpha-glucosidase (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for α-Portugal The detection of polyglycoside enzymatic activity.
Embodiment 10
The inventive method is used for detecting the feasibility analysis experiment of activity of beta-glucosidase.
By 20 μ L 0.05mg mL-1Beta-glucosidase enzyme solutions are added to 600 μ L and contain 10mM p-amino phenyl- β-D- pyrrole Glucopyranoside glycosides, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record Its uv-visible absorption spectra.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.From fig. 10 it can be seen that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid P-amino phenyl- β-D- glucopyranoside (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+ E, when () or beta-glucosidase (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for β-Portugal The detection of polyglycoside enzymatic activity.
Embodiment 11
The inventive method is used for detecting the feasibility analysis experiment of alpha-Mannosidase activity.
By 20 μ L 0.1mg mL-1Alpha-Mannosidase solution is added to 600 μ L and contains 10mM p-amino phenyl- α-D- pyrrole Mutter mannoside, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record Its uv-visible absorption spectra.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.It can be seen from figure 11 that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid P-amino phenyl- α-D- mannopyranose glycosides (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+ E, when () or alpha-Mannosidase (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for α-sweet The detection of dew glycosidase activity.
Embodiment 12
The inventive method is used for detecting the feasibility analysis experiment of beta-Mannosidase activity.
By 20 μ L 0.1mg mL-1Beta-Mannosidase solution is added to 600 μ L and contains 10mM p-amino phenyl- β-D- pyrrole Mutter mannoside, 0.3mM Cu2+, in the detection liquid of 2mM bicinchoninic acid, after resulting solution is incubated 15min at 25 DEG C, record Its uv-visible absorption spectra.
In feasibility analysis experiment, the scarce component of institute is substituted with same volume buffer solution.It can be recognized from fig. 12 that in detection liquid Middle all components all in the presence of (a), detection liquid has very strong absworption peak at～562nm；And be not added with when detecting in liquid P-amino phenyl- β-D- mannopyranose glycosides (b), bicinchoninic acid (c), bicinchoninic acid and Cu2+All it is not added with (d), Cu2+ E, when () or beta-Mannosidase (f), it no substantially absorbs at～562nm.As can be seen here, the inventive method can be used for β-sweet The detection of dew glycosidase activity.

Claims (9)

1. a kind of method of detection hydrolytic enzyme activities is it is characterised in that comprise the following steps：Will be containing substrate, Cu2+With two quinoline The detection liquid of formic acid and hydrolysis enzyme solutions incubation to be measured, generate Cu+- bicinchoninic acid compound, measures reactant liquor at 562nm Optical absorption intensity, according to the calibration curve relation of optical absorption intensity and hydrolytic enzyme activities, be calculated hydrolysis enzyme solutions to be measured The activity of middle hydrolase；Described substrate is irreducibility substrate, generates reproducibility product with hydrolysis enzyme reaction to be measured.
2. the method for detection hydrolytic enzyme activities according to claim 1 is it is characterised in that described hydrolase is selected from alkalescence Phosphatase, Soil neutral phosphatase, acid phosphatase, alpha-galactosidase, beta galactosidase, alpha-glucosidase, β-glucose Glycosides enzyme, alpha-Mannosidase or beta-Mannosidase.
3. the method for detection hydrolytic enzyme activities according to claim 1 is it is characterised in that described hydrolase is alkaline phosphorus Sour enzyme, Soil neutral phosphatase or acid phosphatase, substrate is ascorbic acid -2- phosphoric acid.
4. the method for detection hydrolytic enzyme activities according to claim 1 is it is characterised in that described hydrolase is α-gala Glycosidase, substrate is p-amino phenyl- α-D- galactopyranoside.
5. the method for detection hydrolytic enzyme activities according to claim 1 is it is characterised in that described hydrolase is β-gala Glycosidase, substrate is p-amino phenyl- β-D- galactopyranoside.
6. the method for detection hydrolytic enzyme activities according to claim 1 is it is characterised in that described hydrolase is α-grape Glycosidase, substrate is p-amino phenyl- α-D- glucopyranoside.
7. the method for detection hydrolytic enzyme activities according to claim 1 is it is characterised in that described hydrolase is β-grape Glycosidase, substrate is p-amino phenyl- β-D- glucopyranoside.
8. the method for detection hydrolytic enzyme activities according to claim 1 is it is characterised in that described hydrolase is α-sweet dew Glycosidase, substrate is p-amino phenyl- α-D- mannopyranose glycosides.
9. the method for detection hydrolytic enzyme activities according to claim 1 is it is characterised in that described hydrolase is β-sweet dew Glycosidase, substrate is p-amino phenyl- β-D- mannopyranose glycosides.
CN201611025014.4A 2016-11-22 2016-11-22 Method for detecting hydrolase activity Pending CN106442484A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090155828A1 (en) * 2001-09-06 2009-06-18 The Burnham Institute For Medical Research Methods of detecting prostate cancer
JP2012044878A (en) * 2010-08-24 2012-03-08 Toyo Univ Carbon allotrope supplied with dna and enzyme
CN105259168A (en) * 2015-10-15 2016-01-20 南京理工大学 Method for measuring alkaline phosphatase activity
CN105543335A (en) * 2016-01-08 2016-05-04 南京理工大学 Measurement method of acid phosphatase activity

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090155828A1 (en) * 2001-09-06 2009-06-18 The Burnham Institute For Medical Research Methods of detecting prostate cancer
JP2012044878A (en) * 2010-08-24 2012-03-08 Toyo Univ Carbon allotrope supplied with dna and enzyme
CN105259168A (en) * 2015-10-15 2016-01-20 南京理工大学 Method for measuring alkaline phosphatase activity
CN105543335A (en) * 2016-01-08 2016-05-04 南京理工大学 Measurement method of acid phosphatase activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ELISABETH GARCIA ET.AL.: ""ASSESSMENT OF ENDO- 1,4-BETA-D-GLUCANASE ACTIVITY BY A RAPID COLORIMETRIC ASSAY USING DISODIUM 2,2’-BICINCHONINATE"", 《JOURNAL OF FOOD BIOCHEMISTRY》 *
李菡等: "《生物化学实验技术原理和方法》", 31 December 2013, 中国农业出版社 *
贲长恩等: "《组织化学》", 30 November 2001, 人民卫生出版社 *

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