CN105842219B - A kind of Fluorescence Increasing type protein tyrosine kinase activity analysis method of tyrosinase auxiliary - Google Patents
A kind of Fluorescence Increasing type protein tyrosine kinase activity analysis method of tyrosinase auxiliary Download PDFInfo
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Abstract
The invention discloses a kind of Fluorescence Increasing type protein tyrosine kinase activity analysis methods of tyrosinase auxiliary; this method first mixes the peptide substrate of the corresponding fluorescent marker of target tyrosine protein kinase; make tyrosine in polypeptide that phosphorylation occur; phenolic hydroxyl group on non-phosphorylated tyrosine residues can be oxidized to adjacent benzoquinones and quench the fluorescence of substrate by tyrosinase; and tyrosine phenolic hydroxyl group will not be oxidized by phosphate group protection on phosphorylated substrate, not will produce quenching effect to the fluorescent molecular marked on polypeptide.Therefore, under the booster action of tyrosinase, pass through the enhancing situation of fluorescence signal in detection architecture, you can realize the quantitative analysis of protein tyrosine kinase activity.The present invention can carry out the measurement of fluorescence signal after only need to mixing protein kinase reaction system with tyrosinase, the rapid sensitive that protein tyrosine kinase activity is realized using easy operating procedure is analyzed.The present invention can be used for the screening of tyrosine protein kinase inhibitor simultaneously.
Description
Technical field
The invention belongs to biomolecule detection technical fields, and in particular to a kind of to utilize tyrosinase to fluorescent peptide substrates
It is real to the interception of tyrosinase oxidability after the oxidability and tyrosine residue phosphorylation of upper tyrosine residue
The method of existing protein tyrosine kinase activity analysis.
Background technology
Protein phosphorylation plays an important role during cell signalling.The metabolism of cell, growth, hair
It educates, most of life processes such as apoptosis, all there is close contact with the phosphorylation of protein.Protein kinase is regulation protein phosphorus
The biomolecule of the most critical of acidification.γ-phosphate group on adenine triphosphate nucleosides (ATP) can be transferred to kinases by it
On the serine of substrate protein or polypeptide, threonine or tyrosine residue.The protein phosphorylation of human body about 30% is by kinases
Regulation and control.On the one hand, kinases adjusts the activity of downstream albumen by regulating and controlling phosphorylation；On the other hand, by by protein
Phosphorylation step by step, signal amplify step by step, finally cause cell effect.In protein kinase family, the work of tyrosine protein kinase
With being even more important, the exception of many kinds of protein tyrosine kinase activities is unbalance often with the growth of malignant tumour, shifts and is metabolized
It is closely related.
In view of tyrosine protein kinase and tumorigenic substantial connection, can quickly, it is accurate, delicately detect tyrosine
The activity of protein kinase is of great significance to finding and effectively controlling malignant tumour growth early.In addition, research and development are with junket ammonia
Pka acid is target spot, can efficiently inhibit the active drug of tyrosine protein kinase, is Effective Regulation malignant
Increase, treatment disease provides a kind of efficient approach.Therefore, rapid sensitive is established, tyrosine easy to operate, cheap
Protein kinase activity analysis method is basis and the key of Related Research Domain.
Up to the present, protein tyrosine kinase activity analysis method has following several：(1) radioactive labeling techniques, γ
-32P-ATP labelling techniques are all kinds of protein kinases of detection, include the standard method of tyrosine kinase activity.But there are many method
Disadvantage：Cumbersome, time-consuming, radioactive pollution etc..(2) detection technique based on Immune discrimination.Using to tyrosine phosphorylation
Affinity antibody of the site with specific recognition effect is to work as in conjunction with a variety of detection techniques such as fluorescence, electrochemistry for recognition component
Very active research direction in arogenic acid protein kinase activity analysis field.Although such method achieved it is great into
Exhibition, but there are still many deficiencies：Antibody marker preparation process is complicated, and activity is easy by environment, response matrix, operation
Etc. factors influence, detection process requires harsh and of high cost.(3) inspection of the colour developing for sensor mechanism is assembled with gold nanoparticle
Survey method.The features such as, excellent in optical properties good using gold nanoparticle bio-compatibility, many researchers have built based on gold
The visible protein kinase activity assay of nanoparticle aggregation colour developing.Although detection method is easy, the aggregation of gold nanoparticle
It is vulnerable to the interference of many factors such as media environment such as acidity, ionic strength, coexisting substances, easy tos produce the signal of false positive,
It is extremely limited in terms of inhibition and drug screening.
Accordingly, it is considered to tyrosine protein kinase detection to low cost, the demand of simple operations and practicability etc., reason
The protein tyrosine kinase activity detection method thought should break away from the dependence to antibody identification and radioactive labeling techniques, and meet high
The features such as imitating, be sensitive, easy to operate, cheap, applied widely.
Deliver content
Technical problem to be solved by the present invention lies in providing, one kind is easy to operate, at low cost, is not necessarily to expensive instrument
Efficiently, the method for High Sensitive Analysis protein tyrosine kinase activity.
Technical solution is made of following step used by solving above-mentioned technical problem：
1, the peptide substrate of the corresponding fluorescent marker of the tyrosine protein kinase of known activity is mixed and carries out phosphoric acid
Change reaction；
2, the fluorescence intensity of detection architecture after mixing the solution after phosphorylation reaction in step 1 with tyrosinase is drawn
The standard curve that fluorescence intensity changes with protein tyrosine kinase activity；
3, the corresponding fluorescence intensity of tyrosine protein kinase sample to be measured is detected according to above-mentioned steps 1 and 2, according to standard song
The quantitative analysis of protein tyrosine kinase activity in sample to be tested can be realized in line.
In above-mentioned steps 1, fluorescent marker in the fluorescent marker peptide substrate be fluoresceins, rhodamine,
Any one or cyanine dye or fluorescent marker in Coumarins and its derivative are semiconductor or carbon fluorescence quantum
Or gold, silver fluorescence nano cluster, specifically such as：Carboxyl tetramethylrhodamine, two chloro- 6- Fluoresceincarboxylic acids of 2,7- dimethyl -4,5-, four
Chloro- 6- Fluoresceincarboxylic acids, chlordene -6- methylfluoresceins, texas Red, 3H- indole cyanine dyes etc. or derivatives thereof；Described
Tyrosine protein kinase is to refer to by the kinases of tyrosine protein phosphorylation, specifically such as：Src kinases series, Abl, Csk,
Fes, EGFR, TrkA, Flt3 etc..
Beneficial effects of the present invention are as follows：
1, present invention firstly provides Fluorescence Increasing type (turn-on) analysis methods mediated using tyrosinase to detect junket ammonia
Pka acid activity.Tyrosinase can be by the phenol hydroxyl in the corresponding peptide substrate of tyrosine protein kinase on tyrosine residue
Base is oxidized to adjacent benzoquinones structure, and the fluorescent dye marked on adjacent benzoquinones peptide substrate has an efficient quenching ability, and tyrosine egg
The phenolic hydroxyl group on tyrosine residue after white kinase catalytic phosphorylation has modified phosphate group, and tyrosinase cannot be oxidized,
And then the fluorescence of fluorescent dye can not be quenched.Therefore, the present invention utilizes tyrosinase to tyrosine residue on fluorescent peptide substrates
Oxidability and tyrosine residue phosphorylation after to the interception of tyrosinase oxidability, by detection architecture
The enhancing situation of fluorescence signal, you can realize the quantitative analysis of protein tyrosine kinase activity.
2, the present invention establish protein tyrosine kinase activity analysis method operation it is extremely simple, only need to by protein kinase with
System after the polypeptides reactive of fluorescent marker directly mixed with tyrosinase and detection architecture fluorescence signal, be one kind i.e.
Mixed i.e. survey type fluorescence analysis method, has on-radiation, Antibody independent, without using the additional sensing element such as nano-particle
The outstanding features such as part and rapid sensitive, stability are good, operating procedure is simple, many advantages such as at low cost, easy to detect,
Overcome radiological hazard present in conventional radioactive mass signatures, antibody identification and gold nanoparticle colorimetric method, need it is special
Industry personnel operation, time-consuming, of high cost, the reagent storage time is short, easily by external interference the deficiencies of, have in common chemistry and
Biology laboratory can carry out.
3, protein tyrosine kinase activity analysis method of the present invention is also applied for the screening of kinases inhibitor.
Description of the drawings
Fig. 1 be in embodiment 1 reaction system fluorescence signal with the fluorescence spectrum of tyrosine protein kinase Src activity changes
Figure.
Fig. 2 be in embodiment 1 fluorescence intensity of the reaction system at 583nm with tyrosine protein kinase Src activity changes
Canonical plotting.
Fig. 3 be in embodiment 2 reaction system fluorescence signal with the fluorescence spectrum of tyrosine protein kinase Src activity changes
Figure.
Fig. 4 be in embodiment 2 fluorescence intensity of the reaction system at 583nm with tyrosine protein kinase Src activity changes
Canonical plotting.
Fig. 5 is the fluorescence spectra that fluorescence intensity changes with inhibitor PP2 concentration.
Fig. 6 is the curve graph that 583nm peak fluorescence intensities change with the logarithm of inhibitor PP2 concentration.
Specific implementation mode
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to
These embodiments.
Embodiment 1
By taking tyrosine protein kinase Src as an example, activity test method is as follows：
1, by the polypeptide of 2 μ L, 25 μm of ol/L carboxyl tetramethylrhodamines label, (by gill biochemistry Shanghai, Co., Ltd carries
Amino acid sequence for, polypeptide is RRRRIYGEFKK) aqueous solution, 3 μ L 1mmol/L ATP aqueous solutions and different volumes 10 ×
10-3It is 7.5 that μ g/ μ L-Tyr protein kinases Src (enzymatic activity used is 1020U/mg) solution, which is distributed to 25mmol/L pH value,
HEPES buffer solutions (MgCl containing 10mmol/L2) in, final volume is 20 μ L, makes tyrosine protein kinase Src in mixed system
Activity be respectively 0,0.5 × 10-4、1×10-4、2×10-4、3×10-4、4×10-4、5×10-4、6×10-4、7×10-4、8
×10-4、9×10-4、10×10-4、15×10-4μ g/ μ L incubate 60 minutes for 37 DEG C on constant temperature culture oscillator, carry out phosphoric acid
Change reaction.
2, tyrosinase aqueous solution, 40 μ of 3 μ L 2kU/mL are added into the solution after step 1 phosphorylation reaction
The Tris-HCl buffer solutions that L250mmol/L pH value is 6.6, are settled to 200 μ L, then on constant temperature culture oscillator with water
37 DEG C incubate 40 minutes, carry out tyrosinase oxidation reaction.After having reacted, with luminoscope (excitation wavelength 554nm, emission maximum
Wavelength 583nm, entrance slit 2.0nm, exit slit 1.0nm) measure reaction system fluorescence intensity.The fluorescence of reaction system is believed
Number see Fig. 1 with the spectrogram of tyrosine protein kinase Src activity changes, and draws at 583nm fluorescence intensity with tyrosine protein
The standard curve of kinases Src activity changes (see Fig. 2).As shown in Figure 2, fluorescence intensity and tyrosine protein kinase Src at 583nm
Activity 0~2 × 10-4μ g/ μ L and 2 × 10-4~9 × 10-4It is in good linear pass respectively in the active sections μ g/ μ L two
System, the activity of wherein tyrosine protein kinase Src is 0~2 × 10-4The linear equation in μ g/ μ L activity section is：Y=7080X+
34936,2 × 10-4~9 × 10-4The linear equation in μ g/ μ L activity section is：Y=30318X-13247, Y represents body in formula
The fluorescence intensity of system, X represent tyrosine protein kinase Src activity, and correlation coefficient r value is 0.99329,0.99824 respectively.Thus
It is found that the fluorescence intensity of system and the active linear relationships of tyrosine protein kinase Src are good.
3, the corresponding fluorescence intensities of tyrosine protein kinase Src in sample to be tested are detected according to above-mentioned steps 1 and 2, according to
The active quantitative analyses of tyrosine protein kinase Src in sample to be tested can be realized in standard curve.
Embodiment 2
In order to further increase the Activity determination range of tyrosine protein kinase Src, the present invention is by improving peptide substrate
Concentration carries out phosphorylation reaction, has widened the active detection dynamic ranges of tyrosine protein kinase Src, specific experiment process is such as
Under：
1, by the polypeptide of 2 μ L, 100 μm of ol/L carboxyl tetramethylrhodamines label, (by gill biochemistry Shanghai, Co., Ltd carries
Amino acid sequence for, polypeptide is RRRRIYGEFKK) aqueous solution, 3 μ L 1mmol/L ATP aqueous solutions and different volumes 10 ×
10-3It is 7.5 that tyrosine protein kinase Src (enzymatic activity used is 1020U/mg) solution of μ g/ μ L, which is distributed to 25mmol/L pH value,
HEPES buffer solutions (MgCl containing 10mmol/L2) in, final volume is 20 μ L, makes tyrosine protein kinase in mixed system
The activity of Src is respectively 0,2 × 10-4、5×10-4、7.5×10-4、10×10-4、12.5×10-4、15×10-4、20×10-4μ
G/ μ L incubate 60 minutes for 37 DEG C on constant temperature culture oscillator, carry out phosphorylation reaction.
2, tyrosinase aqueous solution, 40 μ of 3 μ L 2kU/mL are added into the solution after step 1 phosphatase reaction
The Tris-HCl buffer solutions that L250mmol/L pH value is 6.6, are settled to 200 μ L, then on constant temperature culture oscillator with water
37 DEG C incubate 40 minutes, carry out tyrosinase oxidation reaction.After having reacted, with luminoscope (excitation wavelength 554nm, emission maximum
Wavelength 583nm, entrance slit 2.0nm, exit slit 1.0nm) measure reaction system fluorescence intensity.The fluorescence of reaction system is believed
Number see Fig. 3 with the spectrogram of tyrosine protein kinase Src activity changes, and draws at 583nm fluorescence intensity with tyrosine protein
The standard curve of kinases Src activity changes (see Fig. 4).From fig. 4, it can be seen that fluorescence intensity and tyrosine protein kinase Src at 583nm
Activity 2 × 10-4~15 × 10-4The linear equation in μ g/ μ L activity section is：Y=28844X+231274, Y is represented in formula
The fluorescence intensity of system, X represent tyrosine protein kinase Src activity, and correlation coefficient r value is 0.99418.
3, the corresponding fluorescence intensities of tyrosine protein kinase Src in sample to be tested are detected according to above-mentioned steps 1 and 2, according to
The active quantitative analyses of tyrosine protein kinase Src in sample to be tested can be realized in standard curve.
The peptide substrate of different types of tyrosine protein kinase effect has specificity, therefore is directed to specific tyrosine
Protein kinase, the present invention need to only choose corresponding polypeptide and carry out fluorescent marker, according to above-mentioned similar detection process
Realize the activity analysis of specific tyrosine kinase.
Embodiment 3
Protein tyrosine kinase activity analysis method of the present invention is also applied for the screening of kinases inhibitor.It uses below
For the Src inhibitor PP2 (3- (4- chlorphenyls) -1- tertiary butyl -1H- pyrazolos [3,4-D] pyrimidine -4- amine) known, this is verified
Feasibility of the method in inhibitor screening, specific experiment process are as follows：
1, by 2 μ L 7 × 10-3Tyrosine protein kinase Src (enzymatic activity used the be 1020U/mg) solution of μ g/ μ L and not
The polypeptide marked with the inhibitor PP2 aqueous solutions of concentration, 2 μ L, 25 μm of ol/L carboxyl tetramethylrhodamines is (by gill biochemistry Shanghai
Co., Ltd provides, and the amino acid sequence of polypeptide is RRRRIYGEFKK) aqueous solution and 3 μ L1mmol/L ATP aqueous dispersions exist
HEPES buffer solution (the MgCl containing 10mmol/L that 25mmol/L pH value is 7.52) in, final volume is 20 μ L, is made in mixed system
The final concentration of inhibitor PP2 be respectively 0nmol/L, 1nmol/L, 5nmol/L, 10nmol/L, 50nmol/L, 100nmol/L,
200nmol/L、500nmol/L、1μmol/L、5μmol/L.The mixed system is incubated 60 for 37 DEG C on constant temperature culture oscillator
Minute, carry out phosphorylation reaction.
2, tyrosinase aqueous solution, 40 μ of 3 μ L 2kU/mL are added into the solution after step 1 phosphorylation reaction
The Tris-HCl buffer solutions that L250mmol/L pH value is 6.6, are settled to 200 μ L, then on constant temperature culture oscillator with water
37 DEG C incubate 40 minutes, carry out tyrosinase oxidation reaction.After having reacted, with luminoscope (excitation wavelength 554nm, emission maximum
Wavelength 583nm, entrance slit 2.0nm, exit slit 1.0nm) measure reaction system fluorescence intensity.The fluorescence of reaction system is believed
Number see Fig. 5 with the spectrogram of inhibitor PP2 concentration variation, and draws the fluorescence intensity of reaction system and inhibitor PP2 583nm at
The logarithmic relationship curve of concentration (see Fig. 6).
By Figures 5 and 6 as it can be seen that under fixed tyrosine protein kinase Src concentration conditions, with the increasing of inhibitor PP2 concentration
Add, the fluorescence intensity of system gradually weakens.It follows that the increase of inhibitor PP2 concentration improves inhibition tyrosine protein kinase
The active abilities of enzyme Src cause the substrate polypeptide ratio not being phosphorylated to increase, after tyrosinase oxidation, fluorescence dye
The fluorescence intensity of material continuously decreases.This is the result shows that the method for the present invention can be used in the screening of tyrosine protein kinase inhibitor.
Claims (2)
1. a kind of Fluorescence Increasing type protein tyrosine kinase activity analysis method of tyrosinase auxiliary, it is characterised in that it is under
State step composition：
（1）It is anti-that the peptide substrate of the corresponding fluorescent marker of the tyrosine protein kinase of known activity is mixed into progress phosphorylation
It answers, wherein the fluorescent marker in the peptide substrate of the fluorescent marker is carboxyl tetramethylrhodamine, 2,7- dimethyl -4,5-
Two chloro- 6- Fluoresceincarboxylic acids, four chloro- 6- Fluoresceincarboxylic acids, chlordene -6- methylfluoresceins, texas Red, 3H- indoles cyanines dye
Any one in material；
（2）By step（1）The fluorescence intensity of detection architecture after solution after middle phosphorylation reaction is mixed with tyrosinase is drawn glimmering
The standard curve that luminous intensity changes with protein tyrosine kinase activity；
（3）According to above-mentioned steps（1）With（2）The corresponding fluorescence intensity of tyrosine protein kinase sample to be measured is detected, according to standard
The quantitative analysis of protein tyrosine kinase activity in sample to be tested can be realized in curve.
2. the Fluorescence Increasing type protein tyrosine kinase activity analysis method of tyrosinase auxiliary according to claim 1,
It is characterized in that：The step（2）In, it is added 10 according to every micromolar fluorescent marker peptide substrate4~105U tyrosinases.
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| CN109001177A (en) * | 2018-06-14 | 2018-12-14 | 福建师范大学 | The detection method of tyrosine in a kind of urine |
| CN111678900B (en) * | 2020-06-30 | 2023-03-21 | 东南大学 | Cell membrane and cell wall fluorescence labeling method based on tyrosinase catalysis and application |
| CN112255208B (en) * | 2020-10-09 | 2023-01-10 | 安阳工学院 | Compound for detecting tyrosinase and application thereof |
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