CN104730049A - Method for testing ion transmission condition of peptide fragment in simulated environment - Google Patents
Method for testing ion transmission condition of peptide fragment in simulated environment Download PDFInfo
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- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 claims abstract description 80
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Abstract
The invention belongs to the technical field of biology and particularly relates to a method for testing an ion transmission condition of a peptide fragment in a simulated environment. The testing method is a fluorescent spectrometry; and ions are metal ions. The testing method comprises the following six steps: preparing a buffering solution; preparing a standard sample stock solution of aureomycin, preparing a phospholipid monolayer vesicle without the peptide fragment, carrying out fluorescence detection on the phospholipid monolayer vesicle without the peptide fragment, preparing a phospholipid monolayer vesicle with the peptide fragment, and carrying out the fluorescence detection on the phospholipid monolayer vesicle with the peptide fragment. Aiming at the peptide fragment of a fourth membrane-spanning protein region, a testing method and a testing condition are further improved; by virtue of the testing method, the defects that the operation is complicated, the specialty is high, the cost is high and the requirements on instruments and operators are relatively high in the prior art are overcome; and the ion transmission condition of the fourth membrane-spanning protein region can be relatively conveniently researched, and a transmission function of a whole solute transportprotein transmission body is achieved.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method of ion transmission situation of test peptides section in simulated environment.
Background technology
Genome sorting data discloses 1/4th memebrane proteins in the protein decoded at DNA, and memebrane protein is the protein that a class has special construction, and it is embedded in immobilized artificial membrane, is in cell and extraneous junction section.Memebrane protein is divided into peripheral protein and the large class of integrated protein two substantially.The memebrane protein running through whole Lipid bilayer membranes is also called transmembrane protein.Memebrane protein controls the basic activity process of many life entities, as cell growth, division and tune is died, the transmission of the various signal of iuntercellular, the sense of smell sense of taste etc. of life entity.Memebrane protein forms the acceptor of various nerve signal molecule, hormone and substrate, forms the transmission ion channel of ion and the transporter of material.Memebrane protein plays an important role in Disease epizootic and in new drug development, in new drug development, about has the drug target of 70% to be memebrane protein.Solute Transport Protein S lc11a1 (being also Nramp1) is exactly a member in these important membrane albumen, is made up of 548 residues, and some infectious diseases and the auto-immune disease of it and the mankind are closely related.Solute Transport albumen has the peptide molecule of typical integral membrane protein feature, has in 10-12 the hydrophobic transmembrane supposed, a 1-2 N-glycosylated kytoplasm outer ring-like structure and a kytoplasm the upper structure with transport protein feature highly retained of evolving.
In glycosylated born of the same parents' outer ring-like structure and several born of the same parents, the phosphorylation position in unknown loop district has transmission Fe
2+, Zn
2+, and Mn
2+deng the function of metallic ion, it is a kind of important metallic ion transmission body.Wherein the 4th transmembrane protein district (TM4) participates in the metalloform-selective transmission path defining proton coupling, G169D sudden change causes the function transmitting metallic ion to completely lose or some lost, Slc11a1 Gene regulation mammal is to the resistivity of pathogenic infection, and the G169D sudden change in the 4th cross-film district will cause reduction or the forfeiture of macrophage opposing multiple pathogens infection ability.As can be seen here, the ion transmission situation of research the 4th transmembrane protein district functional peptide fragment can disclose the transfer function of overall Solute Transport albumen transmission body, and this research contributes to the function that we are familiar with intact proteins.Slc11a1 plays an important role in the defence of some infectious diseases at mammal opposing courses of infection and the mankind as a member of Slc11 family, us not only can be helped to understand the transporting mechanism of Slc11a1 transmission body to the announcement of its structure and orientation, and also have potential meaning in the treatment of disease and the exploitation of new drug.
In order to better observe the ion transmission situation of the 4th transmembrane protein district functional peptide fragment, in prior art, occur that a large amount of ion for observation the 4th transmembrane protein district functional peptide fragment exports the method for situation.Such as voltage clamping observation method, single channel recording observation method etc.
Voltage clamping observation method, generally speaking, the change of film to certain ion permeability is the function of film potential and time.By forming tight sealing-in between glass microelectrode and cell membrane, utilizing electronics technologies to apply a transmembrane voltage and film potential is fixed on a certain numerical value, the time dependent dynamic process of gas current under this film potential condition can be measured.Utilize the solution composition of medicine or change intraor extracellular, other ion channels were lost efficacy, the functional parameter of certain studied ion channel can be measured, analyze the stable state of gas current and dynamics and the relation between film potential, ion concentration etc., the conductance of deducibility this kind of passage, activation and deactivation rate, ion selectivity etc., and can measure and the characteristic of gating current of analysis channel.But the ion transmission situation in the method observation the 4th transmembrane protein district is more complicated, test findings also needs the result of the ion transmission situation calculating and more just can draw the 4th transmembrane protein district functional peptide fragment further.
Single channel recording observation method, also known as diaphragm clamp technology, is adsorbed in cell surface with special glass micro pipette, makes it the sealing of formation 10 ~ 100G Ω, is μm 2 magnitudes, only has minority ion channel in interior by the Small diaphragm-piece area isolated.Then carry out voltage clamping to this diaphragm, can measure the electric current of pA (10-12 ampere) magnitude that single ion channel opens produces, this channel opener is a kind of stochastic process.By observing the curent change of single channel opener and closedown, directly can obtain the function parameters such as the current amplitude distribution of various ion channel opens, open probability, open life-span distribution, and analyze they and the relation between film potential, ion concentration etc.Also the diaphragm of suction pipe absorption can be separated from cell membrane, carry out experimental study in modes such as outside the lateral of film or the inner side of film are outside.This technology to the voltage clamping of cellule, to change inside and outside film solution composition and apply medicine all very convenient.Applied voltage clamper or single channel recording technology, can use the medicine of various concentration respectively at different time, different parts (inside film or outside), study their impacts on the various function of passage.In conjunction with the understanding to drug molecular structure, not only can understand medicine and toxin in depth to the mechanism of humans and animals physiological function, the information such as type and conformation of channel function subunit can also be obtained from molecular level, above-mentioned technology is all carry out in active somatic cell, have complicated operation, professional height, cost high, all higher shortcoming is required to instrument and operating personnel.
Summary of the invention
In order to solve the problems of the technologies described above, the present invention adopts the method for the ion transmission situation of fluorescent spectrometry test peptides section in simulated environment, solve complicated operation in prior art, professional height, cost high, all higher shortcoming is required to instrument and operating personnel.
Fluorescent spectrometry is commonly used in the research of protein-small molecule interaction.The much informations such as the many polarity environment of information residing for the change of binding constant, binding site number, binding site, acting force type, protein molecule structure in interaction and protein about protein and Small molecular effect can be obtained by the mensuration (fluorescence intensity, quantum yield, fluorescence lifetime, fluorescence polarization, fluorescent quenching) to some fluorescence parameters.Fluorescence spectrum is widely used in the research of memebrane protein.Utilize the method for fluorescent quenching can determine the impact of the change of the degree of depth that transmembrane protein is imbedded in film and external environmental condition on protein position.The ion transmission situation that the present invention will adopt fluorescent spectrometry to test transmembrane protein functional peptide fragment in simulated environment, for the 4th transmembrane protein district, further retrofit testing method and test condition, make method of testing of the present invention solve complicated operation in prior art, professional height, cost high, all higher shortcoming is required to instrument and operating personnel, be more convenient for further investigation the 4th transmembrane protein district functional peptide fragment ion transmission situation, and disclosed overall Solute Transport albumen transmission body transfer function.
Technical scheme of the present invention is:
A method for the ion transmission situation of test peptides section in simulated environment, described method of testing is fluorescent spectrometry, and described ion is metallic ion, said method comprising the steps of:
Step one: prepare buffer solution: by deionized water secure ph scope at the sodium hydrogen phosphate-citric acid solution 200 ~ 300ml of 3 ~ 7, and add sodium chloride;
Step 2: the standard specimen storing solution preparing aureomycin: diluted by aureomycin ultrapure water, makes its concentration be 0.05g/L ~ 0.2g/L, with ultrasonic dissolution, seals for subsequent use;
Step 3: the phospholipid monolayer vesica of peptide section is not added in preparation: be that 0.5 ~ 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 ~ 150 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 15 ~ 25h, standard specimen storing solution and the buffer solution of aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle,
Step 4: fluoroscopic examination: selective excitation wavelength is 280 ~ 380nm, the phospholipid monolayer vesica fluorescence spectrum figure not adding transmembrane protein prepared by measuring process three, records its maximum emission wavelength λ
max1; Bivalent metal ion is joined in the solution of step 3 and be prepared into liquid A to be measured, measure liquid A fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max2; After being greater than 24h standing time, measure the fluorescence spectrum figure of liquid A to be measured, record its maximum emission wavelength λ
max3;
Step 5: the phospholipid monolayer vesica of peptide section is added in preparation: prepare peptide storing solution, be that 0.5 ~ 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 ~ 150 μ l, add the peptide storing solution that volume is 15 ~ 30 μ l, shake up and be blown into nitrogen until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 15 ~ 25h, standard specimen storing solution and the buffer solution of aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle,
Step 6: fluoroscopic examination: selective excitation wavelength is 280 ~ 380nm, the phospholipid monolayer vesica fluorescence spectrum figure of interpolation peptide section prepared by measuring process five, records its maximum emission wavelength λ
max4; Bivalent metal ion is joined in the solution of step 5 and be prepared into liquid B to be measured, measure liquid B fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max5; After it is stable, measures the fluorescence spectrum figure of liquid B to be measured, records its maximum emission wavelength λ
max6, compare λ
max1~ λ
max6numerical result.
DMPG (GLYCEROL,DIMYRISTOYL PHOSPHATIDYL) is made up of two alkyl chains of a negative ion head base and 14 carbon.Under physiological condition, the head base of anionic membrane causes cell membrane electronegative.The electronegative characteristic of cell membrane plays important effect in many life processes, and therefore anionic liposome is widely used as model in biological study.The stability, size, phase in version behavior etc. of many physical propertys as vesica of DMPG vesica are affected by the external environment larger.GLYCEROL,DIMYRISTOYL PHOSPHATIDYL is mainly present on prokaryotic film and bacterium as anionic membrane, in water, go into vesica.CTC (aureomycin) molecular formula C
22h
23clN
2o
8, be a kind of bivalent metal ion sensitive fluorescence group, have fluorescence to produce, add when there being bivalent metal ion and fashionablely with it chelating occurs, fluorescence intensity is changed.
Described metallic ion is bivalent metal ion.
Preferably, the pH value range in described step one is 4 ~ 6; The addition of described sodium chloride is 0.500 ~ 1.000g.
Preferably, the preparation method of the standard specimen storing solution of the aureomycin in described step 2 is: adopt electronic balance accurately to take aureomycin standard model 0.500 ~ 2.000mg, use ultrapure water in brown volumetric flask constant volume to 10mL, ultrasonic dissolution, be mixed with the storing solution of 0.05g/L ~ 0.2g/L, it is for subsequent use that sealing is placed in 4 DEG C of refrigerators.
Preferably, the standard specimen Stock concentrations of the aureomycin in described step 2 is 0.1g/L.
Preferably, the excitation wavelength in described step 4 is 300 ~ 350nm.
Preferably, the phospholipid monolayer vesica not adding peptide section is prepared: be that 0.5 ~ 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality in described step 3, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 ~ 150 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 20 ~ 25h, standard specimen storing solution and 1.0 ~ 4.0ml buffer solution of aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, in described step 5, the phospholipid monolayer vesica of peptide section is added in preparation: be that to add volume be prepared into peptide storing solution in 0.5 ~ 1.5ml hexafluoroisopropanol solution to 1.00 ~ 3.00mg peptide by quality, be that 0.5 ~ 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2
:1 mixed solvent 80 ~ 150 μ l, add the peptide storing solution that volume is 15 ~ 30 μ l, shake up and be blown into nitrogen until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 20 ~ 25h, in sample hose, add standard specimen storing solution and 1.0 ~ 4.0ml buffer solution of aureomycin, ultrasonic in a water bath, obtain monolayer vesicle, make aureomycin be dispersed in the inside and outside of monolayer vesicle.
More preferably, in described step 3, the phospholipid monolayer vesica of peptide section is not added in preparation: be that 1.2mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 100 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 24h, standard specimen storing solution and 2.7ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, in described step 5, the phospholipid monolayer vesica of peptide section is added in preparation: be that to add volume be prepared into peptide storing solution in 1ml hexafluoroisopropanol solution to 1.68mg peptide by quality, be that 1.2mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 100 μ l, add the peptide storing solution that volume is 24 μ l, nitrogen is blown into until solvent volatilization is complete after shaking up, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 24h, standard specimen storing solution and 2.7ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle.
Preferably, the excitation wavelength in described step 6 is 300 ~ 350nm.
After liquid B to be measured being greater than 24 standing time in described step 6, measure the fluorescence spectrum figure of liquid B to be measured.
The fluorescence spectrum figure of liquid A more to be measured and liquid B to be measured in described step 6, concrete grammar is: if λ is max
1=λ max
2=λ max
3, prove that bivalent metal ion transmission does not occur liquid A to be measured, if λ is max
1=λ max
2> λ max
3, prove that bivalent metal ion transmission occurs liquid A to be measured; If λ is max
3=λ max
4=λ max
5, prove that bivalent metal ion transmission does not occur liquid B to be measured, if λ is max
4=λ max
5> λ max
6, prove that bivalent metal ion transmission occurs liquid B to be measured; If λ is max
1=λ max
2=λ max
3, λ max
4=λ max
5> λ max
6, prove that described peptide section has the function of transmission bivalent metal ion.
Simulated environment of the present invention is the 4th transmembrane protein district environment of simulation Solute Transport albumen, to measure the ion transmission situation of its peptide section.
Beneficial effect of the present invention: the present invention mainly utilizes fluorescence spectrum research functional peptide fragment to transmit the problem of ion in membrane simulation environment.This method of testing is simple to operate, test result is directly perceived, cost is low, consuming time short, instrument and personnel professional is required low.Can the research of functional peptide fragment transfer function in perfect aspect adventitia simulated environment.
Accompanying drawing explanation
Figure 1 shows that the present invention does not add the phospholipid monolayer vesica schematic diagram of peptide section.
Figure 2 shows that the present invention adds the phospholipid monolayer vesica schematic diagram of peptide section.
Figure 3 shows that phospholipid monolayer capsule that the present invention does not add peptide section adds the aureomycin fluorescence spectrum figure of bivalent metal ion.
Figure 4 shows that phospholipid monolayer capsule that the present invention adds peptide section adds the aureomycin fluorescence spectrum figure of bivalent metal ion.
Figure 5 shows that aureomycin fluorometric investigation that phospholipid monolayer capsule that the present invention adds peptide section adds bivalent metal ion and the contrast spectrogram of aureomycin fluorometric investigation adding bivalent metal ion after 24 hours.
Embodiment
It should be noted that the combination of technical characteristic or the technical characteristic described in following embodiment should not be considered to isolated, they can mutually be combined thus be reached better technique effect.
Embodiment 1
Step one: prepare buffer solution: be the sodium hydrogen phosphate-citric acid solution 200ml of 3 with deionized water secure ph, and add 0.500g sodium chloride;
Step 2: the standard specimen storing solution preparing aureomycin: adopt electronic balance accurately to take aureomycin standard model 0.501mg, use ultrapure water in brown volumetric flask constant volume to 10mL, ultrasonic dissolution, is mixed with the storing solution of 0.05g/L, and it is for subsequent use that sealing is placed in 4 DEG C of refrigerators;
Step 3: the phospholipid monolayer vesica of peptide section is not added in preparation: be that 0.5mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 15h, standard specimen storing solution and 1.0ml buffer solution that volume is 0.2ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 1 is shown in by the phospholipid monolayer vesica schematic diagram not adding peptide section,
Step 4: fluoroscopic examination: selective excitation wavelength is 280nm, the phospholipid monolayer vesica fluorescence spectrum figure not adding transmembrane protein prepared by measuring process three, records its maximum emission wavelength λ
max1; By bivalent metal ion Mn
2+join in the solution of step 3 and be prepared into liquid A to be measured, measure liquid A fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max2; After being greater than 24h standing time, measure the fluorescence spectrum figure of liquid A to be measured, record its maximum emission wavelength λ
max3;
Step 5: the phospholipid monolayer vesica of peptide section is added in preparation: be that to add volume be prepared into peptide storing solution in 0.5ml hexafluoroisopropanol solution to 1.00mg peptide by quality, be that 0.5mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 μ l, add the peptide storing solution that volume is 15 μ l, nitrogen is blown into until solvent volatilization is complete after shaking up, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 15h, standard specimen storing solution and 1.0ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 2 is shown in by the phospholipid monolayer vesica schematic diagram adding peptide section,
Step 6: fluoroscopic examination: selective excitation wavelength is 280nm, the phospholipid monolayer vesica fluorescence spectrum figure of interpolation peptide section prepared by measuring process five, records its maximum emission wavelength λ
max4; By bivalent metal ion Mn2
+join in the solution of step 5 and be prepared into liquid B to be measured, measure liquid B fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max5; After it is stable, measures the fluorescence spectrum figure of liquid B to be measured, records its maximum emission wavelength λ
max6, compare λ
max1~ λ
max6numerical result.Data result is in table 1.
Table 1 embodiment 1 fluorescence data result
From table 1 data, the present embodiment is by Mn
2+ion to join in the solution of step 3 configuration i.e. liquid A to be measured, and metallic ion concentration is in the solution 40uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max1=λ
max2=λ
max3as Fig. 3.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max3still do not change.By Mn
2+ion to join in the solution of step 5 configuration i.e. liquid B to be measured, and metallic ion concentration is in the solution 40uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max4=λ
max5as Fig. 4.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max6there is obvious blue shift, for 417nm is as Fig. 5.Prove that the peptide section of the embodiment of the present invention has the function of transmission bivalent metal ion.
Embodiment 2
Step one: prepare buffer solution: be the sodium hydrogen phosphate-citric acid solution 250ml of 7 with deionized water secure ph, and add 0.731g sodium chloride;
Step 2: the standard specimen storing solution preparing aureomycin: adopt electronic balance accurately to take aureomycin standard model 2.000mg, use ultrapure water in brown volumetric flask constant volume to 10mL, ultrasonic dissolution, is mixed with the storing solution of 0.2g/L, and it is for subsequent use that sealing is placed in 4 DEG C of refrigerators;
Step 3: the phospholipid monolayer vesica of peptide section is not added in preparation:: be that 0.8mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 90 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 25h, standard specimen storing solution and 2.0ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, standard specimen storing solution, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 1 is shown in by the phospholipid monolayer vesica schematic diagram not adding peptide section,
Step 4: fluoroscopic examination: selective excitation wavelength is 380nm, the phospholipid monolayer vesica fluorescence spectrum figure not adding transmembrane protein prepared by measuring process three, records its maximum emission wavelength λ
max1; By bivalent metal ion Cu
2+join in the solution of step 3 and be prepared into liquid A to be measured, measure liquid A fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max2; After being greater than 24h standing time, measure the fluorescence spectrum figure of liquid A to be measured, record its maximum emission wavelength λ
max3;
Step 5: the phospholipid monolayer vesica of peptide section is added in preparation: be that to add volume be prepared into peptide storing solution in 0.9ml hexafluoroisopropanol solution to 1.50mg peptide by quality, be that 0.7mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 90 μ l, add the peptide storing solution that volume is 20 μ l, nitrogen is blown into until solvent volatilization is complete after shaking up, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 25h, standard specimen storing solution and 2.0ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 2 is shown in by the phospholipid monolayer vesica schematic diagram adding peptide section,
Step 6: fluoroscopic examination: selective excitation wavelength is 380nm, the phospholipid monolayer vesica fluorescence spectrum figure of interpolation peptide section prepared by measuring process five, records its maximum emission wavelength λ
max4; By bivalent metal ion Cu
2+join in the solution of step 5 and be prepared into liquid B to be measured, measure liquid B fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max5; After it is stable, measures the fluorescence spectrum figure of liquid B to be measured, records its maximum emission wavelength λ
max6, compare λ
max1~ λ
max6numerical result.Numerical result is in table 2.
Table 2 embodiment 2 fluorescence data result
From table 2 data, the present embodiment is by Cu
2+ion to join in the solution of step 3 configuration i.e. liquid A to be measured, and metallic ion concentration is in the solution 38uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max1=λ
max2=λ
max3, with reference to Fig. 3.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max3still do not change.By Cu
2+ion to join in the solution of step 5 configuration i.e. liquid B to be measured, and metallic ion concentration is in the solution 38uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max4=λ
max5, with reference to Fig. 4.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max6obvious blue shift occurs, and is 416nm, with reference to Fig. 5.Prove that the peptide section of the embodiment of the present invention has the function of transmission bivalent metal ion.
Due to fluorescigenic be aureomycin, λ
maxchange and ionic type have nothing to do, and change not quite so each embodiment does not add peptide section with the fluorescence spectrum figure adding peptide section, and the fluorescence spectrum figure of the present embodiment is with reference to Fig. 3,4 and 5.
Embodiment 3
Step one: prepare buffer solution: be the sodium hydrogen phosphate-citric acid solution 250ml of 4 with deionized water secure ph, and add 0.731g sodium chloride;
Step 2: the standard specimen storing solution preparing aureomycin: adopt electronic balance accurately to take aureomycin standard model 1.000mg, use ultrapure water in brown volumetric flask constant volume to 10mL, ultrasonic dissolution, is mixed with the storing solution of 0.1g/L, and it is for subsequent use that sealing is placed in 4 DEG C of refrigerators;
Step 3: the phospholipid monolayer vesica of peptide section is not added in preparation: be that 1.2mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 100 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 20h, standard specimen storing solution and 2.7ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 1 is shown in by the phospholipid monolayer vesica schematic diagram not adding peptide section,
Step 4: fluoroscopic examination: selective excitation wavelength is 300nm, the phospholipid monolayer vesica fluorescence spectrum figure not adding transmembrane protein prepared by measuring process three, records its maximum emission wavelength λ
max1; By bivalent metal ion Pb
2+join in the solution of step 3 and be prepared into liquid A to be measured, measure liquid A fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max2; After being greater than 24h standing time, measure the fluorescence spectrum figure of liquid A to be measured, record its maximum emission wavelength λ
max3;
Step 5: the phospholipid monolayer vesica of peptide section is added in preparation: be that to add volume be prepared into peptide storing solution in 1ml hexafluoroisopropanol solution to 1.68mg peptide by quality, be that 1.2mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 100 μ l, add the peptide storing solution that volume is 24 μ l, nitrogen is blown into until solvent volatilization is complete after shaking up, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 20h, standard specimen storing solution and 2.7ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 2 is shown in by the phospholipid monolayer vesica schematic diagram adding peptide section,
Step 6: fluoroscopic examination: selective excitation wavelength is 300nm, the phospholipid monolayer vesica fluorescence spectrum figure of interpolation peptide section prepared by measuring process five, records its maximum emission wavelength λ
max4; By bivalent metal ion Pb
2+join in the solution of step 5 and be prepared into liquid B to be measured, measure liquid B fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max5; After it is stable, measures the fluorescence spectrum figure of liquid B to be measured, records its maximum emission wavelength λ
max6, compare λ
max1~ λ
max6numerical result.Data result is in table 3
Table 3 embodiment 3 fluorescence data result
From table 3 data, the present embodiment is by Pb
2+ion to join in the solution of step 3 configuration i.e. liquid A to be measured, and metallic ion concentration is in the solution 42uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max1=λ
max2=λ
max3, with reference to Fig. 3.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max3still do not change.By Pb
2ion to join in the solution of step 5 configuration i.e. liquid B to be measured, and metallic ion concentration is in the solution 42uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max4=λ
max5=λ max
3, with reference to Fig. 4.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max6obvious blue shift occurs, and is 415nm, with reference to Fig. 5.Prove that the peptide section of the embodiment of the present invention has the function of transmission bivalent metal ion.
Due to fluorescigenic be aureomycin, λ
maxchange and ionic type have nothing to do, and change not quite so each embodiment does not add peptide section with the fluorescence spectrum figure adding peptide section, and the fluorescence spectrum figure of the present embodiment is with reference to Fig. 3,4 and 5.
Embodiment 4
Step one: prepare buffer solution: be the sodium hydrogen phosphate-citric acid solution 250ml of 6 with deionized water secure ph, and add 0.731g sodium chloride;
Step 2: the standard specimen storing solution preparing aureomycin: adopt electronic balance accurately to take aureomycin standard model 1.500mg, use ultrapure water in brown volumetric flask constant volume to 10mL, ultrasonic dissolution, is mixed with the storing solution of 0.15g/L, and it is for subsequent use that sealing is placed in 4 DEG C of refrigerators;
Step 3: the phospholipid monolayer vesica of peptide section is not added in preparation: be that 1.5mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 120 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 24h, standard specimen storing solution and 3.5ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 1 is shown in by the phospholipid monolayer vesica schematic diagram not adding peptide section,
Step 4: fluoroscopic examination: selective excitation wavelength is 350nm, the phospholipid monolayer vesica fluorescence spectrum figure not adding transmembrane protein prepared by measuring process three, records its maximum emission wavelength λ
max1; By bivalent metal ion Cd
2+join in the solution of step 3 and be prepared into liquid A to be measured, measure liquid A fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max2; After being greater than 24h standing time, measure the fluorescence spectrum figure of liquid A to be measured, record its maximum emission wavelength λ
max3;
Step 5: the phospholipid monolayer vesica of peptide section is added in preparation: be that to add volume be prepared into peptide storing solution in 1.2ml hexafluoroisopropanol solution to 2.00mg peptide by quality, be that 1.50mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 120 μ l, add the peptide storing solution that volume is 28 μ l, nitrogen is blown into until solvent volatilization is complete after shaking up, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 24h, standard specimen storing solution and 3.5ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 2 is shown in by the phospholipid monolayer vesica schematic diagram adding peptide section,
Step 6: fluoroscopic examination: selective excitation wavelength is 350nm, the phospholipid monolayer vesica fluorescence spectrum figure of interpolation peptide section prepared by measuring process five, records its maximum emission wavelength λ
max4; By bivalent metal ion Cd
2+join in the solution of step 5 and be prepared into liquid B to be measured, measure liquid B fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max5; After it is stable, measures the fluorescence spectrum figure of liquid B to be measured, records its maximum emission wavelength λ
max6, compare λ
max1~ λ
max6numerical result.
Table 4 embodiment 4 fluorescence data result
From table 4 data, the present embodiment is by Cd
2+ion to join in the solution of step 3 configuration i.e. liquid A to be measured, and metallic ion concentration is in the solution 45uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max1=λ
max2=λ
ax3, with reference to Fig. 3.After placing 24h, again measure fluorogram under the same terms, the wavelength X max3 of emission maximum does not still change.By Cd
2+ion to join in the solution of step 5 configuration i.e. liquid B to be measured, and metallic ion concentration is in the solution 45uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max4=λ
max5, with reference to Fig. 4.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max6obvious blue shift occurs, and is 414, with reference to Fig. 5.Prove that the peptide section of the embodiment of the present invention has the function of transmission bivalent metal ion.
Due to fluorescigenic be aureomycin, λ
maxchange and ionic type have nothing to do, and change not quite so each embodiment does not add peptide section with the fluorescence spectrum figure adding peptide section, and the fluorescence spectrum figure of the present embodiment is with reference to Fig. 3,4 and 5.
Embodiment 5
Step one: prepare buffer solution: be the sodium hydrogen phosphate-citric acid solution 300ml of 5 with deionized water secure ph, and add 1.000g sodium chloride;
Step 2: the standard specimen storing solution preparing aureomycin: adopt electronic balance accurately to take aureomycin standard model 1.800mg, use ultrapure water in brown volumetric flask constant volume to 10mL, ultrasonic dissolution, is mixed with the storing solution of 0.18g/L, and it is for subsequent use that sealing is placed in 4 DEG C of refrigerators;
Step 3: the phospholipid monolayer vesica of peptide section is not added in preparation: be that 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 150 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 23h, standard specimen storing solution and 4.0ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 1 is shown in by the phospholipid monolayer vesica schematic diagram not adding peptide section,
Step 4: fluoroscopic examination: selective excitation wavelength is 320nm, the phospholipid monolayer vesica fluorescence spectrum figure not adding transmembrane protein prepared by measuring process three, records its maximum emission wavelength λ
max1; By bivalent metal ion Fe
2+join in the solution of step 3 and be prepared into liquid A to be measured, measure liquid A fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max2; After being greater than 24h standing time, measure the fluorescence spectrum figure of liquid A to be measured, record its maximum emission wavelength λ
max3;
Step 5: the phospholipid monolayer vesica of peptide section is added in preparation: be that to add volume be prepared into peptide storing solution in 1.5ml hexafluoroisopropanol solution to 3.00mg peptide by quality, be that 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 150 μ l, add the peptide storing solution that volume is 30 μ l, nitrogen is blown into until solvent volatilization is complete after shaking up, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 23h, standard specimen storing solution and 4.0ml buffer solution that volume is 0.3ml aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, Fig. 2 is shown in by the phospholipid monolayer vesica schematic diagram adding peptide section,
Step 6: fluoroscopic examination: selective excitation wavelength is 320nm, the phospholipid monolayer vesica fluorescence spectrum figure of interpolation peptide section prepared by measuring process five, records its maximum emission wavelength λ
max4; By bivalent metal ion Fe
2+join in the solution of step 5 and be prepared into liquid B to be measured, measure liquid B fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max5; After it is stable, measures the fluorescence spectrum figure of liquid B to be measured, records its maximum emission wavelength λ
max6, compare λ
max1~ λ
max6numerical result.
Table 5 embodiment 5 fluorescence data result
From table 5 data, the present embodiment is by Fe
2+ion to join in the solution of step 3 configuration i.e. liquid A to be measured, and metallic ion concentration is in the solution 40uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max1=λ
max2=λ
max3, with reference to Fig. 3.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max3still do not change.By Fe
2+ion to join in the solution of step 5 configuration i.e. liquid B to be measured, and metallic ion concentration is in the solution 40uM, with aureomycin generation chelatropic reaction, after it is stable, measures fluorogram, and does not add fluorescence intensity compared with metallic ion and strengthens, but λ
max4=λ
max5, with reference to Fig. 4.After placing 24h, again measure fluorogram under the same terms, the wavelength X of emission maximum
max6obvious blue shift occurs, and is 416nm, with reference to Fig. 5.Prove that the peptide section of the embodiment of the present invention has the function of transmission bivalent metal ion.
Due to fluorescigenic be aureomycin, λ
maxchange and ionic type have nothing to do, and change not quite so each embodiment does not add peptide section with the fluorescence spectrum figure adding peptide section, and the fluorescence spectrum figure of the present embodiment is with reference to Fig. 3,4 and 5.
In sum, the present invention is directed to the peptide section in the 4th transmembrane protein district, further retrofit testing method and test condition, this method of testing solve complicated operation in prior art, professional height, cost high, all higher shortcoming is required to instrument and operating personnel, be convenient to the ion transmission situation of further investigation the 4th transmembrane protein district functional peptide fragment, the research of functional peptide fragment transfer function in quick perfect aspect adventitia simulated environment, and the transfer function being disclosed overall Solute Transport albumen transmission body.
Finally should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention; but not limiting the scope of the invention; although done to explain to the present invention with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to technical scheme of the present invention or equivalent replacement, and not depart from essence and the scope of technical solution of the present invention.
Claims (10)
1. in simulated environment, the ion of test peptides section transmits a method for situation, and it is characterized in that, described method of testing is fluorescent spectrometry, and described ion is metallic ion, said method comprising the steps of:
Step one: prepare buffer solution: by deionized water secure ph scope at the sodium hydrogen phosphate-citric acid solution 200 ~ 300ml of 3 ~ 7, and add sodium chloride;
Step 2: the standard specimen storing solution preparing aureomycin: diluted by aureomycin ultrapure water, makes its concentration be 0.05g/L ~ 0.2g/L, with ultrasonic dissolution, seals for subsequent use;
Step 3: the phospholipid monolayer vesica of peptide section is not added in preparation: be that 0.5 ~ 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 ~ 150 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 15 ~ 25h, standard specimen storing solution and the buffer solution of aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle,
Step 4: fluoroscopic examination: selective excitation wavelength is 280 ~ 380nm, the phospholipid monolayer vesica fluorescence spectrum figure not adding transmembrane protein prepared by measuring process three, records its maximum emission wavelength λ
max1; Bivalent metal ion is joined in the solution of step 3 and be prepared into liquid A to be measured, measure liquid A fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max2; After being greater than 24h standing time, measure the fluorescence spectrum figure of liquid A to be measured, record its maximum emission wavelength λ
max3;
Step 5: the phospholipid monolayer vesica of peptide section is added in preparation: prepare peptide storing solution, be that 0.5 ~ 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 ~ 150 μ l, add the peptide storing solution that volume is 15 ~ 30 μ l, shake up and be blown into nitrogen until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 15 ~ 25h, standard specimen storing solution and the buffer solution of aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle,
Step 6: fluoroscopic examination: selective excitation wavelength is 280 ~ 380nm, the phospholipid monolayer vesica fluorescence spectrum figure of interpolation peptide section prepared by measuring process five, records its maximum emission wavelength λ
max4; Bivalent metal ion is joined in the solution of step 5 and be prepared into liquid B to be measured, measure liquid B fluorescence spectrum figure to be measured, record its maximum emission wavelength λ
max5; After it is stable, measures the fluorescence spectrum figure of liquid B to be measured, records its maximum emission wavelength λ
max6, compare λ
max1~ λ
max6numerical result.
2. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 1, it is characterized in that, described metallic ion is bivalent metal ion.
3. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 1, it is characterized in that, the pH value range in described step one is 4 ~ 6; The addition of described sodium chloride is 0.500 ~ 1.000g.
4. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 1, it is characterized in that, the preparation method of the standard specimen storing solution of the aureomycin in described step 2 is: adopt electronic balance accurately to take aureomycin standard model 0.500 ~ 2.000mg, use ultrapure water in brown volumetric flask constant volume to 10mL, ultrasonic dissolution, be mixed with the storing solution of 0.05g/L ~ 0.2g/L, it is for subsequent use that sealing is placed in 4 DEG C of refrigerators.
5. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 4, it is characterized in that, the standard specimen Stock concentrations of the aureomycin in described step 2 is 0.1g/L.
6. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 1, it is characterized in that, the excitation wavelength in described step 4 is 300 ~ 350nm.
7. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 1, it is characterized in that, the phospholipid monolayer vesica not adding peptide section is prepared: be that 0.5 ~ 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality in described step 3, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 ~ 150 μ l, nitrogen is blown into until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 20 ~ 25h, standard specimen storing solution and 1.0 ~ 4.0ml buffer solution of aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle, in described step 5, the phospholipid monolayer vesica of peptide section is added in preparation: be that to add volume be prepared into peptide storing solution in 0.5 ~ 1.5ml hexafluoroisopropanol solution to 1.00 ~ 3.00mg peptide by quality, be that 0.5 ~ 2.0mg GLYCEROL,DIMYRISTOYL PHOSPHATIDYL puts into sample hose by quality, add chloroform and methyl alcohol volume ratio is 2: 1 mixed solvent 80 ~ 150 μ l, add the peptide storing solution that volume is 15 ~ 30 μ l, shake up and be blown into nitrogen until solvent volatilization is complete after GLYCEROL,DIMYRISTOYL PHOSPHATIDYL dissolves completely, liposome forms the film of layer of transparent in the bottom of pipe, after vacuum drying 20 ~ 25h, standard specimen storing solution and 1.0 ~ 4.0ml buffer solution of aureomycin is added in sample hose, ultrasonic in a water bath, obtain monolayer vesicle, aureomycin is made to be dispersed in the inside and outside of monolayer vesicle.
8. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 1, it is characterized in that, the excitation wavelength in described step 6 is 300 ~ 350nm.
9. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 1, is characterized in that, after liquid B to be measured being greater than 24h standing time in described step 6, measures the fluorescence spectrum figure of liquid B to be measured.
10. the method for the ion transmission situation of a kind of test peptides section in simulated environment according to claim 1, it is characterized in that, the fluorescence spectrum figure of liquid A more to be measured and liquid B to be measured in described step 6, concrete grammar is: if λ is max
1=λ max
2=λ max
3, prove that bivalent metal ion transmission does not occur liquid A to be measured, if λ is max
1=λ max
2> λ max
3, prove that bivalent metal ion transmission occurs liquid A to be measured; If λ is max
3=λ max
4=λ max
5, prove that bivalent metal ion transmission does not occur liquid B to be measured, if λ is max
4=λ max
5> λ max
6, prove that bivalent metal ion transmission occurs liquid B to be measured; If λ is max
1=λ max
2=λ max
3, λ max
4=λ max
5> λ max
6, prove that described peptide section has the function of transmission bivalent metal ion.
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| WO2005056146A2 (en) * | 2003-12-11 | 2005-06-23 | Kazuhiro Imai | Method of detection, separation and identification for expressed trace protein/peptide |
| CN101995430A (en) * | 2010-09-21 | 2011-03-30 | 华南师范大学 | Detecting device and detection method of ion transmembrane migration number tracking |
| CN103868897A (en) * | 2014-01-26 | 2014-06-18 | 中国科学院长春光学精密机械与物理研究所 | Fluorescent biomarker multi-micropore plate self-reference quantitative detection method |
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| US5245551A (en) * | 1991-04-10 | 1993-09-14 | University Of Utah Research Foundation | Method of determining extinction coefficient of fluorescent dye and protein concentration of dye-protein conjugate |
| WO2005056146A2 (en) * | 2003-12-11 | 2005-06-23 | Kazuhiro Imai | Method of detection, separation and identification for expressed trace protein/peptide |
| CN101995430A (en) * | 2010-09-21 | 2011-03-30 | 华南师范大学 | Detecting device and detection method of ion transmembrane migration number tracking |
| CN103868897A (en) * | 2014-01-26 | 2014-06-18 | 中国科学院长春光学精密机械与物理研究所 | Fluorescent biomarker multi-micropore plate self-reference quantitative detection method |
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