CN107312068B - Fluorescent probe for detecting activity of proline isomerase, preparation and application thereof - Google Patents
Fluorescent probe for detecting activity of proline isomerase, preparation and application thereof Download PDFInfo
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Abstract
The invention provides a fluorescent probe for detecting the activity of proline isomerase. The probe has weak fluorescence in physiological environment, can specifically generate configuration inversion under the catalytic action of proline isomerase to generate a product with enhanced fluorescence signal, and can be used for activity evaluation of proline isomerase. Can be applied to the detection of the activity of proline isomerase in the extracting solution of cell or tissue protein; and the application in detecting the activity of proline isomerase in living cells. The fluorescent probe and the detection method provided by the invention have the following beneficial effects: (1) the probe has good stability and can be stored and used for a long time; (2) the detection method is simple and convenient, the detection system is added with the probe to be incubated for 10 minutes to measure the fluorescence intensity, and the result can be given without the assistance of other reagents; (3) in vivo detection of proline isomerase activity in living cells could be achieved, whereas other reported methods could only be used for cell homogenates. The structure of the probe is as follows:
Description
Technical Field
The invention belongs to the field of biological detection, and relates to a fluorescent probe for detecting the activity of proline isomerase, a preparation method thereof and application thereof in detecting the activity of proline isomerase in a biological sample.
Background
Proline is a special amino acid, and the amino group in the structure of proline is disubstituted secondary amine. In the protein secondary structure, the normal alpha helical backbone is disrupted by the presence of proline, which forms the beta turn. The proline isomerase is a highly conserved immunophilin family, which is composed of three subfamilies, namely an FK506 binding Family (FKBPs), a cyclophilins Family (FKBPs) and a parvulins family, and is expressed in various microorganisms, such as prokaryotes and eukaryotes, and animals and plants. In many tissue sites of the human body, expression is also abundant and can regulate various functions of cells, such as calcium-related signal regulation, protein folding and gene expression. The catalytic principle of the proline isomerase is mainly that the proline of a substrate protein is isomerized by acting on the proline of the substrate protein through the active structural domain of the proline isomerase, so that the protein conformation is changed, and the function of the protein is changed.
The storage and release of intracellular calcium signals can lead to the activation of a variety of signaling pathways, involving a variety of response pathways for autophagy, apoptosis and necrosis of cells. While the endoplasmic reticulum serves as a reservoir of intracellular calcium ions, which are regulated by the ryanodine (ryanodine) receptor and inositol triphosphate (IP 3). Meanwhile, related research reports indicate that FKBPs can bind to related receptors on endoplasmic reticulum membrane, regulate calcium release, and thus cause activation of cell signals.
There is increasing evidence that FKBPs are involved in numerous cellular processes, such as the cell cycle, cell survival and apoptotic pathways, particularly in cancer. Because in cancer tissues, the expression of FKBPs is found to be abnormal. Thus, proline isomerase plays an important role in tumor neogenesis, radiotherapy and chemotherapy reactions, and it can inhibit oncogenes or tumors in various tissues. Numerous clinical data also indicate that the proline isomerase FKBPs can be used as biomarkers for cancer diagnosis, treatment and prognosis.
At present, no detection reagent for activity and content of proline isomerase exists in the market and clinically, but the detection for activity of proline isomerase recombinant protein generally adopts a double-enzyme-linked enzyme digestion method for detection, the method mainly combines chymotrypsin digestion with activity of recombinant protein proline isomerase, and meanwhile, the method is only suitable for in vitro detection and cannot be used for detection of living cells and in vivo, so that the application range of the method is limited.
Disclosure of Invention
The invention aims to provide a fluorescent probe for detecting the activity of proline isomerase, which can quickly detect the activity of proline isomerase in vitro and in living cells. Has a structure shown in formula I:
wherein R is1Is hydrogen or a benzene ring;
R2is a fluorophore such as naphthoic acid, fluorescein, rhodamine, BODIPY, etc.
Still another object of the present invention is to provide a method for synthesizing a compound represented by formula I, which comprises the following steps: (1) preparing N-Fmoc-phenylalanyl-p-nitroaniline by taking phenylalanine and p-nitroaniline protected by amino Fmoc as raw materials; (2) preparation of N-terminal R by solid phase synthesis2A modified "alanine-glycine (or phenylalanine) -proline" tripeptide chain; (3) removing Fmoc protecting group from the N-Fmoc-phenylalanyl-p-nitroaniline prepared in the step and then reacting the Fmoc protecting group with the R2And condensing the marked tripeptide chain in a dried dichloromethane solution to obtain the fluorescent probe shown in the formula I.
Still another object of the present invention is to provide the use of the compound represented by formula I for detecting proline isomerase activity in a cell or tissue protein extract, which is achieved by the following steps:
1. tissue sample extraction: 1) collecting a tissue sample of a specific part, and storing the tissue sample at-80 ℃; 2) preparing a lysate (10mM Tris,150mM NaCl,1mM EDTA, pH7.5, adding 0.1mg/ml PMSF, 10 mu g/ml Leuteptin and 20 mu g/ml Aprotitin before use); 3) adding a certain amount of lysate according to the sample mass of 10mg/50 mu l, and grinding and cracking by using a grinding rod; 4) the cells were lysed on ice for 30min, centrifuged at 12000g for 10min, the supernatant was pipetted into a new EP tube, the protein extract was quantitatively analyzed using a kit (Bio-Rad, cat. 5000111), and finally the lysate was supplemented to a final concentration of 1. mu.g/. mu.l.
2. Cell sample extraction: 1) when the cells had grown to 90% density, the supernatant was discarded and 3ml of PBS (8.00g NaCl,0.20g KCl,3.63g Na) was added2HPO4·12H2O,0.24g KH2PO4Adding water to a constant volume of 1000ml), cleaning for 3 times, adding 1.5ml of PBS solution, collecting cells by using a clean cell spatula, sucking the solution into a centrifuge tube, centrifuging for 4min at 1200rpm, discarding all supernatant, keeping bottom cells, and freezing at-80 ℃ for subsequent use; 2) lysates (10mM Tris,150mM NaCl,1mM EDTA, pH7.5) were prepared. Adding PMSF 0.1mg/ml, Leuteptin 10 mug/ml and Aprotitin 20 mug/ml before use); 3) adding a certain amount of lysate according to the number of cells and the ratio of 6 pore plates to 20 mul; 4) the cells were lysed on ice for 30min, centrifuged at 12000g for 10min, the supernatant was pipetted into a new EP tube, the protein extract was quantitatively analyzed using a kit (Bio-Rad, cat. 5000111), and finally the lysate was supplemented to a final concentration of 1. mu.g/. mu.l.
3. Proline isomerase activity assay in tissue/cell samples: 1) preparing a 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution with the concentration of 100mM and the pH value of 8.0; 2) preparing probe I with the concentration of 5mM, and dissolving the probe I in 70% DMSO; 3) in a 96-well all-black plate, the set is HEPES + ddH2O + Probe I, HEPES + ddH2Adding HEPES and ddH into the O + probe I + protein extract in sequence2O, 10 mu g of protein extracting solution and 100 mu L of probe I in total; 4) the fluorescence value was measured every 20s for 10min using a M5 microplate reader (SpectraMax) with shaking 5s before measurement, EX 360 and EM 460. And after the detection is finished, data are exported, and the fluorescence value change trend result is counted.
The fourth purpose of the invention is to provide the application of the compound shown in the formula I in detecting the activity of proline isomerase in living cells. The method is realized by the following steps: the compound of formula I was added to a DMEM high-glucose medium (brand: Gibco, cat # 12800017) to a final concentration of 5. mu.M, incubated at 37 ℃ for 30 minutes, and the fluorescence intensity of the cells was observed and recorded. The fluorescent probe provided by the invention is characterized in that the fluorescent probe only has weak fluorescence in a physiological environment, but can change the configuration under the catalytic action of proline isomerase to generate a strong-fluorescence product, and the fluorescence intensity is positively correlated with the activity of the proline isomerase, so that the specificity detection and quantitative analysis of the proline isomerase are realized.
The fluorescent probe and the detection method provided by the invention have the following beneficial effects: (1) the probe has good stability and can be stored and used for a long time; (2) the detection method is simple and convenient, the detection system is added with the probe to be incubated for 10 minutes to measure the fluorescence intensity, and the result can be given without the assistance of other reagents; (3) in vivo detection of proline isomerase activity in living cells could be achieved, whereas other reported methods could only be used for cell homogenates.
Drawings
FIG. 1 shows that the fluorescence of the HBVP cell protein extract gradually increases after the probe represented by formula Ia is added.
FIG. 2 shows that the fluorescence of the HBVP cell protein extract gradually increases when a probe represented by formula Ib is added.
FIG. 3 shows that intracellular fluorescence gradually increases after the probe represented by formula Ia is added to EA.Hy926 cell culture medium.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1: synthesis of Probe molecule Ia
Removing Fmoc protecting group from N-Fmoc-phenylalanyl-p-nitroaniline, dissolving the Fmoc protecting group in a dried dichloromethane methane solution, adding equivalent alanine-phenylalanine-proline tripeptide chain marked by N, N-dimethylnaphthoic acid and equivalent EDC, condensing HOBt, and separating a prepared liquid phase to obtain the compound shown in the formula Ia. MS: 798.3550.
example 2: synthesis of Probe molecule Ib
The N-Fmoc-phenylalanyl-p-nitroaniline removes Fmoc protecting groups, is dissolved in a dried dichloromethane methane solution, and is added with an equivalent of an alanine-glycine-proline tripeptide chain marked by N, N-dimethylnaphthoic acid and an equivalent of EDC and HOBt for condensation, and the product is separated by a preparation liquid phase to obtain the compound shown in the formula Ia. MS: 722.3333.
example 3: probe molecule Ia for detecting proline isomerase activity in HBVP cell protein extracting solution
Preparing a cell protein extracting solution: preparing cell lysate (10mM Tris,150mM NaCl,1mM EDTA, pH7.5), adding 100. mu.l lysate to each bottle of cells, adding corresponding protease inhibitor Cocktail, performing lysis on ice for 30min, centrifuging at 12000g for 10min, sucking supernatant into a new EP tube, performing quantitative analysis on protein extract by using a kit (Bio-Rad company, Cat. 5000111), and finally supplementing the lysate to the final concentration of the protein extract of 1. mu.g/. mu.l.
And adding Ia into phosphate buffer salt solution to enable the final concentration to be 10 micromoles, adding 20 microliters of protein extracting solution, and placing a fluorescence spectrophotometer to record the change rule of the fluorescence emission intensity at 460nm along with time under the 363nm excitation condition. The experimental results show that: the fluorescence value of the proline isomerase fluorescent probe can be improved by adding the protein extract, so that the fact that the protein extract contains proline isomerase is proved, and the proline isomerase fluorescent probe can change the fluorescence value to respond to the proline isomerase fluorescent probe is proved. See fig. 1.
Example 4: probe molecule Ib for detecting activity of proline isomerase in HBVP cell protein extracting solution
Ib is added into phosphate buffer salt solution to enable the final concentration to be 10 micromole, 20 microliter of protein extracting solution is added, and a fluorescence spectrophotometer is arranged to record the change rule of the fluorescence emission intensity at 460nm along with time under the 363nm excitation condition. The experimental results show that: the fluorescence value of the proline isomerase fluorescent probe can be improved by adding the protein extract, so that the fact that the protein extract contains proline isomerase is proved, and the proline isomerase fluorescent probe can change the fluorescence value to respond to the proline isomerase fluorescent probe is proved. See fig. 2.
Example 5: probe molecule Ia detection of proline isomerase in EA.Hy926 live cell
When the cells grew to 90% density, they were passaged to a glass petri dish at a ratio of 1: 5. After 48 hours, adding the compound shown in the formula I into the cell culture medium to ensure that the final concentration is 5 mu M, and uniformly mixing; and (3) using a fluorescence microscope, starting a 405nm laser, collecting a fluorescence signal of the compound shown in the formula I, and recording the fluorescence of the cells in real time. The results are shown in FIG. 3.
Claims (4)
1. A fluorescent probe for detecting the activity of proline isomerase is characterized by having a structure shown in a formula I:
wherein R is1Is hydrogen or a benzene ring;
R2is a fluorophore selected from naphthoic acid; the fluorescent probe is realized by the following steps:
(1) preparing N-Fmoc-phenylalanyl-p-nitroaniline by taking phenylalanine and p-nitroaniline protected by amino Fmoc as raw materials; (2) preparation of N-terminal R by solid phase synthesis2Modified "alanine-glycine or phenylalanine-proline" tripeptide chains;
(3) removing Fmoc protecting group from the N-Fmoc-phenylalanyl-p-nitroaniline prepared in the step and then reacting the Fmoc protecting group with the R2And condensing the marked tripeptide chain in a dried dichloromethane solution to obtain the fluorescent probe shown in the formula I.
2. Use of a compound of formula I according to claim 1 for the preparation of a reagent for the detection of proline isomerase activity in a tissue protein extract; the method is characterized by comprising the following steps:
(1) tissue sample extraction: a) collecting a tissue sample of a specific part, and storing the tissue sample at-80 ℃; b) preparing a lysis solution; c) adding lysis solution according to the sample mass of 10mg/50 mul, and grinding and lysing by using a grinding rod; d) cracking on ice for 30min, centrifuging at 12000g for 10min, sucking supernatant into a new EP tube, performing quantitative analysis on protein extract by using a kit of Bio-Rad company, and finally supplementing lysate until the final concentration of the protein extract is 1 mug/mul; the lysis solution is 10mM Tris,150mM NaCl,1mM EDTA, pH7.5, and PMSF 0.1mg/ml, Leuteptin 10 μ g/ml and Aprotinin 20 μ g/ml are added before use;
(2) cell sample extraction: a) when the cells grow to 90% density, discarding the supernatant, adding 3ml of PBS solution for cleaning for 3 times, finally adding 1.5ml of PBS solution, collecting the cells by using a clean cell spatula, sucking the solution into a centrifuge tube, centrifuging at 1200rpm for 4min, discarding all the supernatant, reserving the cells at the bottom, and freezing and storing at-80 ℃ for subsequent use; b) preparing a lysis solution; c) adding lysis solution according to the number of cells and the volume of 6 pore plates/20 mul; d) cracking on ice for 30min, centrifuging at 12000g for 10min, sucking supernatant into a new EP tube, performing quantitative analysis on protein extract by using a kit of Bio-Rad company, and finally supplementing lysate until the final concentration of the protein extract is 1 mug/mul; the PBS solution was: 8.00g NaCl,0.20g KCl,3.63g Na2HPO4•12H2O，0.24g KH2PO4Adding water to a constant volume of 1000 ml; the prepared lysate is 10mM Tris,150mM NaCl,1mM EDTA, pH7.5, and PMSF 0.1mg/ml, Leuteptin 10 μ g/ml and Aprotinin 20 μ g/ml are added before use;
(3) proline isomerase activity assay in tissue/cell samples: a) preparing a 4-hydroxyethyl piperazine ethanesulfonic acid solution with the concentration of 100mM and the pH = 8.0; b) preparing probe I with the concentration of 5mM, and dissolving the probe I in 70% DMSO; c) in a 96-hole full black plate, 4-hydroxyethyl piperazine ethanesulfonic acid + ddH is arranged2O + Probe I, 4-hydroxyethyl piperazine Ethanesulfonic acid + ddH2Adding 4-hydroxyethyl piperazine ethanesulfonic acid solution and ddH into O + probe I + protein extract2O, 10 mu g of protein extracting solution and 100 mu L of probe I in total; d) shaking 5s before detection by using M5 multifunctional microplate reader, and setting EX =36And 0, EM =460, detecting the fluorescence value once every 20s for 10min, deriving data after detection is finished, and counting the result of the change trend of the fluorescence value.
3. Use of a compound of formula I according to claim 1 for the preparation of a reagent for the detection of proline isomerase activity in living cells.
4. The use according to claim 3, characterized by the fact that it is achieved by the following steps: adding the compound of the formula I into a DMEM high-sugar medium to enable the final concentration to be 5 mu M, incubating for 30 minutes at 37 ℃, and observing and recording the fluorescence intensity of cells; the fluorescence intensity of the compound in the formula I is positively correlated with the activity of the proline isomerase, so that the specificity detection and quantitative analysis of the proline isomerase are realized.
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