CN112986426A - Analysis method for in vitro screening enzyme capable of catalyzing and cracking homocysteine - Google Patents
Analysis method for in vitro screening enzyme capable of catalyzing and cracking homocysteine Download PDFInfo
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Abstract
The invention relates to the technical field of drug analysis methods, in particular to an analysis method for in vitro screening of an enzyme capable of catalyzing and cracking homocysteine, which comprises the reaction conditions of oxidized homocysteine and the enzyme to be screened, sample pretreatment before chromatographic analysis and a high performance liquid chromatography analysis method for measuring homocysteine. The invention saves the tedious steps of plasma sample pretreatment, can rapidly and accurately preliminarily screen the enzyme capable of catalyzing and cracking the homocysteine by using the screening system, provides a new thought and a new method for developing potential drugs for preventing and treating hyperhomocysteinemia, and provides reference and reference for screening potential drugs for preventing and treating hyperhomocysteinemia in vitro; based on the existing high performance liquid chromatography technology for measuring homocysteine, the high performance liquid chromatography method for detecting homocysteine is established based on the advantages of wide application of an ultraviolet detector and high detection sensitivity, and is accurate, sensitive, rapid and simple.
Description
Technical Field
The invention relates to the technical field of drug analysis methods, in particular to an analysis method for in vitro screening of an enzyme capable of catalyzing and cracking homocysteine.
Background
Homocysteine (Hcy), otherwise known as Homocysteine, is an intermediate product of methionine and cysteine metabolism in vivo. Homocysteine exists in two forms in plasma, one is reduced form ([ H ] -Hcy) with sulfydryl in the structure, and the other is oxidized form ([ O ] -Hcy) with the structure formed by disulfide bonds in the reduced form. In vivo, about 99% of homocysteine exists in oxidized form in protein-bound form ([ O ] -Hcy), or in free oxidized form, and about 1% of homocysteine exists in reduced form ([ H ] -Hcy).
When the homocysteine level in the plasma is higher than 15 mu mol/L, hyperhomocysteinemia can be diagnosed, and the high-level homocysteine can directly or indirectly cause vascular endothelial cell injury, enhance the platelet function activity, promote thrombosis, influence the oxidation process of low-density lipoprotein, induce and promote the proliferation of vascular smooth muscle cells through various ways, and is known to be one of independent risk factors of cardiovascular and cerebrovascular diseases. Therefore, the search for potential drugs for reducing homocysteine in vivo has great significance for the diagnosis and the prevention of cardiovascular system diseases. The enzyme capable of catalyzing and cracking the homocysteine can reduce the concentration of the homocysteine through the catalytic cracking effect of the enzyme, and can be used as one of the types of the medicines for preventing and treating hyperhomocysteinemia.
The homocysteine can be detected by a radioimmunoassay, a fluorescence polarization immunoassay, an enzyme-linked immunosorbent assay, a cyclic enzyme method, a high-performance liquid phase fluorescence detection method, a high-performance liquid phase electrochemical detection method, an amino acid analyzer detection method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method and the like, wherein the high-performance liquid phase fluorescence detection method and the cyclic enzyme method are most commonly used. Because the existing form of homocysteine in vivo has oxidized form, reduced form and protein conjugate form thereof, the high performance liquid analysis method determines that the homocysteine is total homocysteine in blood plasma, a reducing agent is added to convert the oxidized homocysteine into the reduced homocysteine, then a sulfydryl derivatization agent is used for pre-column derivatization, the characteristic that the derivatized homocysteine has fluorescence is utilized, and a fluorescence detector is used for detection.
Disclosure of Invention
The invention provides an analysis method for screening an enzyme capable of catalytically cracking homocysteine in vitro, which overcomes the defects of the prior art, and adopts oxidized homocysteine to directly participate in the screening of the enzyme capable of catalytically cracking homocysteine for the first time, thereby omitting the complicated pretreatment step (namely the pretreatment of a plasma sample) for screening the enzyme capable of catalytically cracking by using the plasma sample (such as a homocysteine plasma sample), so that the analysis method can quickly and accurately screen the enzyme capable of catalytically cracking homocysteine for the first time, provide a new thought and a new method for developing potential drugs for preventing and treating hyperhomocysteinemia, and provide reference and reference for screening potential drugs for preventing and treating hyperhomocysteinemia in vitro.
As known to those skilled in the art, a plasma sample is complex to process, has a plurality of interference factors and is not beneficial to quickly screening the catalytic cracking action of enzyme on homocysteine, and because 99 percent of human plasma is oxidized homocysteine, the purchased homocysteine reagent is oxidized homocysteine, and the purchased homocysteine reference substance is reduced homocysteine, the oxidized homocysteine is reacted with the enzyme to be screened, and then the reaction product is reduced and derivatized to be used as a sample solution; phosphate buffer solution is added into a reaction system to be used as blank control solution after replacing enzyme to be screened for reaction; preparing a standard curve by using reduced homocysteine as a reference substance after derivatization; the method adopts high-phase liquid chromatography for determination, can quickly determine whether the enzyme to be screened has catalytic cracking effect on homocysteine, can research the catalytic kinetics of the enzyme on homocysteine catalytic cracking, and can be used as a new high-performance liquid detection method of homocysteine in the future. The technical scheme of the invention is realized by the following measures: an assay method for in vitro screening of an enzyme capable of catalytically cleaving homocysteine comprising the steps of:
(1) the oxidized homocysteine solution reacts with the enzyme solution to be screened, and the reaction temperature is as follows: 20 ℃ to 40 ℃, reaction time: 5min to 60min, and cooling to room temperature after the reaction is finished;
(2) adding a reducing agent into the solution obtained after the reaction in the step (1) for reduction reaction, wherein the ratio of the reducing agent to the oxidized homocysteine is 1: 1-1: 8, and the reduction reaction temperature is as follows: 25 ℃ to 50 ℃, reduction reaction time: reducing the temperature to room temperature after the reduction reaction is finished for 10min to 60 min;
(3) adding a derivatization agent into the solution obtained after the reduction reaction in the step (2) for derivatization reaction, wherein the ratio of the derivatization agent to the reduced homocysteine is 1: 1-1: 16, the temperature of the derivatization reaction is 60-100 ℃, the time of the derivatization reaction is 50-100 min, and the solution is cooled for 10min after the derivatization reaction is finished;
(4) and (3) filtering the solution obtained by cooling in the step (3) by using the filtered solution as a sample solution (a test solution) after reaction, sequentially treating enzymes to be screened by using a phosphate buffer solution instead of the enzymes to be screened in the steps (1) to (3) to obtain a blank control solution, using the blank control solution as a sample solution before reaction, and respectively performing high performance liquid analysis on the sample solution before and after reaction to obtain a result of whether the enzymes to be screened can catalyze and crack homocysteine.
The steps (1) to (3) of the analysis method of the present invention provide reaction conditions for homocysteine and an enzyme to be screened and a sample pretreatment method before chromatographic analysis.
The following is further optimization or/and improvement of the technical scheme of the invention:
in the step (1), the reaction temperature of the oxidized homocysteine solution and the enzyme solution to be screened is preferably 35 ℃, and the reaction time of the oxidized homocysteine solution and the enzyme solution to be screened is preferably 10 min.
The preferable ratio of the reducing agent to the oxidized homocysteine is 1: 2; the reducing agent is preferably tris (2-carboxyethyl) phosphine hydrochloride (TCEP).
In the step (2), the reduction reaction temperature is preferably 37 ℃ and the reduction reaction time is preferably 10 min.
In the step (3), the ratio of the derivatization agent to the reduced homocysteine is preferably 1:8, the derivatization reaction temperature is preferably 70 ℃, and the derivatization reaction time is preferably 60 min; the derivative is preferably 7-fluorobenzofurazan-4-ammonium sulfate (SBD-F).
When the high performance liquid analysis is carried out, the solution obtained after the derivatization reaction of the step (3) of the reduced homocysteine is used as a control solution.
In the step (1), the oxidized homocysteine solution and the enzyme solution to be screened are obtained by dissolving in a phosphate buffer solution.
In the step (4), the high performance liquid chromatography adopts reversed phase liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column adopts a C18 chromatographic column, a mobile phase consists of acetate buffer solution and methanol, gradient elution is carried out, an ultraviolet detector is used for detecting, and an external standard method is adopted for carrying out quantitative analysis: in particular, the amount of the solvent to be used,
the invention also provides a high performance liquid chromatography method for measuring homocysteine, and the chromatographic conditions are as follows:
a chromatographic column: chromatographic column with octadecylsilane chemically bonded silica as filler, specification (4.6mm × 250mm, 5 μm);
mobile phase: phase A is acetate buffer solution with pH value of 4.5, and phase B is methanol;
gradient elution: the volume change of the phase B is 0-16min and 2-5 percent;
flow rate: 0.8ml/min to 1.2ml/min, preferably 0.8 ml/min;
column temperature: 25 ℃ to 35 ℃, preferably 25 ℃;
sample introduction amount: 20 to 50 μ L, preferably 20 μ L;
a detector: an ultraviolet detector;
detection wavelength: 350nm to 400nm, preferably 384 nm.
The invention has the beneficial effects that: the complex steps of plasma sample pretreatment are omitted, and the enzyme capable of catalyzing and cracking homocysteine can be rapidly and accurately preliminarily screened by using the screening system; based on the existing high performance liquid chromatography technology for measuring homocysteine, the high performance liquid chromatography method for detecting homocysteine is established based on the advantages of wide application of an ultraviolet detector and high detection sensitivity, and is accurate, sensitive, rapid and simple.
Drawings
FIG. 1 is a standard curve of derivatized [ H ] -Hcy.
FIG. 2 is a high performance liquid chromatogram before and after the reaction of allinase and [ O ] -Hcy.
FIG. 3 shows the effect of substrate concentration on the enzymatic reaction rate (pH5.5) in alliinase catalyzed cleavage [ O ] -Hcy reaction.
FIG. 4 shows the effect of substrate concentration on the enzymatic reaction rate (pH6.8) in alliinase catalyzed cleavage [ O ] -Hcy reaction.
FIG. 5 is a straight-line graph (pH5.5) fitted to the reciprocal of the respective reaction rates for different substrate concentrations in the allinase catalyzed lysis [ O ] -Hcy reaction.
FIG. 6 is a straight-line graph (pH5.5) fitted to the reciprocal of the respective reaction rates for different substrate concentrations in the alliinase catalyzed lysis [ O ] -Hcy reaction.
In Table 2, curve A is the peak of the chromatogram of the HPLC of the solution before the reaction of [ O ] -Hcy with allinase, and curve B is the peak of the chromatogram of the solution after the reaction of [ O ] -Hcy with allinase.
Detailed Description
The present invention is not limited by the following examples, and specific embodiments may be determined according to the technical solutions and practical situations of the present invention.
In order to make the present invention be better applied to screening enzymes capable of degrading Hcy, the technical solution of the present invention is further described in detail below with reference to specific examples, and the following described examples are only some examples, but not all examples, for screening enzymes using the present invention.
The allinase used in the following examples was allinase lyophilized powder produced by Xinjiang Enlexin pharmaceutical Co.
The reagents used in the following examples, such as [ H ] -Hcy control and [ O ] -Hcy, are commercially available.
The invention is further described below with reference to the following examples:
example 1: screening catalytic cracking action of allinase on homocysteine
The experimental steps are as follows:
1 preparation of solution
Precisely weighing 27mg of a [ H ] -Hcy reference substance, placing the reference substance in a 100mL volumetric flask, and dissolving the reference substance by using a phosphate buffer solution to fix the volume to obtain a reference substance stock solution of 320 mu mol/L; dissolving [ O ] -Hcy in phosphate buffer solution to obtain 1mmol/L [ O ] -Hcy solution.
Placing appropriate amount of alliinase in a measuring flask, adding pH 7.0 phosphate buffer solution, vortex to dissolve, and shaking to obtain alliinase solution.
2 chromatographic conditions
A chromatographic column: waters Xbridge C18(4.6 mm. times.250 mm, 5 μm), detection wavelength: 384nm, mobile phase: phase a, acetate buffer at pH 4.5; phase B, methanol; gradient elution: the volume change of the phase B is 0-16min 2% to 5%; the flow rate was 0.8mL/min, the column temperature was 25 ℃ and the amount of sample was 20. mu.L.
3 preparing a standard curve
Diluting the [ H ] -Hcy reference substance stock solution with phosphate buffer solution to obtain 4, 10, 20, 40, 80 and 160 mu mol/L standard solutions, respectively measuring the standard solutions with different concentrations, adding SBD-F working solution, carrying out water bath reaction at 70 ℃ for 60min, cooling for 10min, filtering the solution, respectively injecting the solution into a liquid chromatograph for measurement, and drawing a standard curve by taking derivatization [ H ] -Hcy as an abscissa and taking a peak area as an ordinate, wherein the standard curve equation is Y-6034.9X-73769, r-0.9990, and the linearity is good in the range of concentration from 4 mu mol/L to 160 mu mol/L (see figure 1).
4 sample pretreatment
0.01 mL, 0.02 mL, 0.03 mL, 0.04 mL, 0.05 mL and 0.06mL of 1mmol/L [ O ] -Hcy solution are respectively added into 0.4mL of allinase solution and reacted in 35 ℃ water bath for 10 min; adding TCEP solution 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06mL respectively, and reacting in water bath at 37 deg.C for 10 min; and then adding SBD-F stock solutions of 0.12, 0.24, 0.36, 0.48, 0.60 and 0.72mL respectively, reacting in a water bath at 70 ℃ for 60min, and cooling for 10min to obtain a test solution for later use. And phosphate buffer solution is added into the reaction system to replace allinase solution to serve as blank control.
5 determining whether allinase has catalytic cracking effect on homocysteine
The test solution prepared after the sample pretreatment is used as the solution after the reaction of allinase and [ O ] -Hcy, the blank control is the solution before the reaction of allinase and [ O ] -Hcy, high performance liquid chromatography (see figure 2) is carried out, and the contents of [ H ] -Hcy are respectively measured, and the results are shown in the following table 1.
As can be seen from Table 1, the allinase can reduce the content of [ H ] -Hcy with different concentrations after the reaction, which indicates that the allinase has a certain catalytic cracking effect on Hcy.
Example 2: catalytic kinetics of allinase on homocysteine
1 Effect of reaction time on enzymatic reactions
0.4mL of alliinase solution is added with 0.1mL of 1mmol/L O-Hcy solution, the mixture is immediately placed in a water bath at 35 ℃ for accurate reaction for 5min, 10min and 15min, the microporous membrane is filtered after the treatment according to the sample pretreatment method, 20 mu L of the mixture is injected into a liquid chromatograph, the content of H-Hcy in a sample is calculated, the optimal reaction time is determined, and the result is shown in Table 2 below.
As shown in Table 2, the reaction time was selected to be 10min because the conversion rate did not change with the increase of the reaction time of [ O ] -Hcy with allinase.
2O-Hcy and allinase
Measuring 0.1mL of [ O ] -Hcy and 0.4mL of allinase solution with different concentrations, immediately placing the samples in a water bath at 35 ℃ for accurate reaction for 10min, filtering the samples by a microporous filter membrane after treatment according to a sample pretreatment method, injecting 20 mu L of the samples into a liquid chromatograph, calculating the content of [ H ] -Hcy in a sample to be tested, determining the optimal proportion of the [ O ] -Hcy and the allinase, and obtaining the results shown in the following table 3.
The data in Table 3 show that [ O ] -Hcy: allinase 1: at 6.0U, the alliinase of 6.0U can catalyze and crack 1 microgram of [ O ] -Hcy, and the conversion rate can reach more than 80%.
Influence of 3 substrate ([ O ] -Hcy) concentration on the initial velocity of the enzymatic reaction
0.4mL of alliinase solution is added with [ O ] with different concentrations]0.1mL of-Hcy solution is immediately placed in a 35 ℃ water bath for accurate reaction for 10min, the solution is filtered by a microporous filter membrane after being treated according to a sample pretreatment method, 20 mu L of the solution is injected into a liquid chromatograph, and a chromatogram is recorded; another take [ H]Hcy control solutions, determined in the same way. Calculating [ H ] in the sample by peak area according to external standard method]The Hcy content, the reaction rate being calculated as follows, the reaction rate V being plotted against the corresponding substrate concentration, a trend line being drawn in the region of the straight line, the slope of which is the initial rate of the enzymatic reaction (V)0)。
When the pH of the phosphate buffer solution is 5.5, the slope of the trend line is an initial velocity, i.e., V, in the range of 0 to 129.25. mu. mol/L00.09 μmol/(L · min) (see fig. 3).
When the pH of the phosphate buffer solution is 6.8When the gradient of the trend line is within the range of 0-64.78 mu mol/L, the initial velocity is V00.09 μmol/(L · min) (see fig. 4).
Determination of kinetic constant of 4 alliinase
With the reciprocal of the reaction rate as the ordinate, [ H ]]The reciprocal of the Hcy concentration is plotted on the abscissa using the double reciprocal plot method (Lineweaver-Burk), the K of the enzymem＝4710μmol/L，Vmax384.62 μmol/(L · min), regression equation (see fig. 5): Y-12.248X-0.0026 (r-0.9997). When the pH of the phosphate buffer is 6.8, the K of the enzymem＝316.78μmol/L，Vmax22.03 μmol/L · min, regression equation (see fig. 6): Y-14.382X-0.0454 (r-0.9994).
The above examples demonstrate that the analysis method of the present invention can rapidly and accurately screen out the enzyme having catalytic cracking effect on homocysteine, and research the catalytic kinetics thereof, and has the advantages of simple operation and accurate result, and has important significance for screening potential drugs capable of preventing and treating hyperhomocysteinemia.
The technical characteristics form an embodiment of the invention, which has strong adaptability and implementation effect, and unnecessary technical characteristics can be increased or decreased according to actual needs to meet the requirements of different situations.
TABLE 1 catalytic cleavage of oxidized homocysteine by allinase
TABLE 2 results of the influence of reaction time on the enzymatic reaction
| Reaction time/min | Conversion rate/% | RSD/％ |
| 5 | 50.46 | 1.41 |
| 10 | 50.95 | 1.96 |
| 15 | 49.66 | 1.29 |
TABLE 3 influence of the ratio of [ O ] -Hcy, allinase on the conversion
| Proportioning (mug: U) | Conversion (%) | RSD(％) |
| 1：0.8 | 25.38 | 1.15 |
| 1：1.2 | 35.54 | 1.71 |
| 1：2.4 | 46.17 | 2.20 |
| 1：4.8 | 77.32 | 1.68 |
| 1：6.0 | 82.78 | 0.25 |
| 1：8.0 | 80.59 | 1.42 |
| 1：10.0 | 82.37 | 1.99 |
Claims (9)
1. An analytical method for in vitro screening of an enzyme capable of catalytically cleaving homocysteine, comprising the steps of:
(1) the oxidized homocysteine solution reacts with the enzyme solution to be screened, and the reaction temperature is as follows: 20 ℃ to 40 ℃, reaction time: 5min to 60min, and cooling to room temperature after the reaction is finished;
(2) adding a reducing agent into the solution obtained after the reaction in the step (1) for reduction reaction, wherein the ratio of the reducing agent to the oxidized homocysteine is 1: 1-1: 8, and the reduction reaction temperature is as follows: 25 ℃ to 50 ℃, reduction reaction time: reducing the temperature to room temperature after the reduction reaction is finished for 10min to 60 min;
(3) adding a derivatization agent into the solution obtained after the reduction reaction in the step (2) for derivatization reaction, wherein the ratio of the derivatization agent to the reduced homocysteine is 1: 1-1: 16, the temperature of the derivatization reaction is 60-100 ℃, the time of the derivatization reaction is 50-100 min, and the solution is cooled for 10min after the derivatization reaction is finished;
(4) and (3) taking the solution obtained by cooling in the step (3) as a sample solution after reaction, sequentially treating the enzyme to be screened with a phosphate buffer solution to obtain a blank control solution after the treatment in the steps (1) to (3), taking the blank control solution as a sample solution before reaction, and respectively carrying out high performance liquid analysis on the sample solution before and after the reaction to obtain a result of whether the enzyme to be screened can catalyze and crack homocysteine.
2. The assay method for in vitro screening of an enzyme that can catalyze the cleavage of homocysteine according to claim 1 wherein in step (1) the reaction temperature of the oxidized homocysteine solution with the enzyme solution to be screened is preferably 35 ℃ and the reaction time of the oxidized homocysteine solution with the enzyme solution to be screened is preferably 10 min.
3. The assay for in vitro screening of enzymes catalyzing the cleavage of homocysteine according to claim 1 or 2 characterised in that the reducing agent and the oxidized homocysteine are preferably 1: 2; the reducing agent is preferably tris (2-carboxyethyl) phosphine hydrochloride.
4. The assay method for in vitro screening of an enzyme that can catalyze the cleavage of homocysteine according to claim 1, 2 or 3 wherein in step (2) the reduction reaction temperature is preferably 37 ℃ and the reduction reaction time is preferably 10 min.
5. The assay method for in vitro screening of an enzyme that can catalyze the cleavage of homocysteine according to claim 1 or 2 or 3 or 4, wherein in step (3), the ratio of derivatizing agent to reduced homocysteine is preferably 1:8, the temperature of derivatization is preferably 70 ℃, and the time of derivatization is preferably 60 min; the derivative agent is preferably 7-fluorobenzofurazan-4-ammonium sulfate.
6. The assay method for in vitro screening of an enzyme that catalyzes the cleavage of homocysteine according to claim 1 or 2 or 3 or 4 or 5 wherein in step (4) the HPLC assay is reversed phase LC assay and the chromatographic conditions are: the chromatographic column adopts a C18 chromatographic column, the mobile phase consists of acetate buffer solution and methanol, gradient elution is carried out, an ultraviolet detector is used for detecting, and an external standard method is adopted for carrying out quantitative analysis.
7. The assay method for in vitro screening of an enzyme that can catalyze the cleavage of homocysteine according to claim 6, wherein the detection wavelength is 350nm to 400nm, the pH of the mobile phase acetate buffer is 4.5, the volume change of methanol at 0min to 16min is 2% to 5%, the flow rate is 0.8mL/min to 1.2mL/min, and the column temperature is 25 ℃ to 35 ℃.
8. The method for in vitro screening of an enzyme that catalyzes the cleavage of homocysteine according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7 wherein the reduced homocysteine is subjected to the derivatization reaction of step (3) to obtain a solution as a control solution in the HPLC analysis.
9. The method for in vitro screening of an enzyme that catalyzes the cleavage of homocysteine according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 wherein in step (1) the oxidized homocysteine solution and the enzyme solution to be screened are both dissolved in phosphate buffered saline.
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| US20050260250A1 (en) * | 2004-05-24 | 2005-11-24 | Ott David M | Medicinal products incorporating bound organosulfur groups |
| CN108362795A (en) * | 2018-02-08 | 2018-08-03 | 杭州佰辰医学检验所有限公司 | Content of homocysteine rapid detection method in dried blood spot |
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| US20050260250A1 (en) * | 2004-05-24 | 2005-11-24 | Ott David M | Medicinal products incorporating bound organosulfur groups |
| CN108362795A (en) * | 2018-02-08 | 2018-08-03 | 杭州佰辰医学检验所有限公司 | Content of homocysteine rapid detection method in dried blood spot |
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