Method for determining disulfide bond content by oxidation-reduction potentiometric titration method

Abstract

The invention belongs to the technical field of detection methods, and particularly discloses a method for determining the content of disulfide bonds by an oxidation-reduction potentiometric titration method. The method comprises the following steps that a sample to be detected is dissolved in a solvent, a solution to be detected is obtained, and the sample to be detected contains disulfide bonds; taking potassium permanganate as a titrating solution, titrating the to-be-detected solution by adopting a titration method, and calculating the content of the disulfide bonds in the to-be-detected sample according to the volume of the titrating solution consumed during titration of a jump point. According to the method, the potassium permanganate titration solution is adopted to perform oxidation-reduction titration determination on the cystine compound, the potassium permanganate titration solution can be used as an indicator and has color change in the titration process, so that the titration end point is judged by utilizing a jump point, and the volume of the titration solution consumed when the jump point is titrated is recorded; and calculating the content of the disulfide bond in the sample to be detected. The determination method is simple and easy to implement and low in detection cost; and the kit has good specificity and sensitivity, and can meet daily detection requirements.

Description

Technical Field

[0001] The invention belongs to the technical field of detection methods, and in particular relates to a method for determining disulfide bond content, and specifically relates to a method for determining disulfide bond content by redox potential titration. Background Art

[0002] A disulfide bond is a covalent bond between two sulfur atoms, which plays an important role in many biological molecules. Therefore, determining the existence and quantity of disulfide bonds is of great significance for studying the structure and function of biological molecules.

[0003] At present, there are many methods for determining disulfide bonds, such as disulfide bond reduction method, mass spectrometry, ultraviolet spectroscopy and nuclear magnetic resonance method. Among them: disulfide bond reduction method is a commonly used method for determining disulfide bonds. This method uses a reducing agent to reduce the disulfide bond to two sulfur atoms, and then uses a chemical method to determine the number of sulfur atoms. Although this method is simple and easy, it cannot distinguish disulfide bonds in different positions. Mass spectrometry is a highly sensitive method for determining disulfide bonds. This method uses a mass spectrometer to analyze biological molecules and can directly determine the existence and number of disulfide bonds. The advantage of this method is high sensitivity, but the disadvantage is that it requires expensive equipment and professional technology. Ultraviolet spectroscopy is a conventional method for determining disulfide bonds. This method uses the absorption characteristics of disulfide bonds in biological molecules to determine the existence and number of disulfide bonds by measuring the position and intensity of the absorption peak by an ultraviolet spectrometer. This method is simple and easy, but requires high-purity samples and professional technology. Nuclear magnetic resonance is a high-resolution method for determining disulfide bonds. This method uses a nuclear magnetic resonance instrument to analyze biological molecules, can directly determine the presence and number of disulfide bonds, and can distinguish disulfide bonds at different positions. The advantages of this method are high resolution and sensitivity, but the disadvantages are high equipment cost and difficult operation. In summary, the existing methods for determining disulfide bonds have certain defects and cannot be applied to daily detection needs.

[0004] Therefore, further simplifying the operating steps, reducing the detection cost, and better meeting daily detection needs are of great practical value for the determination of disulfide bonds. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a method for determining the content of disulfide bonds by redox potential titration, which utilizes the structural characteristics of disulfide bonds, uses potassium permanganate as a titrant, and adopts redox titration to quickly and accurately determine the content of disulfide bonds in a sample to be tested.

[0006] The inventive concept of the present invention is: a compound containing a disulfide bond (such as a cystine compound) contains a covalent bond formed by oxidation of two sulfhydryl groups, namely a disulfide bond. According to the properties of the disulfide bond, it can be oxidized to form a sulfone or reduced to form a sulfhydryl group. Therefore, the content of the disulfide bond in the compound can be calculated by consuming the volume of the titration solution. The present invention uses potassium permanganate titration solution to titrate the cystine compound. Since the potassium permanganate titration solution itself can be used as an indicator, there is a color change during the titration process, but since the disulfide bond in the cystine compound is weak in reducibility, the color change is not obvious. Therefore, the redox potential titration method can be used to judge the titration end point using the jump point, and the content of the disulfide bond in the sample to be tested can be calculated by recording the volume of the titration solution consumed when the titration jump point is reached.

[0007] The reaction principle during the titration process is as follows:

[0008]

[0009] In order to solve the above technical problems, the present invention provides a method for determining the disulfide bond content, comprising the following steps:

[0010] (1) dissolving a sample to be tested in a solvent to obtain a solution to be tested, wherein the sample to be tested contains a disulfide bond;

[0011] (2) Using potassium permanganate as a titrant, the test solution is titrated by titration, and the content of disulfide bonds in the test sample is calculated based on the volume of the titrant consumed at the titration jump point.

[0012] In some embodiments of the present invention, the sample to be tested includes at least one of cystine, cystine dimethyl ester, L-cystine dimethyl ester, and cystine dimethyl ester dihydrochloride.

[0013] In some embodiments of the present invention, the sample to be tested is L-cystine dimethyl ester.

[0014] In some embodiments of the present invention, the concentration of potassium permanganate is 100 μmol / L.

[0015] In some embodiments of the present invention, the pH value of the solvent ranges from 6.4 to 7.4.

[0016] In some embodiments of the present invention, the solvent includes water. Studies have found that when other solvents other than water are used, such as dimethyl sulfoxide, fluorescence will occur during the dripping process, affecting the evaluation of the dripping point endpoint; when water is used as the solvent, no fluorescence will occur.

[0017] In some embodiments of the present invention, the water is ultrapure water. In addition to water molecules, there are no other impurities in the ultrapure water, which can ensure the accuracy of the measurement.

[0018] In some embodiments of the present invention, the titration method is redox potentiometric titration. Potentiometric titration is a method for determining the titration endpoint by measuring the potential change. This method does not require accurate measurement of the electrode potential value, so the influence of temperature and liquid junction potential is not important, which is conducive to the accuracy of the titration.

[0019] In some embodiments of the present invention, the detection limit concentration of the test solution is 5 mg / mL, that is, when the concentration of the test solution is above 5 mg / mL, the equivalence point can be accurately identified.

[0020] In some embodiments of the present invention, in step (1), the dissolution method is ultrasonic dissolution to fully dissolve the sample to be tested.

[0021] In some embodiments of the present invention, step (2) further includes a blank titration step before titrating the test solution. The blank titration step is: using potassium permanganate as a titrant, titrating the solvent by titration to determine the sensitivity of the method.

[0022] Compared with the prior art, the above technical solution of the present invention has at least the following technical effects or advantages:

[0023] The present invention uses potassium permanganate titration solution to perform redox potential titration determination on cystine compounds. Since the potassium permanganate titration solution itself can be used as an indicator, there is a color change during the titration process, and the titration end point is determined by using the jump point, and the volume of the titration solution consumed when the titration jump point is recorded to calculate the content of disulfide bonds in the sample to be tested. The determination method is simple and easy to implement, with low detection cost; and has good specificity and sensitivity, and can meet daily detection needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison chart of the titration results for the comparative experiment. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with the examples, so that the technical personnel of the relevant technical field can understand the present invention. It is necessary to point out here that the examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. The non-essential improvements and adjustments made to the present invention by the skilled person in the relevant field according to the above invention content should still belong to the scope of protection of the present invention. At the same time, the raw materials mentioned below that are not described in detail are all commercially available products; the process steps or preparation methods that are not mentioned in detail are all process steps or preparation methods known to those skilled in the art.

[0026] Example 1

[0027] A method for determining disulfide bond content by redox potential titration comprises the following steps:

[0028] (1) Take 0.1037 g of L-cystine dimethyl ester and accurately weigh it, put it in a 250 mL beaker, add 100 mL of ultrapure water, and use ultrasound to completely dissolve the L-cystine dimethyl ester to obtain a test solution;

[0029] (2) using a redox potential titration method with 100 μmol / L potassium permanganate as a titrant; firstly performing a blank titration with 100 mL of ultrapure water as a solvent, and then titrating the test solution prepared in step (1), and recording the volume of the titrant consumed at the titration jump point; and calculating the disulfide bond content in the test sample by the volume of the titrant consumed at the titration jump point.

[0030] Example 2

[0031] The method for determining the disulfide bond content by redox potential titration method in Example 1 was referred to, except that the amount of L-cystine dimethyl ester was changed to 0.3093 g.

[0032] Example 3

[0033] The method for determining the disulfide bond content by redox potential titration method in Example 1 was referred to, except that the amount of L-cystine dimethyl ester was changed to 0.5070 g.

[0034] The measurement results of Examples 1-3 are shown in Table 1.

[0035] Table 1:

[0036]

[0037] As shown in Table 1, as the amount of L-cystine dimethyl ester increases, the volume of potassium permanganate titration solution consumed also increases, and is positively correlated. Moreover, a jump point can be generated when the amount of L-cystine dimethyl ester is about 0.1g, indicating that potassium permanganate titration solution has good sensitivity.

[0038] Comparative Example 1

[0039] A method for determining disulfide bond content by redox potential titration comprises the following steps:

[0040] (1) Take 0.1018 g of L-cystine dimethyl ester and accurately weigh it, put it in a 250 mL beaker, add 100 mL of ultrapure water, and use ultrasound to completely dissolve the L-cystine dimethyl ester to obtain a test solution;

[0041] (2) Using redox potential titration, using sodium thiosulfate with a concentration of 0.05 mol / L as the titrant; firstly, using 100 mL of ultrapure water as the solvent for blank titration, and then titrating the test solution prepared in step (1), and recording the equivalence point and the volume of the consumed titrant.

[0042] Comparative Example 2

[0043] The method for determining the disulfide bond content by redox potential titration method in Comparative Example 1 was referred to, except that the amount of L-cystine dimethyl ester was changed to 0.3016 g.

[0044] Comparative Example 3

[0045] The method for determining the disulfide bond content by redox potential titration method in Comparative Example 1 was referred to, except that the amount of L-cystine dimethyl ester was changed to 0.4132 g.

[0046] The results of comparative examples 1-3 are shown in Table 2.

[0047] Table 2:

[0048]

[0049] As shown in Table 2, although the disulfide bond is oxidizing, the reduction of L-cystine dimethyl ester by titration with 0.05 mol / L sodium thiosulfate cannot indicate the end point and qualitatively characterize the disulfide bond.

[0050] Comparative Example 4

[0051] A method for determining disulfide bond content by redox potential titration comprises the following steps:

[0052] (1) Take 0.1121 g of L-cystine dimethyl ester and accurately weigh it, put it in a 250 mL beaker, add 100 mL of ultrapure water, and use ultrasound to completely dissolve the L-cystine dimethyl ester to obtain a test solution;

[0053] (2) Using redox potential titration, using hydrogen peroxide with a concentration of 100 μmol / L as the titrant; firstly performing a blank titration using 100 mL of ultrapure water as the solvent, and then titrating the test solution prepared in step (1), and recording the equivalence point and the volume of the consumed titrant.

[0054] Comparative Example 5

[0055] The method for determining the disulfide bond content by redox potential titration method in Comparative Example 4 was referred to, except that the amount of L-cystine dimethyl ester was changed to 0.3043 g.

[0056] Comparative Example 6

[0057] The method for determining the disulfide bond content by redox potential titration method in Comparative Example 4 was referred to, except that the amount of L-cystine dimethyl ester was changed to 0.5021 g.

[0058] Comparative Example 7

[0059] The method for determining the disulfide bond content by redox potential titration method in Comparative Example 4 was referred to, except that the amount of L-cystine dimethyl ester was changed to 1.0039 g.

[0060] The measurement results of Comparative Examples 4-7 are shown in Table 3.

[0061] Table 3:

[0062]

[0063] As shown in Table 3, when the amount of L-cystine dimethyl ester is above 0.5g, there is a sudden jump when using 100μmol / L hydrogen peroxide titration solution, and the volume of the titration solution consumed is positively correlated with the sample weight, that is, the volume of the titration solution consumed increases as the sample weight increases. When the amount of L-cystine dimethyl ester is below 0.5g, the equivalence point cannot be identified. Compared with the results in Table 1, it can be seen that the sensitivity of 100μmol / L potassium permanganate titration solution is higher than that of 100μmol / L hydrogen peroxide titration solution.

[0064] Comparative experiment

[0065] The titration process using ultrapure water and dimethyl sulfoxide as solvents is as follows:

[0066] (1) Take 100 mL of ultrapure water and place it in a 250 mL conical flask. Add 0.5 mL of 100 μmol / L potassium permanganate titration solution, shake well, and observe the phenomenon.

[0067] (2) Measure 100 mL of dimethyl sulfoxide into a 250 mL conical flask, add 0.5 mL of 100 μmol / L potassium permanganate titration solution, shake well, and observe the phenomenon.

[0068] The titration results of the above comparative experiment are as follows Figure 1 As shown, from Figure 1 It can be seen that when the solvent used for titration is dimethyl sulfoxide, the solution shows obvious fluorescence phenomenon, which affects the judgment of the results, while no fluorescence phenomenon occurs when ultrapure water is used as the solvent.

[0069] For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, without having to go through creative labor. Therefore, simple improvements made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the protection scope of the present invention. The above embodiments are preferred embodiments of the present invention, and all processes similar to the present invention and equivalent changes made should belong to the protection scope of the present invention.

Claims

1. A method for determining disulfide bond content, characterized in that: The following steps are involved: (1) dissolving a sample to be tested in a solvent to obtain a solution to be tested, wherein the sample to be tested contains a disulfide bond; (2) Using potassium permanganate as a titrant, the test solution is titrated by titration, and the content of disulfide bonds in the test sample is calculated based on the volume of the titrant consumed at the titration jump point.

2. The method for determining disulfide bond content according to claim 1, characterized in that: The sample to be tested includes at least one of cystine, cystine dimethyl ester, L-cystine dimethyl ester, and cystine dimethyl ester dihydrochloride.

3. The method for determining disulfide bond content according to claim 1, characterized in that: The concentration of potassium permanganate is 100 μmol / L.

4. The method for determining disulfide bond content according to claim 1, characterized in that: The pH value of the solvent is in the range of 6.4-7.

4.

5. The method for titrating disulfide bond content according to claim 1 or 4, characterized in that: The solvent includes water.

6. The method for titrating disulfide bond content according to claim 5, characterized in that: The water is ultrapure water.

7. The method for determining disulfide bond content according to claim 1, characterized in that: The titration method is redox potential titration.

8. The method for determining disulfide bond content according to claim 1, characterized in that: The detection limit concentration of the test solution is 5 mg / mL.

9. The method for determining disulfide bond content according to claim 1, characterized in that: In step (1), the dissolution method is ultrasonic dissolution.

10. The method for determining disulfide bond content according to claim 1, characterized in that: In step (2), a blank titration step is also included before titrating the test solution.

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