A method for quantifying the concentration of cystine and cysteine in a mixed solution

Abstract

This invention belongs to the field of environmental chemistry and relates to a method for quantitatively analyzing the concentrations of cystine and cysteine ​​in a mixed solution. Based on the theoretical characteristics of the reaction between the disulfide bond in cystine, a representative amino acid, and the thiol group in cysteine, and persulfate, this invention expands the detection capability of the traditional 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), providing support for the quantitative analysis of cystine and cysteine ​​concentrations and the study of their transformation processes.

Description

Technical Field

[0001] This invention belongs to the field of environmental chemistry and proposes a method for quantitatively determining the concentrations of cysteine ​​and homocysteine ​​in a mixed solution. Based on the reaction characteristics of the disulfide bond in cysteine ​​and the thiol group in cysteine ​​with persulfate, and combined with 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), this invention elucidates the detection and calculation methods for determining the concentrations of homocysteine ​​and cysteine ​​by using a difference method. Background Technology

[0002] Because cysteine ​​and cystine have simple chemical structures and do not contain light-absorbing groups such as carbon-carbon double bonds or benzene rings, commonly used spectroscopic methods are insufficient for quantifying them. If high-performance liquid chromatography (HPLC) is used, which is expensive, derivatization of cysteine ​​is necessary. Although enzyme-linked immunosorbent assay (ELISA) kits can quantify cysteine, this method suffers from drawbacks such as a narrow quantitative range, long processing time, and high cost. Based on the characteristics of the thiol group in cysteine, its abundant electrons make it a nucleophile, with electron acceptors that can be various oxidants, including substances containing disulfide bonds (RSSR, such as cystine). Therefore, 5,5-dithiobis-(2-nitrobenoic acid) (DTNB) is used commercially as an electrophile to quantitatively detect cysteine ​​concentration (Equation 1). However, this technique can only measure the concentration of cysteine ​​and cannot determine the concentration of its derivative, cystine, thus failing to reveal the reasons for amino acid inversions. Therefore, there is an urgent need in the market for a convenient and inexpensive technology to quantify the concentration of cysteine ​​and its oxidation product cystine.

[0003] DTNB+RSH→TNB(1)

[0004] The thiol group is an electron-donating functional group, and the number of electrons transferred can be used to quantify cysteine. Potassium persulfate (PMS), as an oxidizing agent, can specifically react with substances containing thiol groups and disulfide bonds (Equation 2), revealing a stoichiometric ratio of 1:3 between cysteine ​​and PMS. Similarly, for thiol-based amino acid derivatives such as cysteine, this chemical equation (Equation 3) shows a stoichiometric ratio of 1:5 between cysteine ​​and PMS.

[0005]

[0006] For a mixed solution containing both cysteine ​​and cystine: Since cysteine ​​and cysteine ​​have similar structures, the disulfide bond of cysteine ​​can also act as a nucleophile. The chemical reaction formula between cysteine ​​and PMS differs from that between cysteine ​​and PMS (Equation 3), and PMS can react simultaneously with a mixed solution of cysteine ​​and cysteine. In this case, the decrease in PMS reflects the total amount of cysteine ​​and cysteine. Combining the reaction relationship between DTNB and cysteine, the concentration of cysteine ​​can be obtained (Equation 1). Then, by comparing this difference with the decrease in PMS, the concentration of cysteine ​​can be obtained. Summary of the Invention

[0007] The purpose of this invention is to provide a low-cost and easy-to-operate method for quantifying cystine and homocysteine ​​concentrations, providing insights for studying homocysteine ​​concentration and the process of its conversion to cystine.

[0008] To address the aforementioned problems in the prior art, this invention proposes the following technical solution, which mainly includes the following steps:

[0009] 1) Reaction of DTNB with cysteine: Upon mixing with a cysteine ​​solution, colorless DTNB transforms into the colored product TNB, which exhibits a maximum absorption peak at 412 nm. Therefore, this method can be used to quantify the concentration of cysteine ​​in the presence of cystine.

[0010] 2) Detection of PMS concentration: After mixing the PMS solution with the colorimetric reagent and developing for 15 minutes, the resulting solution was pale yellow. The absorbance of the solution was then measured using UV-Vis at a wavelength of 352 nm. The colorimetric reagent was a mixed solution of NaHCO3 and KI. Different concentrations of PMS were prepared and their corresponding absorbances were measured to obtain a standard curve of PMS concentration versus absorbance.

[0011] 3) Reaction of PMS with cysteine ​​and cystine: Excess PMS at a known concentration reacts with cysteine ​​and cystine after mixing.

[0012]

[0013] Since there are two sets of chemical reactions involved, the amount of PMS consumed after the complete reaction of cysteine ​​can be calculated based on the cysteine ​​concentration obtained in 1). Subtracting the total PMS consumption from the total PMS consumption gives the total PMS and cysteine ​​consumption, thus yielding the concentration of cysteine ​​in the solution.

[0014] c[PMS] 总消耗量 =c[PMS] 反应前 -c[PMS] 反应后

[0015] c[PMS] 半胱氨酸= c[cysteine] * 3

[0016]

[0017] Combining the PMS method of this invention with the existing DTNB method enables the quantitative and accurate determination of the concentrations of cystine and cysteine ​​in a mixed solution.

[0018] Beneficial effects:

[0019] This invention provides an efficient and inexpensive method for the quantitative determination of cystine and cysteine ​​concentrations in a mixed solution. Taking cysteine ​​and cystine, two common amino acids, as examples, this invention elucidates the method for quantitative detection and calculation of cysteine ​​and its derivative concentrations by utilizing the reaction characteristics of thiol groups and disulfide bonds with PMS. This expands the detection capabilities of the DTNB method and provides a guarantee for studying the conversion process of cystine and cysteine. Attached Figure Description

[0020] Figure 1 The theoretical and measured concentration relationship of cystine.

[0021] Figure 2 The process of detecting and calculating the concentrations of cysteine ​​and cystine in a mixed solution. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments and accompanying drawings. The following examples simulate the quantitative detection process of cysteine ​​and cystine in a mixed solution. The embodiments of the present invention are provided to enable those skilled in the art to better understand the present invention and do not constitute any limitation on the present invention.

[0023] Example 1

[0024] This embodiment illustrates the usage method of the present invention based on the theoretical and actual concentration relationship of cystine obtained by PMS method. The specific steps are as follows:

[0025] 1) Preparation of the colorimetric reagent: Dissolve 0.4g KI and 0.02g NaHCO3 in 4mL of ultrapure water and let stand for 15min to obtain one sample of colorimetric reagent. Repeat this step to prepare multiple samples of colorimetric reagent.

[0026] 2) PMS concentration and absorbance: Prepare an effective concentration of 0-100 μmol / L. -1 PMS solutions of different concentrations (0.5 mL) were prepared and mixed with a colorimetric reagent. After color development for 15 min, the absorbance was measured using UV-Vis, and a standard curve was plotted for the effective concentration of PMS and absorbance.

[0027] 3) Reaction of PMS and amino acids: Prepare an effective concentration of 800 μmol / L -1PMS solutions containing phosphate buffer (10 mM, pH 7.0) and concentrations ranging from 40 to 120 μmol / L. -1 An amino acid solution containing phosphate buffer (10 mM, pH 7.0) was prepared. The PMS solution and amino acids were mixed 1:1 for 30 min, and 0.5 mL of the mixture was added to the colorimetric reagent for 15 min. The absorbance was then measured using UV-Vis.

[0028] 4) The cystine concentration was obtained by subtracting the PMS concentration before and after the reaction:

[0029]

[0030] The theoretical and measured concentration relationship of cystine is shown in [reference needed]. Figure 1 .

[0031] Example 2

[0032] This embodiment uses a mixed solution of cysteine ​​and cystine for quantitative detection to illustrate the method of using the present invention. The specific steps are as follows:

[0033] 1) Standard curve of cysteine ​​and DTNB: Prepare a concentration of 400 μmol / L -1 DTNB solutions containing phosphate buffer (10 mM, pH 7.0) and concentrations ranging from 40, 80, 120, 160, 200, to 240 μmol / L. -1 A cysteine ​​solution containing phosphate buffer (10 mM, pH 7.0) was prepared. The DTNB solution and cysteine ​​solution were mixed 1:1 and the absorbance was measured at 412 nm using UV-Vis after 10 min. A standard curve of cysteine ​​concentration versus absorbance was established.

[0034] 2) Detection of cysteine ​​concentration in the mixed solution: concentration range of 40, 80, 120, 160, 200, and 240 μmol / L. -1 A mixed solution of cysteine ​​and cystine containing phosphate buffer (10 mM, pH 7.0) was prepared. The mixture was then mixed again with the DTNB solution from step 1) at a 1:1 ratio for 15 min. The absorbance was measured at 412 nm using UV-Vis, and the cysteine ​​concentration was calculated using a standard curve.

[0035] 3) Preparation of PMS colorimetric reagent: Dissolve 0.4g KI and 0.02g NaHCO3 in 4mL of ultrapure water and let stand for 15min to obtain one colorimetric reagent. Repeat this step to prepare multiple colorimetric reagents.

[0036] 4) PMS concentration and absorbance: Prepare an effective concentration of 0-100 μmol / L. -1PMS solutions of different concentrations (0.5 mL) were prepared and mixed with a colorimetric reagent. After color development for 15 min, the absorbance was measured using UV-Vis, and a standard curve was plotted for the effective concentration of PMS and absorbance.

[0037] 5) Calculation of total PMS consumption: Prepare an effective concentration of 1800 μmol / L. -1 A PMS solution containing phosphate buffer (10 mM, pH 7.0). The mixed solutions of cystine and cysteine ​​at different concentrations from step 2) were mixed again with the PMS solution at a 1:1 ratio and reacted for 30 min. 0.5 mL of the mixture was then added to the colorimetric reagent and allowed to develop color for 15 min. The absorbance was measured using UV-Vis, and the total PMS consumption was determined.

[0038] 6) Calculation of cysteine ​​concentration: The cysteine ​​concentration obtained in step 2) is used to calculate the dose of PMS consumed by cysteine.

[0039] 5) The total PMS consumption minus the PMS consumption of cysteine ​​gives the amount of PMS consumed by cysteine, thus yielding the concentration of cysteine ​​in the solution:

[0040] c[PMS] 总消耗量 =c[PMS] 反应前 -c[PMS] 反应后

[0041] c[PMS] 半胱氨酸 = c[cysteine] * 3

[0042]

[0043] The detection and calculation process of cysteine ​​and cystine concentrations in the mixed solution is as follows: Figure 2 As shown in the figure. The experimental results for each step are shown in the table below:

[0044]

[0045] The results of implementing this plan are as follows:

[0046] Depend on Figure 1 It can be seen that this method can effectively detect cystine with a stoichiometric ratio of 1:5 with PMS, and establish a good relationship between theoretical and measured concentrations. Figure 2 This provides a basis for the quantification of mixed solutions of cysteine ​​and cystine. In summary, this invention can accurately and efficiently quantify the concentrations of cysteine ​​and cysteine ​​in mixed solutions, thus providing a basis for studying the conversion process between cysteine ​​and cysteine.

[0047] It should be understood that the embodiments and examples discussed herein are merely illustrative and may be modified or altered by those skilled in the art, and all such modifications and alterations should fall within the scope of protection of the appended claims.

Claims

1. A method for quantitatively determining the concentrations of cystine and cysteine ​​in a mixed solution, comprising the following steps: Step 1: DTNB reacts with cysteine; after mixing with cysteine ​​solution, colorless DTNB is converted into the colored product TNB, and TNB has a maximum absorption peak at 412 nm; therefore, this method can be used to quantify the concentration of cysteine ​​in the presence of cysteine. Step 2: Detection of PMS concentration; After mixing the PMS solution with the colorimetric reagent and developing the color for 15 minutes, the resulting solution is pale yellow. The absorbance of the solution is then detected using UV-Vis at a wavelength of 352 nm. The colorimetric reagent is a mixture of 4 mL of 0.02 g NaHCO3 and 0.4 g KI, and the volume of PMS to be quantified is 0.5 mL. Different concentrations of PMS are prepared and their corresponding absorbances are measured to obtain a standard curve of PMS concentration versus absorbance. Step 3: PMS reacts with cysteine ​​and cystine; an excess of PMS at a known concentration reacts with cysteine ​​and cystine after mixing. Since there are two sets of chemical reactions involved, the amount of PMS consumed after the complete reaction of cysteine ​​can be calculated based on the cysteine ​​concentration obtained in step one. The total PMS consumption minus the consumption of PMS and cysteine ​​gives the total consumption of PMS and cysteine, thus yielding the concentration of cysteine ​​in the solution. c[PMS] 总消耗量 =c[PMS] 反应前 -c[PMS] 反应后 ; c[PMS] 半胱氨酸 = c[cysteine] * 3; 2. The method for quantitatively measuring the concentrations of cystine and cysteine ​​in a mixed solution according to claim 1, characterized in that: In step one, the concentration of DTNB is 400 μmol / L. -1 .

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