Method for quantification of amino acid using iodine titration

Abstract

To solve the following problem that: in a quantification method for amino acid employing a ninhydrin reaction, oxidation reaction reduces the yield of Ruhemann's purple, thus making correlation between amino acid concentration and coloration difficult, and making quantification difficult for a solution containing multiple kinds of amino acids.SOLUTION: Provided is a method for calculating an amino acid concentration, the method comprising: mixing a Leu solution, which is an amino acid solution to which an iodine solution having an iodine concentration [I2]0 at any temperature T of 40°C or higher is added, with a ninhydrin solution at the same temperature and maintaining the temperature; at any time t at which browning of iodine is visually observable, immediately cooling the mixed solution to 5°C or lower; and substituting the iodine concentration [I2]t of the mixed solution at the time t, calculated by redox titration, and [I2]0 into the following formula (1) to calculate the amino acid concentration. [I2]0-[I2]t=(1-e-kt)[Amino acid]0 (1), where [Amino acid]0 represents an initial amino acid concentration (mol / L) in the mixed solution, and k represents a constant dependent on the ninhydrin concentration (mol / L) and the temperature condition T.SELECTED DRAWING: Figure 19

Description

Technical Field

[0001] The present invention causes a decomposition reaction of an amino acid that suppresses the formation of Ruhemann's purple by a ninhydrin reaction to which an iodine solution is added, determines the decrease in the amount of iodine that coincides with the decomposition amount of the amino acid at this time by iodine titration, and based on the ninhydrin reaction rate caused by the type and concentration of the amino acid, calculates the amino acid concentration from the decrease in the amount of iodine. The present invention relates to a method for quantifying amino acids.

Background Art

[0002] When a ninhydrin solution is added to an amino acid solution and heated, Ruhemann's purple is formed and exhibits a bluish purple color. This reaction is called the ninhydrin reaction and is used for the detection of amino acids, proteins, and various peptides. Further, the higher the amino acid concentration, the greater the amount of Ruhemann's purple generated in the ninhydrin reaction and the darker the color development. Therefore, an amino acid quantification device using a colorimetric method capable of quantifying amino acids from the degree of this color development is widely used.

[0003] In addition, since the color development time indicated by the time from the start time of the reaction in the ninhydrin reaction to the minimum value of the time when the color development of Ruhemann's purple generated in the reaction can be visually recognized is determined by the amino acid solution concentration, it is also possible to quantify amino acids from the color development time using the reaction rate equation of the ninhydrin reaction.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Since the intermediate product of the ninhydrin reaction (reduced ninhydrin) and the Ruhemann's purple produced in the reaction are easily air-oxidized, the amount of Ruhemann's purple produced in the reaction does not necessarily indicate the amount of amino acid decomposition. Therefore, treatments such as adding an antioxidant to increase the yield of this Ruhemann's purple are required. For this reason, the amino acid concentration and the degree of color development of Ruhemann's purple cannot be quantified by a mathematical formula, and amino acids are empirically quantified by a colorimetric method. When quantifying amino acids by the colorimetric method, there is a problem that it is difficult to compare the amino acid concentration and the degree of color development of Ruhemann's purple due to the decrease in the yield of Ruhemann's purple caused by this oxidation reaction. Furthermore, it is difficult to quantify an amino acid mixed solution containing a plurality of types of amino acids without separating them by this colorimetric method.

[0006] In the ninhydrin reaction, by measuring a very short time until the moment when the color development of Ruhemann's purple can be visually recognized for the first time, amino acids can be quantified using this time that has a correlation with the amino acid concentration as an index. However, the problem is that there is no objectivity in measuring the moment of color development visually, and a clearer index is required.

Means for Solving the Problems

[0007] When the ninhydrin reaction is caused in the presence of iodine, the ninhydrin concentration in the reaction solution is kept constant, and although the production of Ruhemann's purple is suppressed, amino acids are decomposed. At this time, a reaction mechanism of the primary amino acid decomposition reaction in which the amount of iodine reduction coincides with the amount of amino acid decomposition was found.

[0008] The present invention mixes a Leu solution, which is an amino acid, to which an iodine solution having an iodine concentration [I2]0 at an arbitrary temperature condition T of 40 °C or higher is added, with a ninhydrin solution having a known concentration at the same temperature condition T, and at the same time, maintains the temperature condition. At an arbitrary time t when the brown color of iodine in the mixed solution can be visually recognized, the mixed solution is immediately cooled to 5 °C or lower, and the iodine concentration [I2] of the mixed solution at the time t calculated by redox titration t and a method for calculating the amino acid concentration by substituting [I2]0 into the following formula (1). [I2]0 - [I2] t =(1 - e -kt )[Amino acid]0 ··· (1) In the formula, [Amino acid]0 is the initial concentration of amino acid in the mixed solution (mol / L), k is the ninhydrin concentration (mol / L), and is a constant depending on the temperature condition T. [Advantages of the Invention]

[0009] The present invention enables a primary decomposition reaction of an amino acid that suppresses the formation of Ruhemann's purple while maintaining a constant ninhydrin concentration by causing a ninhydrin reaction in the presence of iodine to oxidize an intermediate product (reduced ninhydrin). The amount of amino acid can be quantified simply by determining the decrease in the amount of iodine equal to the amount of decomposed amino acid by iodine titration. There is an effect that amino acids can be easily quantified without the need for expensive analytical equipment.

[0010] Furthermore, since the present invention is a method for quantifying an amino acid based on the reaction rate of the primary decomposition reaction of the amino acid, by using the difference in the reaction rate of the ninhydrin reaction depending on the type of amino acid, even in an amino acid mixed solution containing multiple types of amino acids, each amino acid can be simultaneously quantified. [Brief Description of the Drawings]

[0011]

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Embodiments for Carrying Out the Invention

[0012] The ninhydrin reaction is a reaction in which a mixed solution of ninhydrin and an amino acid turns purple when heated, and it is widely known as a qualitative analysis method for amino acids (Figure 1). To cause the ninhydrin reaction, it is necessary to increase the reaction rate, and a certain reaction temperature is required. Many amino acids such as leucine hardly react at room temperature and react at 40 °C or higher. Also, the ninhydrin reaction does not occur under temperature conditions of 5 °C or lower. This reaction mechanism is as follows: First, in the first step, ninhydrin and the amino acid react to form a reduced ninhydrin, which is an intermediate product. Next, in the second step, the reduced ninhydrin and the unreacted ninhydrin condense to form Ruhemann's purple and exhibit color (Figure 2).

[0013] It was conceived that if the ninhydrin reaction is caused in the presence of an oxidizing agent, the reduced ninhydrin in Figure 2 is oxidized, suppressing the formation of Ruhemann's purple (the reaction does not proceed to the second step), and a reaction mechanism in which the amino acid is decomposed can be realized. At this time, if a relationship is shown such that the decomposition amount of the amino acid can be replaced by the decrease amount of the oxidizing agent, the amino acid can be quantified using the decrease amount of this oxidizing agent and the reaction rate formula of the ninhydrin reaction.

[0014] First, hydrogen peroxide was used as the oxidizing agent. Under the temperature condition of 60 °C, 10 mL of a 0.15 mol / L leucine solution to which 1 mL of distilled water and 1 mL of 30% hydrogen peroxide solution were respectively added was added with 10 mL of a 0.05 mol / L ninhydrin solution to cause the ninhydrin reaction, and it was observed whether hydrogen peroxide could oxidize the reduced ninhydrin and decompose the amino acid without generating Ruhemann's purple. As shown in Figure 3, it was found that Ruhemann's purple was not generated in the presence of hydrogen peroxide.

[0015] However, under the temperature condition of 60 °C, 1 mL of 3% hydrogen peroxide solution was added to 20 mL of a 0.05 mol / L ninhydrin solution, and the absorbance at 350 nm was measured immediately after mixing and after 5, 10, and 15 minutes to examine the reaction between hydrogen peroxide and ninhydrin. The absorbance of ninhydrin (350 nm) decreased with respect to the reaction time (Figure 4). That is, it is considered that hydrogen peroxide did not act on the reduced ninhydrin but directly acted on ninhydrin.

[0016] Using the self-made polarimeter described below, at a temperature of 60°C, 70 mL of 0.05 mol / L ninhydrin solution was added to 140 mL of 0.15 mol / L leucine solution to which 5 mL of 30% hydrogen peroxide solution was added. Immediately after mixing and at 10, 20, and 30 minutes later, the reaction solutions were quickly cooled to 5°C or lower to suppress the reaction, and the polarizations of the reaction solutions were measured. It was found that there was no change in the polarization with respect to the reaction time (Figure 5), and it was also found that the amino acids were not decomposed. With the addition of hydrogen peroxide having a strong oxidizing power, it was found that the ninhydrin reaction itself did not occur in the first place, and the target reaction mechanism conceived in

[0013] could not be established.

[0017] The self-made polarimeter was used to directly measure the amino acid concentration (Figure 6). Both ends of the pipe filled with the amino acid solution were closed with culture test tubes, and two polarizing plates were arranged on the left and right, respectively. The light irradiated from the LED light source and transmitted through the amino acid solution was measured with an illuminometer. The polarizing plate on the illuminometer side was fixed, and the polarizing plate on the LED light source side was rotated. The position where the two polarizing plates were orthogonal was set to 0°, and the angles were changed to -25°, -20°, -15°, -10°, -5°, 0°, 5°, 10°, 15°, 20°, 25°. The illuminance with respect to the rotation angle of the polarizing plate was measured.

[0018] The relationship between the illuminance and the rotation angle measured using the self-made polarimeter for distilled water and 0.15 mol / L threonine solution is shown (Figure 7). Distilled water was darkest near 0° where the two polarizing plates were orthogonal, showing illuminance symmetric about the y-axis. For the threonine solution, the darkest position shifted to the left with respect to distilled water. Here, the polarization that can be obtained with this self-made polarimeter is defined (Figure 7). The relationship between the rotation angle of the polarizing plate and the illuminance was approximated by a quadratic function, and the x at which the illuminance was minimized, that is, the axis -b / 2a of the quadratic function, was taken as the polarization.

[0019] Based on the definition of the specific rotation, when measuring the specific rotation of solutions of various amino acids (Ala, Val, Thr, Cys, Leu, Ile, Phe, Arg), different specific rotations are shown for different types of amino acids, and the same tendency is observed even when changing the concentration of the amino acid (Figure 8). It is considered that different three-dimensional structures of amino acids have unique specific rotations. Furthermore, when examining the relationship between the amino acid concentration and the specific rotation, for all amino acids, the specific rotation showed a proportional relationship with the concentration (Figure 9). The amino acid concentration can be directly measured from the specific rotation. However, this method for quantifying amino acids using the specific rotation has problems such as the need for a large amount of sample solution, difficulty in measuring low concentrations due to small specific rotations depending on the type of amino acid, and inability to quantify when multiple types of amino acids are mixed.

[0020] Next, iodine, which has a relatively weak oxidizing power, was used as the oxidizing agent, and a verification was carried out in the same way to see if a reaction could be found that oxidizes reduced ninhydrin by the intended reaction mechanism conceived in

[0013] and suppresses the formation of Ruhemann's purple. A 0.2 mol / L I3 solution prepared by dissolving 25.38 g of iodine (molecular weight 253.8) and 50.76 g of potassium iodide (molecular weight 166) in a 500 mL volumetric flask was diluted and used as the iodine solution for each experiment. Figure 10 shows the change over time of the ninhydrin reaction solution to which iodine solutions of each concentration were added. Under the temperature condition of 60 °C, the change over time of the reaction solution obtained by adding 20 mL of a 0.05 mol / L ninhydrin solution to 20 mL of a 0.15 mol / L leucine solution to which 1 mL of an iodine solution of each concentration was added was observed. In the presence of iodine, none of them proceed to the second-stage reaction. It can also be seen that the higher the concentration of the added iodine solution, the longer the time it takes to stop at the first-stage reaction. That is, if an excessive amount of iodine is added to the ninhydrin reaction solution, the formation of Ruhemann's purple can be suppressed. - The change over time of the ninhydrin reaction solution to which iodine solutions of each concentration were added is shown in Figure 10. Under the temperature condition of 60 °C, the change over time of the reaction solution obtained by adding 20 mL of a 0.05 mol / L ninhydrin solution to 20 mL of a 0.15 mol / L leucine solution to which 1 mL of an iodine solution of each concentration was added was observed. In the presence of iodine, none of them proceed to the second-stage reaction. It can also be seen that the higher the concentration of the added iodine solution, the longer the time it takes to stop at the first-stage reaction. That is, if an excessive amount of iodine is added to the ninhydrin reaction solution, the formation of Ruhemann's purple can be suppressed.

[0021] As envisioned, it was confirmed in the following experiment whether iodine actually acts on reduced ninhydrin to suppress the formation of Ruhemann's purple. First, at a temperature of 60 °C, 1 mL of 0.2 mol / L iodine solution was added to 5 mL of ninhydrin solutions with concentrations of 0.05, 0.025, and 0.0125 mol / L respectively. At each reaction time, 1 mL of 1 mol / L sodium thiosulfate solution was added to decolorize the iodine, and the absorbance at 350 nm was measured. When examining the action of iodine on ninhydrin, the absorbance (350 nm) of ninhydrin showed a constant value with respect to the reaction time (Figure 11). It is considered that iodine does not act on ninhydrin.

[0022] Next, at a temperature of 60 °C, 30 mL of 0.2 mol / L iodine solution was added to 140 mL of 0.15 mol / L leucine solution and threonine solution respectively. After 0, 10, 20, and 30 minutes, 20 mL of 1 mol / L sodium thiosulfate solution was added to decolorize the iodine, and the specific rotation of the solution at each reaction time was measured with a self-made polarimeter. When examining the action of iodine on amino acids, the specific rotation did not change similarly (Figure 12), confirming that the added iodine does not act on amino acids either. That is, iodine does not act directly on ninhydrin and amino acids, but acts on reduced ninhydrin, indicating the possibility of finding the target reaction mechanism by adding iodine.

[0023] At this time, to investigate whether amino acid decomposition actually occurred, 70 mL of 0.05 mol / L ninhydrin solution was added to 140 mL of 0.15 mol / L leucine solution to which 30 mL of 0.2 mol / L iodine solution was added under the temperature condition of 60 °C, and the change in specific rotation over time was measured and examined using a self-made polarimeter. 5 mL of 30% hydrogen peroxide solution was added to decolorize the ninhydrin, 20 mL of 2 mol / L sodium thiosulfate solution was added to decolorize the iodine, and the buffer solution was adjusted to pH 5. As a result, the absolute value of the specific rotation measured by the self-made polarimeter of the ninhydrin reaction solution added with iodine solution decreased with respect to the reaction time (Figure 13). Different from the case where hydrogen peroxide solution was added as the oxidant in

[0016] , it was confirmed that amino acids were decomposed. That is, the reaction envisioned in

[0013] occurs upon the addition of iodine.

[0024] Furthermore, the change over time in the concentration of ninhydrin in the ninhydrin reaction solution in the presence of iodine was examined under temperature conditions of 60°C. To 14 mL of a 0.15 mol / L glycine solution to which 3 mL of a 0.2 mol / L iodine solution was added, 7 mL of 0.01, 0.02, or 0.05 mol / L ninhydrin solution was added to prepare reaction solutions. After 0, 10, 20, and 30 minutes, the reaction solutions were immediately quenched to 5°C or lower, 2 mL of a 1 mol / L sodium thiosulfate solution was added to decolorize the iodine coloration, and the absorbance at 350 nm was measured. At this time, glycine, in which the resulting aldehyde is colorless, was used as the amino acid so that the coloration of the aldehyde, which is a by-product generated in the ninhydrin reaction in Fig. 2, would not affect the absorbance measurement. As a result, it was found that the absorbance (350 nm) of ninhydrin did not change (Fig. 14). That is, it is considered that a loop occurs in which the reduced ninhydrin oxidized by iodine returns to the original ninhydrin. While maintaining this constant ninhydrin concentration, the reaction in which the amino acid is decomposed was defined as the primary amino acid decomposition reaction (Fig. 15).

[0025] As shown in Fig. 15, one molecule of reduced ninhydrin is produced per molecule of amino acid. Also, since reduced ninhydrin and iodine react one-to-one, it was predicted that the decrease in the amount of amino acid would be the same as the decrease in the amount of iodine. Therefore, the rate of change in the amino acid concentration calculated from the optical rotation measured in the experiment of was compared with the rate of change in the iodine concentration determined by the following method under the same experimental conditions. A reaction solution obtained by adding 7 mL of 0.05 mol / L ninhydrin solution to 140 mL of 0.15 mol / L leucine solution to which 30 mL of 0.2 mol / L iodine solution was added was rapidly cooled to 5°C or lower immediately after 0, 10, 20, and 30 minutes to suppress the decomposition of amino acids, and redox titration was performed with 0.1 mol / L sodium thiosulfate solution to measure the decrease in the amount of iodine. Since the ninhydrin concentration is kept constant in this primary amino acid decomposition reaction, it can be any concentration, but it is a factor that determines the measurement conditions (reaction time and reaction temperature). Also, if the decomposition of amino acids is stopped by cooling, the redox titration may be performed at any time in this state. At this time, in order to correct the influence of iodine volatilization, the same operation was performed on a mixed solution of 10 mL of purified water and 2 mL of 0.2 mol / L iodine solution, and the titration value of the reaction solution was subtracted from the titration value to calculate the iodine concentration purely decreased by the reaction. As a result, the rate of change in the amino acid concentration calculated from the optical rotation and the rate of change in the iodine concentration under the same experimental conditions showed extremely close values (Fig. 16). That is, the decomposition rate of amino acids can be replaced by the decrease rate of iodine. That is, the decomposition amount of amino acids can be calculated by iodine titration.

[0026] According to Fig. 17, a theory for quantifying amino acids from the reaction rate of the primary amino acid decomposition reaction was established. Since the ninhydrin concentration is constant, the decomposition rate v of amino acids can be expressed by Equation (1). k is a reaction rate constant that depends on the type of amino acid and the reaction temperature, including the initial ninhydrin concentration that is kept constant during the reaction. When this is transformed and integrated, Equation (2) is obtained. For the reaction time t, log e[Amino acids] are in a linear relationship, and k can be calculated from the slope. The linear relationship obtained from the experiment is shown in a graph (Figure 18). From the slope, k at each temperature can be determined. Here, Equation ▲2▼ is transformed into Equation ▲3▼. Since the amount of iodine decrease and the amount of amino acid decomposition are equal, Equation ▲4▼ holds. That is, from k, t, and the amount of iodine decrease by iodine titration, the initial concentration of amino acid [amino acid]0 in the reaction solution can be obtained, so the amino acid concentration used in the measurement can be calculated. In the case of a mixed solution, a system of simultaneous equations of Equation ▲4▼ can be set up under conditions corresponding to the number of amino acids, and each amino acid can be quantified in the same way.

[0027] The amino acid primary decomposition reaction was caused by the method of Figure 19, and it was verified whether amino acids could really be quantified by the above theory. [I2]0 represents the concentration immediately before the reaction, and [I2] t represents the concentration obtained by subtracting the amount of iodine decreased in the reaction. Therefore, the amount of iodine volatilized by heating was measured in advance and corrected for each. An arbitrary reaction temperature and reaction time were determined, and the reaction solution was rapidly cooled to 5°C or lower immediately at that reaction time to suppress the reaction. Then, the amount of iodine decreased in the reaction was determined by redox titration with a sodium thiosulfate solution as a reducing agent, and the amino acid concentration was calculated based on the above theory. As shown in the table, the concentration of the standard solution used for verification and the calculated concentration showed close values in all concentration ranges (Figure 20). Furthermore, in the mixed solution, similar close values were shown for any mixing ratio (Figure 20), and it was verified that quantification was possible even in the mixed state. Based on the theory so far, amino acids can be quantified only by titrating the remaining iodine amount.

Industrial Applicability

[0028] Ordinarily, in the ninhydrin reaction, it is common to add an antioxidant to increase the yield of Ruhemann's purple. In contrast, in the present invention, conversely, by adding iodine, which is an oxidizing agent, a primary decomposition reaction is carried out while keeping the ninhydrin concentration constant, enabling the decomposition amount of the amino acid to be determined only by iodine titration. Currently, amino acids are quantified mainly for the amino acid composition analysis of proteins by various amino acid quantification methods. This is an essential analysis especially in the medical field. Compared with the conventional methods, the present invention can easily quantify amino acids even in a mixed state only by iodine titration. It is expected to become one of the options for amino acid quantitative analysis methods in the future.

Claims

【Claim 1】 The iodine concentration [I ２ ０ of an iodine solution is added to a Leu solution, which is an amino acid, and a ninhydrin solution with a known concentration at the same temperature condition T are mixed, while maintaining the temperature condition T. At any time t when the brown color of iodine in the mixed solution can be visually recognized, the mixed solution is immediately cooled to 5°C or lower, and the iodine concentration [I ２ ｔ and [I ２ ０ are substituted into the following formula (1) to calculate the amino acid concentration. [I ２ ０ −[I ２ ｔ =(1−e －ｋｔ )[Amino acid] ０ ...(1)​​ where [amino acid] ０ is the initial concentration of amino acid in the mixed solution (mol / L), and k is a constant that depends on the ninhydrin concentration (mol / L) and the temperature condition T.

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