SU1386883A1 - Method of determining l-amino acids - Google Patents

Description

(21) 4120397 / 31-13

(22) 07/02/86

(46) 04/07/88. Bul № 13

(71) Institute of Biochemistry of the Academy of Sciences of the Lithuanian SSR

(72) Yu. Y. Kulis, M.V. Pesl kene and .V.-S.A. Laurinavicius

(53) 663.15 (088.8)

(56) White W. G., Guilbault G. G. Lysine specific enzyme electrode for grays and foodstuffs. - Anal. Chem, 1978, V. 50, p. 1481-14.

laniello R.M., Jacynyh A.M., Immobilized Enzyme Chemically Modified Electrode as an Amperometrie Sensor. - Anal. Chem, 1981, v. 53, p. 2090-2095.

(54) METHOD FOR DETERMINING L-AMINO ACID LOTS

(57) The invention relates to the investigation and analysis of materials by determining the current and can be used to determine the L-amino acid. The purpose of the invention is to simplify the process and increase the selectivity of the determination of L-amino acids. For this, peroxidase is introduced into the immobilized membrane of L-amino acid oxidase, electrolysis is carried out in a weakly alkaline buffer solution in the presence of potassium ferrocyanide at a concentration of 1-2 mM. with OB on a graphite electrode without prior chemical modification with respect to the Ag / AgCl reference electrode and the concentration of L-amino acids is determined by the increase in the cathode current. 1 dw., 5 tab.

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The invention relates to electrochemical methods for the analysis of materials, namely, a method for quantifying L-amino acids by measuring the current during electrolysis during enzymatic reactions.

The aim of the invention is to simplify the process and increase the selectivity of the determination of L-amino acids.

The drawing shows a diagram of a measuring cell for implementing the method.

The method involves the electrolysis of the test substance in a weakly alkaline phosphate buffer using a graphite electrode coated with an immobilized oxidase membrane.

H Oj + fFe (CN) + 2E peroxidase 2 D (CN). (one)

The amount of peroxidase added to the membrane must be such that the reaction, which can be catalyzed by peroxidase according to equation (1), is fast, i.e. did not limit the overall process. For this, a 10–50-fold excess of peroxidase is sufficient compared to the L-amino acid oxidase in activity. Going beyond the upper limit is not required and is not economically feasible. In addition, as the concentration of the enzyme increases, the membrane thickness increases, as a result, the response of the electrode increases and its sensitivity decreases.

The reduction of the formed ferrocyanide on a graphite electrode at О В leads to the generation of a cathode current, the value of which is proportional to the concentration of the L-amino acid.

The proposed method does not require chemical modification of the graphite electrode, thus simplifying the process. The increase in selectivity is ensured by the fact that electrolysis is carried out at 0 V, and at this potential, the main electrochemically active substances are not oxidized and the magnitudes of the background currents when introducing samples of real samples do not exceed 1%. Therefore, the concentrations of L-amino acids in real media are determined using a calibration graph for a pure solution.

The measuring cell consists of a body I made of organ0

L-amino acids, and a reference electrode from Ag / AgCl and measurement of the current in the cell during interaction of the enzyme with the substrate, potassium ferrocyanide at a concentration of 1-2 mM is introduced into the buffer solution before electrolysis, peroxide is added to the immobilized L-amino acid oxidase membrane. A graphite electrode is used without chemical modification, the electrolysis is carried out at 0 V and the concentration of L-amino acids is determined by the increase in the cathode current.

In the enzyme membrane containing the L-amino acid oxidase and peroxidase, in addition to the oxidation of the L-amino acid, the transformation occurs according to scheme E.

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of the glass and containing the inlet and outlet nozzles for washing and filling the cell with a buffer solution, and a measuring chamber 2 with a magnetic stirrer 3, into which a working graphite electrode 4, covered with an immobilized membrane 5 and an Ag / AgCl comparison electrode 6, is placed, and which contains an upper opening for the introduction of the sample. Washing and filling the measuring chamber with a buffer solution is carried out using a two-channel peristaltic pump. Worker and auxiliary electrodes are attached to a recorder with a recorder -.

A 0.05 ml L-amino acid solution is injected into a 1 ml measuring cell containing a buffer solution with potassium ferrocyanide. When the enzyme interacts with the substrate, cell parameters are measured. After analysis, the cell is washed for 1.5 minutes with a stream of buffer, after which the next measurement can be carried out.

Example 1. The determination of the concentration of L-leucine.

For the preparation of the transfer membrane, 3 mg of L-amino acid oxidase, 10 mg of peroxidase and 16 mg of serum albumin are dissolved in 0.2 ml of 0.01 M phosphate buffer pH 7.5, 0.1 M KCl. The mixture is treated with 5 µl of 5% glutaraldehyde and poured onto a 6 cm dialysis membrane. A membrane dried at 4 C, 50 µm thick, is put on - directly on the graphite electrode and attached with a rubber ring.

Determination of L-leucine using the prepared enzyme electrode is carried out in 0.01 M phosphate buffer pH 7.5, containing 0.1 M KCl and 2 mM potassium ferrocyanide, the potential of the graphite electrode is 0.0 V relative to the Ag / AgCl electrode.

These changes in the cathode current when the concentration of L-leucine added is changed are given in Table. I.

From tab. 1, it can be seen that the straightness of the calibration graph is observed up to 0.8 mM L-leucine.

Example 2. Determining the concentration of various amino acids.

The enzyme membrane and the enzyme electrode are made analogously to Example 1. The current in the buffer solution containing different amino acids is determined: 0.01 M phosphate buffer pH 8.0, 0.1 M KCl, potassium ferrocyanide concentration 1.5 mM, amino acid concentration 0, 5 mM The conditions of determination are as in Example I.

The results are presented in table. 2

From tab. 2 that the L-amino acid L-leucine is the best substrate for the oxidase, however L-methionine, L-ji-phenyl-alanine, L-tryptophan, L-isoleucine, L-arginine can be determined. D-Anino acids, L-alanine L-asparagine, L-threonine and L-proline practically do not interfere with the determination.

Example 3. Determination of cell current dependence on potassium ferrocyanide concentration w and pH of the buffer solution.

The catalytic membrane and the truss electrode are made as in Example 1. The concentration of L-leucine is determined depending on the concentration of potassium ferrocyanide in the tuber solution pH 7.8, the concentration of L-leucine 1.0 mM. The conditions of determination are as in Example 1.

The data are shown in Table. 3,

From tab. 3, it can be seen that the cell current depends on the concentration of potassium ferrocyanide in the buffer solution.

With an increase in the concentration of the mediator to 1 M, the current increases, and at a concentration of the compound above 1 1, it does not change. Thus, the method is implemented when the concentration of the mediator is higher than 1 mM, and with

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the aim of saving the reagent is an optimal concentration of 1 mM.

The electrode readings depend on the pH of the buffer solution. So, if there is 0.6 mM L-leucine in the cell at pH 6.0, the cell current is 390 pA, at pH 6.5 - 450 nA, at pH 70 - 485 nA, at pH 7.5 - 480 nA, at pH 8.0-- 482 nA, at pH 8.5 - 465 nA, at pH 9.0 - 436 nA. Thus, the pH range of 7-8 is optimal.

Example 4. Determining the concentration of L-leucine in microorganism growing media.

The catalytic membrane was made as in Example 1. Microorganism growth media were prepared by dissolving appropriate amounts of L-leucine in culture media. Measurements are made as for pure solutions, the concentration of L-leucine is determined by the calibration graph for pure solutions. The conditions of determination are as in Example 1.

The measurement data are presented in Table. four.

From tab. 4 that the determination accuracy (i.e., the coefficient of variation) does not exceed 3%. The residual current in the medium does not exceed 0.5-1.0%, since the substances in real solutions are electrochemically inactive at zero potential of the graphite electrode.

Example 5. Determination of the stability of the electrode in time.

The enzyme electrode was made as in Example 1. The concentration of L-leucine was 0.5 mM. The conditions of determination are as in Example 2.

The results of the dependence of the current on the duration of the preservation of the electrode are presented in Table. five.

From tab. 5, it can be seen that the operability of the electrode is maintained for 2 months.

Compared with the known, the proposed method of determining L-amino acids is simpler, more expressive and selective, since there are no pre-processing operations for samples, chemical modification of the electrode, and no determination of the effect of other substances that are in biological real media. The speed of analysis is especially important in the microbiological industry with continuous monitoring of the level of L-amino acids.

Claims (1)

Invention Formula 13868836 The following determination of the concentration of amino acids to increase the current, characterized in that, in order to simplify the process and increase the selectivity, before the electrophore of determining L-amino acid-sweat, by lysis, the ferrocum electrolysis of the studied potassium cyanide is introduced into a buffer solution in substances in a buffer solution with an immobilized oxidase membrane using a graphite electrode ™ JQ L-amino acids are additionally introduced with a membrane coated with an immobilized peroxidase, while electrolysis of an L-amino acid oxidase lot and DUT zero electrical potential relative trodes comparison of Ag / AgCl with post-Tel'nykh reference electrode. Table I Current, mA 0.107 0.220 0.326 0.432 0.538 0.700 0.830 0.905 Current, mA 0.21 0.36 0.55 0.66 0.73 0.80 0.82 0.80 0.81 0.81 Current, mA 464 480 472 410 365tl5 330t30 320tlO 220 145 134 130 (29 128 125 123 Table 4