SU1317027A1 - Method of quantitative determination of adenine nucleotides - Google Patents

Abstract

This invention relates to the field of biochemical analysis. The aim of the invention is to reduce the detection limit of adenine nucleotides and reduce the consumption of enzymes. The method is carried out using a co-immobilized luciferase system, adenylate kinase and pyruvate kinase. As a source of luciferase and adenylate kinase, crude light bulbs of Luciola mingrelica light are used. The mass ratio of the light bulbs is bright:: pyruvate kinase: bromine cyanerpharosis is 50: (3-12) :( 50-100). The method allows to reduce the consumption of expensive high-grade enzymes and to increase the sensitivity of the determination. The method can be used in medicine, food industry and in scientific research. (L

Description

113

The invention relates to a biochemical analysis, in particular to a method for determining adenine nucleotides: adenosine 5-monophosphate (AMP), adenosine-3-diphosphate (ADP) and adenosine-5-triphosphate (ATP).

The purpose of the invention is to reduce the detection limit as well as reduce the consumption of enzymes in the determination of adenine nucleotides.

The method is as follows.

Luciola mingrelica light extract containing both enzymes containing both enzymes is used as a source of lyudinerase and adenylate kinase, which is obtained by extraction with 0.1 M phosphate buffer containing 0.2 M NaCl and 2 mM EDTA, pH 8.0 for 12 hours at 4 ° С when the weight ratio of the light bulb is light - buffer is from 1:40 to 1:10, the light bulb extract is light and the coarsely purified rabbit muscle pyruvate kinase is co-mobilized on bromo cyanactivated sepharose. During immobilization, 50 mg / mp of light bulbs are used, the concentration of pyruvatkinase varies from 3 to 12 mg / ml and the carrier from 50 to 100 mg / ml. At pyruvate kinase concentrations below 3 mg / ml, preparations with low pyruvate kinase activity are obtained, which reduces the rate of conversion of AMP and ADP to ATP and leads to an increase in reagent consumption and analysis time. At concentrations of pyruvate kinase above 12 mg / ml, the preparations have low luciferase activity, which reduces the sensitivity of the determination of nucleotides. When using the concentration of bromiancyan-activated sepharose when immobilizing below 50 mg / ml, expensive enzymes are lost due to their incomplete binding to the carrier, and when using above 100 mg / ml of sepharose, drugs with low luciferase, adenylate kinase and pyruvate kinase activities are obtained that do not give high sensitivity in the analysis.

The increase in the sensitivity of the determination of ATP and ADP with the help of a co-mobilization, calling for the multienzyme system as compared to the soluble one, is apparently due to an increase in the local concentration of enzymes and products near the surface of the carrier. This increases the stationary speed of fer0272

in the immobilized multienzyme system and facilitates the diffusion transfer of substrates and products of one enzymatic reaction to the active center of another. In the co-immobilized multienzyme system, the influence of side factors is reduced due to the spatial separation of the enzymes on the carrier,

 O decreases the inhibitory effect of pyruvate kinase on the luciferase reaction, which is observed when working with soluble enzymes. In addition, due to the local concentration 5 or near the carrier, formed with ATP undergoes less hydrolysis than it would have been in solution. All these factors provide a much greater efficiency of the co-mobilized enzyme system than the soluble one.

Analysis of adenine nucleotides using the co-immobilized luciferase, adenylate kinase and pyruvate kinase preparation is carried out as follows:

AMP + ctf (1)

in A gt i pyruvate kinase - ADP + phosphoenolpyruvate

25

ATP + pyruvate

(2)

35

ATP + luciferin +0

luciferase

AMF +

+ oxyluciferin + inorganic phosphate + + CO2 + li)

. (3)

ATP is determined with this drug.

The known method of reaction (1), Co-immobilized with luciferase adenylate kinase and pyruvate kinase, do not affect the detection sensitivity. The intensity of radiation is directly proportional to the concentration of ATP,

To determine ADP, it is first converted to ATP by reaction (2) while incubating with co-immobilized enzymes and phosphoenolpyruvate for 1 minute. The resulting ATP is determined similarly. For this. after adding a luciferin-containing buffer to the mixture, radiation is recorded, the intensity of which is proportional to the concentration of ADP in the system. The unknown concentration of ADP is determined from the calibration straight.

To determine AMP, it is converted to ATP by the reactions (1 and 2) of incubus.

first for 30 minutes with a multienzyme preparation and cytidine-5-triphosphate (CTF), and then 1 minute with phosphoenolpyruvate. The resulting ATP is determined as indicated. After the luciferin-containing buffer is added, a signal is recorded that is proportional to the content of AMP in the system. The unknown concentration of AMP is determined from a calibration straight,

Using the proposed co-immobilized enzyme preparation, it is possible to determine both the total content of adenine nucleotides in a sample, and the content of each of them separately, and for the determination of AMP and ADP, it is necessary to preliminarily separate ATP from other adenine nucleotides, since the content of ATP in biological samples, As a rule, several times higher than AMP and ADP. The separation of ATP is carried out using ion-exchange chromatography on DEAE-cellulose 3 e.

The method is illustrated by the following examples.

Example 1. Preparation of co-immobilized enzyme preparations

50 mg of light bulbs are extracted with 1 ml of 0.1 M Na-phosphate buffer; 0.2 M NaCl; 2 mM EDTA; pH8.0 at 4 ° C for 12 hours. 1 ml of the extract is incubated with 50 mg of swollen bromocyansepharose and 7 mg of pyruvate kinase for 24 hours at A C. The precipitate is washed 5 times alternately with 5 ml of 0.02 M Na-acetate buffer (1 M, NaCl, pH 4.0) and 5 ml of tris. -acetate buffer (1 M NaCl, pH 8.5), and then 5 times in 10 ml of distilled water and 5 times in 5 ml of 0.1 M tu is-acetate buffer, pH 7.6, 2 mM EDTA. The concentration of the obtained suspension of immobilized enzymes is 10 mg / ml, the luciferase activity in the preparation is 42000 mV / mg of carrier, and the activity of pyruvate kinase is 12 U / mg of carrier. ATP Definition

A 1 ml solution in a mass cuvette (containing 1 mg / ml co-immobilized enzyme suspension, 1 mM luciferin, 2 mM EDTA, 10 mM MgS04 in 0.1 M tris-acetate buffer pH 7.8 is placed in a luminometer and measured background luminescence, which for this preparation does not exceed 0.1 mV. Then 50 µl of ATP solution of a known concentration of 0274 is added to this solution.

centration, do not interrupt registration of the glow. The difference between the intensity of the signal in the presence of ATP and the background signal is proportional to the concentration of ATP. The experiments are repeated for ATP solutions with ATP concentrations in the same range in which measurements are intended. On the basis of the data obtained, a graph is plotted of the dependence of the bioluminescence intensity on the ATP concentration. For the resulting preparation, co-immobilized enzymes are directly proportional to the dependence observed in the range

5 ATP concentrations of 10 pM-0.10 mM. The concentration of ATP in an unknown sample is determined from the calibration curve obtained. The limit of ATP detection with this reagent is 10 pM,

Determination of ADP, K 0.7 ml 0.1 M Tris-acetate buffer pH 7.8, located in a plastic 5-cuvette for a luminometer and containing 2 mM EDTA, 10 mM MgS04, 20 mM potassium acetate and 5 mM phosphoenol- pyruvate, 50 µl of a solution of ADP of known concentration and 0.1 ml, suspension of co-immobilized enzymes (10 mg / ml) in the same buffer are added. After incubating the mixture for 1 min at room temperature, the cuvette is placed in a luminometer, add-on. Oj3 ml of luciferin is obtained until a concentration of 1 mM is obtained in the cuvette and a signal is recorded that is directly proportional to the concentration of ADP in the system. The experiments are repeated for ADP solutions with.-concentrations in the same range in which the measurements are supposed to be carried out. On the basis of the data obtained, a calibration graph of the dependence of the bioluminescence intensity 1g on the concentration of ADP is used, which is used to determine the concentration of ADP in an unknown sample. For the resulting preparation of co-immobilized enzymes, it is directly proportional to the dependence-Q capacity is observed in the range of the concentration of ADP 10 pM - 0.10 1. The limit of detection of ADP for this enzyme preparation is 10 mM.

Definition of AMF,

5 To 0.6 ml of 0.1 M Tris-acetate buffer pH 7.8, located in a cuvette, and containing 2 mM EDTA, 10 mM MgSO4, 20 mM potassium acetate and 1 mM CTP, add 50 µl of a solution of AMP known

concentrations and 0 ml of suspension of immobilized enzymes (10 mg / ml) in the same buffer. The mixture was incubated for 30 minutes while stirring, after which 50 µl of 0.1 M phosphoenol-5 pyruvate was added. The cuvette is placed in the phosphor and after 1 min, 0.2 ml of luciferin (its concentration in the cuvette is 1 mM) are introduced and the radiation is recorded. Since the intensity of bioluminescence is proportional to the concentration of AMP in the system, using a known concentration of AMP in the range in which measurements are supposed to be carried out, a calibration graph is constructed. An unknown 15 concentration of AMP is found from the bioluminescence signal using a calibration direct. For this drug, co-immobilized enzymes are directly proportional to the dependence on AMP 20 observed in the range of concentrations of g nM - 0.10 mM. The detection limit for AMP for this enzyme preparation is 1 nM.

Example 2. Preparation of co-immobilized enzyme preparation.

The preparation is also prepared as in Example 1, but during immobilization, 3 mg of pyruvate kinase are used at the same concentrations of all other substances. The resulting preparation possesses a lymphasis activity of 55,000 mV / mg of carrier and a pyruvate kinase activity of 8 U / mg of carrier.

The determination of ATP with the obtained pharmaceutical preparation was also carried out as in Example 1. The range of determined ATP concentrations and the limit of ATP detection are the same as in Example 1.

The definition of ADP is carried out as in Example 1. Sensitivity. analysis does not depend on the concentration used in determining the suspension. / lapazone ATP concentrations determined from this preparation of co-immobilized enzymes are 50 pM - 0.1 mM. 50 pM detection limit

The determination of AMP is also carried out, Q

ak in example 1. The range of determined concentrations of AMP 1.0 nM - 0.1 mM. The detection limit for AMP is 1.0 nM.

Example 3. Preparation of pre-enzyme co-immobilized enzymes.

The preparation is also obtained as in the rimer 1, but during immobilization 12 mg of pyruvate kinase are used at all other oval concentrations and

CONDITIONS, The resulting preparation has a luciferase activity of 35,000 mV / mg carrier and pyruvate kinase activity - 25 U / mg carrier.

Determination of ATP with the obtained multienzyme preparation is carried out as in Example 1. The bioluminescence signal is proportional to the ATP concentration in the range of 50 pM - 0.1 mM. ATP detection limit is 50 pM.

The definition of ADP is carried out as in Example 1. The range of concentrations of ADP determined with this multienzyme preparation is 50 pM -0.1 mM. Detection limit of ADP 50 pM.

The determination of AMP is carried out as in Example 1. The range of AMP concentrations determined with this enzyme preparation is 5 nM - .0.1 mM. Detection limit of AMF 5 nM

Example 4. Obtaining drug coimmobilized enzymes.

The preparation is also prepared as in Example 1, but during immobilization 100 mg of bromo cyanactivated sepharose is used. In the resulting preparation, the luciferase activity is 25,000 mV / mg of carrier and the activity of pyruvate kinase 9 U / mg of carrier.

Determination of ATP with the obtained multienzyme preparation is carried out as in Example 1. The bioluminescence signal is proportional to the ATP concentration in the range of 0.1 nM-0.1 mM. The detection limit of ATP is -0.1 nM.

The determination of ADP is carried out as in Example 1, the range of concentrations of ADP determined with this preparation is 0.1 nM - 0.1 mM, the detection limit of ADP is 0.1 nM.

The determination of AMP is carried out as in Example 1. The range of AMP concentrations determined with this multienzyme preparation is 5 nM-0.1 mM, the detection limit of AMP is 5 nM.

Using the obtained preparations co-immobilized on bromo cyanactized sepharose luciferase, pyruvatkinase and adenylate kinase, it is possible to determine both the sum of all adenine nucleotides in the sample and the content of each of them separately (the latter is real, provided that ATP is separated from AMP and ADP) ,

The best multienzyme preparations that allow to determine all

71

Adenine nucleotides with high sensitivity up to concentrations of 10 pM are preparations in which pyruvate kinase was used in the concentration range from 3 to 12 mg / ml and carrier bromine-activated sepharose in the concentration range 50-100 when immobilizing 50 mg / ml of light bulbs. mg / ml Preparations obtained with lower and higher concentrations of pyruvate kinase and carrier than those indicated in the range are less sensitive and can be used to determine adenine nucleotides with concentrations not lower than 1 nM.

The invention provides an increase in the sensitivity of the method for determining ATP and ADP hundreds of times in comparison with known methods. With the use of the resulting drug-mobilized enzymes, the method of determination of simpletin becomes easier. By reducing the number of operations, the efficiency of the process is increased; its cost is reduced due to the use of untreated enzymes, the possibility of multiple use of a single preparation and the increase in stability of the enzymes after their immobilization. The resulting preparation can be used for scientific purposes to study the metabolism of adenine nuclei. Editor I. Seglnik Order 2394/23

Compiled by L. Borisova

Tehred M. Khodanich Proofreader L. Patay

Circulation 499 Subscription VNIIPI USSR State Committee

for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5

Production and printing company, Uzhgorod, st. Project, 4

7027 .8

Leotides of various biological objects, as well as for determining the activity of ATP, ADP and AMP-dependent enzymes. It can also be used for medical diagnostics, in the food industry and microbiological industry for monitoring the level of adenine nucleotides.

Claims (1)

Invention Formula A method for quantitative determination of adenine nucleotides, involving enzymatic reactions using luciferase, adenylate kinase bulbs, Luciola mingrelica and pyruvate kinase, followed by registration of products by the bioluminescent method, about the fact that in order to reduce the detection limit of adenine nucleotides and reduce consumption enzymes, for carrying out enzymatic reactions using crude luciferase and adenylate kinase of bright light bulbs and coarsely purified pyruvate kinase from cr Olix co-immobilized on bromine cyanasepharose with a mass ratio of light bulbs: pyruvate kinase: bromine sepharose 50: (3-12): (50-100).