RU2694964C2 - Method of measuring catalytic activity (catalytic concentration) of enzymes using methods of mass spectrometry and spectrophotometry with preliminary identification of target analyte and determination of purity of substances - Google Patents

Abstract

FIELD: measurement.

SUBSTANCE: invention relates to clinical diagnostics and biochemistry in terms of creating methods which enable to measure catalytic activity of substances and can be used to identify and determine parameters of catalytic activity of previously unknown biologically active substances. Method of measuring catalytic concentration of enzymes by spectrophotometric measurement of optical density of a product of an enzymatic reaction involves using a tandem quadrupole mass spectrometer with a time-of-flight detector and high-performance liquid chromatograph with spectrophotometric detector, which provide identification of analyte and possibility of measuring catalytic activity of a priori unknown enzyme.

EFFECT: technical result is wider number of analyzed enzymes, high accuracy of measuring previously unknown enzymes, reliable identification and determination of purity of analytes.

1 cl, 1 dwg

Description

The technical field to which the invention relates.

The invention relates to the field of clinical diagnosis and biochemistry, in terms of creating methods to measure the catalytic activity of substances and can be used to identify and determine the parameters of the catalytic activity of previously unknown biologically active substances.

The level of technology

To solve various diagnostic problems, it is necessary to quantify the activity of enzymes in units of catalytic concentration “cata”.

There are a significant number of patented methods and devices for determining the catalytic activity of substances [1-6]. Known methods for determining the activity of enzymes approved by the International Federation of Clinical Chemistry (IFCC). According to the provisions of the method [1], the measurement of α-amylase activity consists in the spectrophotometric measurement of the accumulation of the colored reaction product. The rate of change in optical density is directly proportional to the activity of total α-amylase. 4,6-ethylidene- (G7) -p-nitrophenil- (G1) -α, D-maltoheptaoside (4,6-ethylidene- (G7) -p-nitrophenyl- (G1) -α, D- maltoheptazide; Et-G7PNP). During the reaction, α-amylase cleaves Et-G7PNP to G2PNP, G3PNP, G4PNP, Et-G3, Et-G4 and Et-G5. After that, α-glucosidase hydrolyzes G2PNP, G3PNP and G4PNP to form glucose and the colored substance p-nitrophenol [7]. The closest in technical essence is a method for measuring catalytic activity, given in [1] (hereinafter - the prototype).

The direct optical methods mentioned in the analogues of the invention have limitations associated with the possibility of measuring the catalytic concentration of samples containing a priori known enzymes in a narrow range of the analyzed substances.

This technical problem could not be solved by implementing or using the prototype of the invention in connection with:

- lack of identification of unknown compounds;

- the lack of determination of the purity of the target analyte - enzyme.

Using the invention, the technical problem of identifying unknown compounds is solved using a database of high-resolution mass spectra, followed by determining the purity of the target analyte and measuring its catalytic activity.

Disclosure of the invention

The invention aims to create a method for metrological assurance of measurements of catalytic activity / concentration of "unknown" analytes, to eliminate the restriction in measuring the characteristics of only known enzymes, expanding the possibilities of measuring the characteristics of unknown substances, sharing both methods.

When using the proposed method for patenting are achieved:

- the ability to measure the catalytic activity / concentration of an a priori unknown compound due to its identification, determination of its purity, which allows choosing the optimal conditions for the reaction, determining the reaction product, necessary coenzymes, cofactors;

- improving the accuracy of measurements of unknown compounds (achieved through preliminary identification and determination of the analyte purity).

Thus, the inventive method allows metrologically to provide an analysis of the catalytic concentration of unknown samples. Unknown samples can be solutions, biological fluids, pharmaceutical substances.

Essential features of the invention:

1. Identification of biologically active substances is carried out by a mass spectrometric method using a database of mass spectra.

2. The calculation of the purity of the target analyte is carried out by subtracting from the total mass of the substance the mass of impurities measured by the mass spectrometer.

3. Achievement of the following metrological characteristics of the method:

- measurement range of the catalytic activity (catalytic concentration) of biological substances, (from 7.8⋅10 ^-7 to 9.4⋅10 ^-6 ) cat / dm ³ ,

- extended uncertainty of measurements of catalytic activity (k = 2), (from 1.5 to 15)%.

The technical result of the invention is to increase the number of analyzed enzymes; achieving the measurement accuracy of “unknown” enzymes, guaranteed by IFCC reference methods for a priori known ones; ensuring reliable identification and determination of analyte purity.

This technical result for a priori known identified enzymes is achieved by performing all the procedures prescribed in the measurement procedures. For known and "unknown" substances using a mass spectrometer determine the purity of the starting materials. For "unknown" substances carry out (if necessary) identification of "unknown" analyzed enzyme, determine the quantitative content of the analyte in the sample. High performance liquid chromatograph is used to separate the components of the sample, identify and characterize the analyte. The spectrophotometer allows you to determine the optical density of the substrate or reaction product in the kinetic mode. The measurement of the catalytic activity (catalytic concentration) of substances, the substrates or reaction products of which have absorption in the UV-visible part of the spectrum, is carried out spectrophotometrically.

The implementation of the invention

The invention is as follows:

1. Identification and measurement of the concentration of the “unknown” enzyme

1.1. Carry out the identification of the analyzed enzyme. Prepare an analyte solution in an appropriate solvent. If it is necessary to analyze solid samples, transfer the sample to the solution. A sample is introduced through HPLC into a mass spectrometer, identification is performed and the chemical structure of the substance to be certified is determined by the methods:

- mass spectrometry with electron ionization and identification of mass spectra using electronic libraries,

- quadrupole mass spectrometry with QTOF time-of-flight detector for substances analyzed by HPLC / UV and HPLC / MS / MS.

1.2. Measure the concentration of enzyme in the sample. If necessary, conduct sample preparation, freeing the sample from most impurities.

1.3. Establish certified value of the mass fraction of pure compounds used to measure the catalytic activity of enzymes.

1.3.1. Pre-confirm the chemical structure of the substance being certified by the methods:

- mass spectrometry with electron ionization and identification of mass spectra using electronic libraries,

quadrupole mass spectrometry with QTOF time-of-flight detector for substances analyzed by HPLC / UV and HPLC / MS / MS.

1.3.2. Measure the mass fraction of all impurities by appropriate methods. The mass fraction of the main component is calculated as 100% minus impurities by the formula (1).

where w _OK - the mass fraction of the main component in the measured sample,%;

w _pr - mass fraction of impurities,%.

Among the considered impurities include all possible to find related impurities, reaction inhibitors.

1.4. Determination of a specific system (set of reagents) for the implementation of a specific catalytic reaction with this enzyme:

- determination of the optimal conditions for the reaction - temperature, pH, the presence of coenzymes, stabilizers;

- preparation of an appropriate substrate for the selected catalyst;

- determination of substrate purity (clause 1.1).

1.5. Determination of substrate concentration by HPLC / UV or HPLC / MS / MS.

1.6. Preparation of solutions of reagents, including coenzymes and stabilizers of the catalytic reaction.

1.7. Measurement of the obtained pH value of the reagent solutions and bringing it to the value required for optimal reaction (usually with an absolute error of no more than ± 0.001), with exactly measured solution temperature (37.00 ° C).

1.8. To control the correctness of the preparation of reagent solutions and to control the accuracy of the measurement procedure, a spectrophotometer is used to measure the catalytic concentration of a standard enzyme sample and check their compliance with the values given in the standard sample certificate. A positive result proves that weighed the reagents accurately, all the solutions were prepared correctly, the conditions of preparation and the reaction were observed.

1.9. Measure the rate of change in optical density blank samples (not containing the enzyme), which should not exceed 1% of the rate of change in optical density of the enzyme. This measurement is carried out in order to take into account the catalytic activity of the blank sample, since even in the absence of an enzyme the catalytic reaction can proceed (due to possible contamination of the sample during preparation of solutions or from insufficiently pure solvents, or from the effect of the matrix) at a very low speed. If this value is exceeded, sources of pollution are found and eliminated. Using the graph of the dependence of absorption on time, the rate of change in optical density is calculated by subtracting the values of the tangents of the tilt angles of the approximating direct measurement result and the blank sample (Fig. 1).

1.10. An aliquot of an enzyme sample (volume or sample) needed for analysis is selected.

1.11. For the production of a catalytic reaction, the reacting solutions necessary for a particular enzyme are combined and they withstand the time required to obtain the reaction product for a specific enzyme. The kinetics of the reaction is recorded on a spectrophotometer for a certain time [9].

1.12. The catalytic concentration of the reaction product is determined by measuring the increment of the reaction rate. The catalytic concentration of the enzyme is calculated by the formula (2):

- изменение абсорбции (c^-1) после корректировки скорости изменения оптической плотности холостого реагента;Where

- change in absorption (c ^-1 ) after adjusting the rate of change in the optical density of the blank reagent;

b _enzyme - catalytic concentration of the enzyme (катkat / dm ³ );

k - coefficient of proportionality, depending on the enzyme.

To control the correctness of the preparation of reagent solutions and control the accuracy of the measurement procedure, if a standard sample of the enzyme analyzed is present, a spectrophotometer is used to measure the catalytic concentration of a standard enzyme sample and verify its compliance with the value given in the certificate for the standard sample. In the absence of a standard sample, certified for the preparation procedure are used samples for comparison.

2. Calculation of the measurement uncertainty of the catalytic concentration of the enzyme

2.1. The average value of measurements of catalytic concentration is calculated by the formula (3):

The uncertainty of measurements of type A is calculated by the formula (4):

2.2. The measurement uncertainty of type B, when measuring the catalytic concentration of a known enzyme, generally consists of the following components:

- inaccuracy of measurements of the reaction rate in the presence of a catalyst - measurements made with a spectrophotometer,

- inaccuracy of measurement of the reaction time period,

- inaccuracy of measurements of the volume of reagents dispenser,

- inaccuracy of measurements of the volume of reagents flasks,

- inaccuracy of measurements of mass of reagents,

- inaccuracy of pH measurements of solutions,

- inaccuracy of temperature measurements of solutions,

- inaccuracy of the linear approximation of the graph of the dependence of absorption on time.

Type B uncertainty is estimated by the formula (5):

The total uncertainty is calculated by the formula (6):

Extended uncertainty with a coverage factor of k = 2 is calculated by the formula (7):

2.3. Uncertainty of type B measurements when measuring the catalytic concentration of an “unknown” enzyme generally consists of the same components as given in § 3.2, with the added uncertainty of identifying the enzyme.

Type B uncertainty in this case is estimated by the formula (8):

The total and extended uncertainties are calculated using formulas (6) and (7).

The application of the method of metrological assurance of measurements of catalytic activity / concentration of unknown enzymes with their preliminary identification and determination of the purity of substances allows to expand the number of analyzed samples of substances - enzymes in the biological matrix, makes it possible to measure the catalytic activity / concentration of a priori unknown compound due to its identification, to measure "unknown" compounds with high accuracy achieved by prior identification and leniyu analyte purity.

List of sources:

1. Gerhard Schumann et al. IFCC Primary Reference Procedures for Concentrations of Enzymes at 37 ° C Part 8. Reference procedure for the measurement of the catalytic concentration of α-amylase. Clin Chem Lab Med 2006; 44 (9): 1146-1155.

2. Reagent for determining the activity of total α-amylase: patent No. RU 2417374 C1, Russian Federation: IPC G01N 33/52 Galina Yakovleva, Ksenia A. Cheremisina, applicant and rightholder: Closed Joint-Stock Company Vector-Best.

3. Means and methods for determining the amount of neurotoxin polypeptide and its catalytic and proteolytic activities: patent number 2545783, EP: IPC G01N 33/569, G01N 33/577 Pfayl Michael, Friedrich Joseph, Applicant and copyright holder: MerzPharma GmbH / HK. KgaA.

4. Method for determining non-specific resistance of pathogenic microorganisms to antibiotics on the basis of measuring the catalytic activity of phosphodiesterases that decompose cyclic diguanosine monophosphate: patent No. 2518249, RF: IPC C12Q 1/02, C12Q 1/44, G01N 33/569, G01N 33/573, G01N 33 / 577, Kolesnikov A.V., Kozyr A.V., Shemyakin I.G., Applicant and copyright holder: FGBUN Institute of Bioorganic Chemistry. Academicians M.M. Shemyakin and Yu.A. Ovchinnikov RAS.

5. Mass Spectrometric Determination Of Blood Enzyme Activity: US Patent 2009/0305327 A1 IPC C12Q 1/02, C12Q 1/00, Jochen Franzen, Karsten Michelmann, Markus Kostrzewa, Applicant and copyright holder: Bruker Daltonik GmbH.

6. Method for Assaying Plasma Enzymes in Whole Blood: patent number US 2011/0104712 A1, IPC G01N 33/573, C12M 1/40, Myriam-LaureCubizolles, Marie-LineCosner, MagaliFaivre, PatrikPouteau, applicant and copyright holder: Commissariat a l ' Energie Atomique et aux Energies Alternatives.

7. Kruse-Jarres J.D., Kaiser C., Hafkenscheid J.C.M. et al. Evaluation of a new amylase assay using 4,6-ethylidene- (G7) -1-4-nitrophenil- (G1) -α-D-maltoheptaoside as substrate // J. Clin. Chem. Clin. Biochem. 1988. Vol. 27. P. 103-113.

8. IRMM Catalog. Certified Reference Materials 2017. https://crm.jrc.ec.europa.eu/graphics/cms_docs/rm_catalogue.pdf

9. Siekmarm L. et al. IFCC primary reference procedures for the measurement of enzymes at 37 degrees C. Part 1. Clin Chem Lab Med 2002; 40 (6): 631-4.

Claims (1)

A method for measuring the catalytic concentration of enzymes by spectrophotometric measurement of the optical density of an enzymatic reaction product, characterized by the use of a tandem quadrupole mass spectrometer with a time-of-flight detector and a high-performance liquid chromatograph with a spectrophotometric detector, providing identification of the analyte and the ability to measure the catalytic activity of a priori unknown enzyme.

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