SU1497218A1 - Method of determining activity of glycogenphosphorylase - Google Patents

Abstract

This invention relates to biochemical analysis and can be used to determine serum glycogen phosphorylase. The aim of the invention is to increase the detection sensitivity, shorten the analysis time, extend the range of detectable activities and simplify the process. The invention consists in using a four-enzyme system: phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH ₂ : FMN-oxidoreductase, bacterial luciferase, co-mobilized on BRCN-agarose at PH 7.5 - 8.0, and the registration process is carried out by a luminescent method initial reaction catalyzed by glycogen phosphorylase. 1 tab.

Description

The invention relates to a biochemical analysis, in particular to a method for determining the activity of glycogen phosphorylase, and can be used to determine serum glycogen phosphorylase.

The purpose of the invention is to increase the detection sensitivity, reduce the time of analysis, expand the range of measured activities, simplify the process.

The invention consists in using a four enzyme system, including: phosphoglucomutase. glucose-6-phosphate dehydrogenase, FMN-oxidoreductase and bacterial

luciferase, which are pre-co-mobilized on BrCN agarose.

Co-mobilization of enzymes is carried out by incubating a buffer solution with a pH of 7.5-8.0, containing phosphoglucomutase (20-200 U / mp), glucose-6-phosphate dehydrogenase (10-100 U / ml), NADH: FMN-oxidoreductase (2-20 U / ml) and luciferase (20-200 U / ml) with a suspension of BrCN-agarose.

The indicated range of enzyme activities used for analysis is a necessary condition for achieving a high sensitivity of the analysis.

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Each value of the activities of any of the four enzymes corresponds to certain optimal values of the activities of the three other enzymes that lie strictly within the specified ranges. The use of enzymes with activities lying outside the specified ranges leads to a decrease in the sensitivity of the analysis, since the dependence of the activity of immobilized enzymes on their initial activities has a bell-like character.

For the analysis, an extract of luminous bacteria can be used, obtained by fractionating extracts of bacteria Beneckea harveyi with ammonium sulfate from 50 to 100% saturation,

The proposed method allows the determination of glycogen phosphorylase in the range of activities from 0.01 to 10 U, i.e. the detection limit is reduced by five times in comparison with the known and the range of measurable activities necessary for the purposes of medical diagnostics is observed. Achieved sensitivity increase

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1 mg / mp sodium azide, a suspension is obtained containing the carrier 100 mg / mp, which is stored at. For long-term storage, the suspension is lyophilized in portions of O, 5 ml. The specific activity of the obtained preparations is 40 U / mp agarose.

Example 2 Immobilization was carried out as in Example 1 for. except that the enzyme solution contains 200 U / ml phosphoglucomutase. 100 U / mp glucose-6-phosphate dehydrogenase, 20 E / MJI NADA: FMN oxidoreductase and 200 U / ml bacterial luciferase. The specific activity of the obtained preparations is 30 U / ml agarose.

Example 3 Immobilization was carried out as in Example 1 with the exception that the enzyme solution contains 20 U / mp phosphoglucomutase, 10 U / C glucose-6-phosphate dehydrogenase, 2 U / ml NLDN: FMN-α-oxidoreductase and 20 U / ml of bacterial luciferase. The specific activity of the preparations obtained is 20 U / mp agarose.

Example 4 Immobilization of Pro

analysis is associated, apparently, with an activ- ity 30 t in the same way as in example 1 for

a multienzyme system in a co-mobilized state.

Example 1, Preparation of a co-immunosized four-enzyme system.

Lyophilized BrCN-arapO3y is soaked in distilled water for 3 hours, then washed with 1 mM HCl and O, 1 M Na-phosphate buffer solution, pH 7.8. In a 0.1 M Na-phosphate buffer solution, pH 7.8, an enzyme solution is prepared with a total concentration of .A protein 4-8 mg / ml, which contains 100 U / ml phosphoglucomutase, 50 U / ml glucose- 6-phosphate dehydrogenase, 10 U / ml NADH: FMN oxidoreductase and 100 U / ml bacterial luciferase. BrCN-agarose is added to the resulting solution at a rate of 100 ml / ml, and the resulting suspension is shaken on a shaker for 20 hours and 4 C. The carrier is then washed with a 0.1 M Na-phosphate buffer solution, pH 7.0, containing 0.5 M NaCl, until the enzyme activity disappears in the washing waters. The carrier with the immobilized enzymes is suspended in a 0.5 M tris-acetate buffer solution, rP 6.0, containing

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except that the enzyme solution was prepared in a 0.1 M Na-phosphate buffer solution, pH 7.57, the specific activity of the resulting preparations was 30 U / ml agarose.

Example 5 Immobilization was carried out in the same manner as in Example 1 by determining that the enzyme solution was prepared in 0.1 M Na-phosphate buffer solution, pH 7.0. The specific activity of the resulting preparations was 25 U / ml agarose.

Example 6, Determination of glucogen phosphorylase activity.

When determining the activity of glycogen phosphorylase, the following were successively introduced into the cuvette of the photoluminescence: 1 ml of a 0.05 M Tris-acetate buffer solution, pH 6.8-7.0, 0.05 ml of a glycogen phosphorylase solution, 0.05 ml of a solution, containing 0.06 M magnesium acetate, 30 mg / ml glycogen, 0.15 mM glucose-1,6-diphosphate. The mixture is incubated for 8-12 minutes at. Then, 0.05 ml of the suspension of the co-immobilized four enzyme system prepared as indicated in Example 1, 0.1 ml of 0.3 mM decanal solution in 2% solution are added to the cuvette.

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n-propanol, 0.05 ml of a 30 mM solution of NAD in water, and background luminescence is recorded. 0.1 ml of a solution of 0.15 M phosphate is added and the mixture is incubated for 1 minute. Then, for 2 min, an increase in the intensity of the luminescence with constant stirring is recorded. For the measure of glycogen phosphorylase activity, take the tangent of the angle of inclination obtained by direct (V / min).

This sequence of operations during the analysis, namely the separate addition of glycogen phosphorylase substrates (first glycogen and then after the incubation of inorganic phosphate), is necessary to standardize the conditions for recording the glow. Otherwise, no reproducibility of the experimental data is observed.

The conversion of glycogen phosphorylase activity into international units (µM / min) is carried out according to a calibration chart of the dependence of the luminescence on the concentration of C 1 and glucose-1-phosphate (the product of glycogen phosphorylase reaction).

The calibration graph is direct in the range of glucose-1-phosphate concentrations from 1 nM to 10 µM. Calibration direct is processed by the method of least squares. Get the equation

at 1.00 x - 8.20,

where y is Ig I (luminescence intensity, B);

X - Ig C (glucose-1-phosphate intensity, M).

The calculation of glycogen phosphorylase activity in ME (µM / min) is carried out according to the formula

A k A,

where a is the activity of glycogen phosphorylase, ME;

A - glycogen phosphorylase activity. V / min;

. , o --- i -,

where V is the volume of the reaction mixture,

ml (1.5);

V is the volume of glycogen phosphorylase solution, ml (0.05).

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The activity of the prepared solutions of glycogen-phosphorus are determined. The results are reported on.

Introduced glycogenphospho-signal, rilase, memv / mit)

0.01 0.05 0.10 0.25 0.50 1, 00 5.00 10.00

3.0 5.3 7.5 18 35

62.5 307 613

Poluche1P {yy dan ple processed by the method of least squares. Semi

chile equation: y 61, OAx + 2, A4, where y is the signal (mV / sn); x is the amount of glycogen phosphorylase (HE) entered.

Oylbka definition does not exceed 15%.

Example 7. Determination of glycogen phosphorylase activity B.

The determination of glycogen phosphorylase B activity is carried out in the same way as

and the determination of glycogen phosphorylase activity, due to the claim that the solution containing glycogen, magnesium acetate and glucose-1,6-β-diphosphate, contains another 0.03 M AMP.

A co-immobilized system was prepared as indicated in Example 1. “0.3 U of glycogen phosphorylase was introduced. 0.28E gilcogen phosphorylase was determined. Error determining 10%.

Example 8. Determination of serum glycogen phosphorylase activity.

Serum is passed through a column filled with starch. Starch (20 g) is suspended in 40 ml

0.02 M tris-acetate buffer solution containing 1 mM dithiothreitol and allowed to swell for 2 hours at 4 ° C. 2 ml of a suspension of starch is added to a column (1x5 cm). Then pipette 1 ml of serum. After applying the blood to the column, 2 ml of 0.02 M tris-acetate buffer solution, pH

, then glycogen phosphorus is eluted with 1 ml of 0.05 M tris-acetate buffer solution, pH 7.6, containing 10 mg / ml glycogen and 0.05 mM EDMA at 20 s.

Method for determining tlicogensphosphorylase activity, involving the use of a four-enzyme system containing phosphoglucomut 10

A new batch of starch gel is used to clean up the next normal serum.

The activity of tlicogene phosphorylase in the eluate is determined as described in Example 2, but instead of 1 ml of Tris-acetate buffer solution and 0.05 ml of enzyme solution, 0.5 ml of the eluate from the column and 0.5 ml of Tris-acetate solution are used. , pH 7.0, Used four-ferment-co-immobilized system prepared, kick is indicated in Example 1 o The calculation of glycogen phosphorylase activity in NOT is carried out in the same way as in Example 6, except that the serum volume is 0.5 ml.

Invention Formula | 20

1497218 8

Memory, glucose-6-phosphate dehydrogenase, oxidoreductase and bacterial luciferase, followed by evaluation of bioluminescence results, characterized in that, to increase the sensitivity of certain glycogen phosphorylase, reduce analysis time, extend the range of measured activities, and simplify the process, use enzymes co-immobilized on BrCN-agarose at pH 7.5-8.0, at a phosphogluco-mutase content of 20-200 U / mp, glucose -6-phosphate dehydrogenase 10-100 U / ml, NADH: FMN-oxidoreductase 2-20 U // ml and bacterial luciferase 20 - 200 E / n, the determination is carried out by sequential addition to the analysis of the sample lizipyemo ry glycogen suspension soimmobilizovannyh enzymes and their substrates, and then an inorganic phosphate, and the result was evaluated by the initial rate of reaction catalyzed glIkogenfosfori- lazoy,

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Claims (1)

The claims; 20 A method for determining the activity of glycogeyphosphorylase, involving the use of a four-enzyme system containing phosphoglucomtase, glucose-6-phosphate dehydrogenase, oxidoreductase and bacterial luciferase, followed by evaluation of the results of bioluminescence, characterized in that, in order to increase the sensitivity of the determination of the reduction of the expansion time analysis range, activities and simplification of the process, use enzymes co-immobilized on BrCN agarose at a pH of 7.5-8.0, with a content of f osfoglucomutase 20-200 U / ml, glucose-6-phosphate dehydrogenase 10-100 U / ml, NADH ^: FMN oxidoreductases 2-20 U / ml and bacterial luciferase 20 200 U / mp, determination is carried out by sequential addition to the analyzed sample glycogen suspension of co-immobilized enzymes and their substrates and then inorganic phosphate, and the result is evaluated by the initial reaction rate catalyzed by glycogen phosphorylase.