Method of identifying pyrivate in biological objects

专利摘要:
This invention relates to bio-engineering: and biotechnology, and provides increased stability and reliability of determination results by preventing precipitation and cloud formation. The method consists in that the composition of the reagent for determining the target product includes pyruvate oxidase, characterized with an activity optimum at pH 5.7, an optimum stability at pH 5.6-5.8, a Michaelis constant for pyruvate at 25 C 0.4 mmol / L, for phosphate 2.3 mmol / L, mol.m. 146,000 daltons, when measured in a centrifuge, are derived from strains of the acid-laden bacteria Cacobacillus DSM 2671, Lbc. salivari- U8 DSM 2573, Leuconostoc mesentero- ideB DSM 2572 and / or Streptococcus creraoris DSM 2574. 4 tab.
公开号:SU1322984A3
申请号:SU843706215
申请日:1984-02-24
公开日:1987-07-07
发明作者:Элстнер Эрих；Шлейфер Карл-Хайнц；Гец Фридрих；Зедевиц Барбара；Редер Альберт；Меллеринг Ханс；Зайдель Ханс
申请人:Берингер Маннхайм Гмбх (Фирма)；
IPC主号:

专利说明:

113
This invention relates to biochemistry and biotechnology, and relates to the production of an enzyme suitable for the quantitative determination of pyruvate and pyruvate-forming enzymes.
The purpose of the invention is to increase the stability and reliability of the determination results by preventing the formation of precipitation and cloudiness.
The method for determining pyruvate in biological samples is that pyruvate oxidizing enzyme is included in the reagent for the determination of pyruvate, which is active without the addition of flavin-adrened dinucleotide (FAD), thiamine pyrophosphate (TPP) and bivalent metal ions. Pyruvate oxidase is optimally active at pH 5.7, optimally stable at pH 5.6-5.8 and, most importantly, with the use of the enzyme, the Michaelis constant is 0.4 mmol / l for pyruvate and 2, 3 mmol / l for phosphate mol.m. the enzyme is 146,000 daltons, which is the same as the well-known enzyme (190,000).
The resulting enzyme is more specific to the substrate than the known one.
Characteristics of specificity are presented in table 1.
The effect of inhibitors on the activity of the proposed enzyme used to determine pyruvate is shown in Table 2.
The inhibitory effect of EDTA on the proposed pyruvate oxidase is 1000 times weaker than in the case of the known enzyme, and after several days of dialysis of the enzyme, the addition of TPP FAD and Mp increases by 5-15Z, while the known enzyme is inactive without these activators.
Lactic acid bacteria Lactobacillus plantarum DSM 2571, Lbc strains are used as an enzyme source. Salivarius DSM 2573, Leucososococ mesenteroides DSM 2572 and / or Streptococcus cremoris DSM 2574, stored in the National Collection of Microorganisms.
Enrichment and purification of the enzyme is carried out by conventional biochemical methods. You can achieve 20–30-fold enrichment to a specific activity of 6–9 U / mg protein. The resulting pyruvate oxidase is included in the reagent for the determination of pyruvate in the analyzed samples.
42
The preferred composition of the reagent is 1-50 units / nl pyruvate oxidase, 10-50 mmol / l phosphate, buffer pH 6-8 (any suitable buffering agent buffering in the indicated pH regions in which the enzyme is active and stable). Phosphate buffer and 0.22 U / ml peroxidase (ROD) are well suited. Reagent use
the proposed enzyme provides a higher capacity for storage.
Isolation of pyruvate oxidase (Py-OD) from Lactobacillusplantarum DSM 2571
performed as follows.
1. Cultivation. The culture medium contains, g / l of water: bacto-tryptone-oxoid Yu; yeast extract oxoid 4; x 3 2;
diammonium hydrocitrate 2; tween-80 (polyoxyethylene sorbitan monooleate) 1 ml; MgSO X 7 H, 0 0.2; MnSO x X 0.2; molybdenum sulfide 0,2; lactose 10; pyruvic acid
3.2; pH 7.
150 ml of medium of the indicated composition or the bacteria’s lactic acid, the usual MRS medium are inoculated with cultures by injection and shaken at
28 ° C. After growth
150 ml of this medium are inoculated with 15% of the purified culture and shaken in a 500 ml Erlenmeyer flask at 28 ° C until the completion of the late
lag-phase.
The MRS medium is as follows, g / l: meat extract (MERCK) 2; casein peptone 10; trypish yeast extract 4; glucose 20; tween-80 1 ml; x3 2.5; Na-acetate x 3 5; diammonium citrate 2; MgSO x 7 200 mg; MnSO + X 50 mg. I
2. Distribution. From 50 l of Lactobacillus DSM 2571 culture fluid with 120 units / l of pyruvate oxidase (Py-OD), 300 g of wet mass is collected by centrifugation. 100 g of cells are placed in a glass dinomerant,
after addition of 4% polyethylenimine, centrifuged and the supernatant is processed by fractionation with ammonium sulfate (25-65%) at pH 6.3,
by chromatography using dextran-blaspharosepharose (firm Farmacia), second fractionation with ammonium sulfate at 20-30% (saturation) and by passing through a Sephadex ASA mopeturate sieve (firm LKB
Data enrichment are presented in Table. 3,
The purified enzyme has a specific activity of 5.8 units / mg and contains less than 0.037, Apo-glutamate-pyruvate-transaminase about ketoglutaratoxidase, and also less than 0.002% NADH oxidase.
Pyruvate oxidase activity is determined under the following conditions: + P. + - ILO
Oj Py - OD acetyl-P +
COj -t g 2
The nina contains .200 mmol / l alanine sulfinic acid, under the same conditions as in Example 1, a GOT-containing sample is added. From the change in extinction, the GOT content is calculated for 1 minute.
EXAMPLE 3. Test strips for GPT.
To prepare the GPT 10 test strips, two papers are impregnated.
Enzyme paper: 0.5 mol / l morpholynes tansulfonic acid (potassium hydroxide, pH 6.5 (MORS buffer), 800 mmol / l alanine, 1 mmol / l potassium hydrogen phosphate, 20 000 units / l P0D, 200000 u / l pyruvate oxidase.
With this solution, absorbing paper 50 microns thick is used, with a surface density of
HjO h-4-aminoantipyrine (4-AA) + + 2 hydroxy-3,5-dichlorobenzene sulfonic acid (HDBS) POP quinone dye (546 im) - 2.
. consumed by equimolar co-20 12 g / m, absorptive capacity
50 g of HjO / M, and dried for 5 minutes at (for example, Teebeutelpapier sieve paper from Scholler and Hosch). The paper is then 25 cut into strips with a width of 1 cm.
Indicator paper: 10 mmol / l 4.5- (4-dimethylamino-phenyl) -2- (3,5-dimethoxy-4-hydroxy-11Il) imidazole and 18 mmol / l "-ketoglutaric acid by adding 0 , 05 μmolzo you are dissolved in 100 mmol / l HCl (bupyruvate, respectively, containing the ferric system), pyruvate samples, in a 2 ml cuvette
at a layer thickness of 1 cm and a temperature using this solution, also jj ° (the appropriate adsorbate is fed. After 15 minutes, stopping - 35 paper is fed, dried for an extinction coefficient at 546 nm min. at 30 C and cut into
H, 0, the amount of dye.
1 unit Py-OD 1 µmol conversion of pyruvate in 1 min at 25 C.
Pyruvate determination: 72 mmol potassium phosphate buffer, pH 6.7; 8 mmol 4-AA; 6.8 mmol / l HDBS; 0.01 mmol / l FAD; 2 items POD; 10 units, Py-OD per 1 ml. Start for a sequence to a target value of 1 cm in width. Transparent polycarbonate film with a thickness of 10 mmk together with indicator paper and enzyme Q paper is applied on one side to plastic strips (tapes), strengthened so that the film is on the outside, indicator paper in middle, enzyme paper inside. Alone
6 16.5 cm (µmol measured for pure pyruvate - monosodium salt).
Example 1. Kinetic determination of pyruvate (GPT).
To 2.5 ml of a solution containing mmol / l: KN, PO (pH 6.7) 80; alanine
1 cm wide. Transparent polycarbonate film 10 mm thick with indicator paper and enzyme Q paper is applied on one side to plastic strips (tapes), reinforced so that the film fits outside, indicator paper in the middle, enzyme paper inside . Alone
t-ti-if j l tj r-j l "iVii-A L1 f / v.v / njictnnn
800; N- (3-sulfonyl-4-hydroxy-5-chloro-5 is applied by them with a 15 mm web width of phenyl) -4-aminoantipyrine 0.2; l -ke-glass fiber, 1.5 mm thick, with toglutaric acid 18, and pyruvate oxidase 0.2 u / ml; peroxidase 2 u / ml, add 0.1 ml of the sample. After 3 minutes, the extinction change in 1 minute was determined. The measurement temperature is 25 ° C, the wavelength is 546 nm, it is 19.6, the total volume is 2.6 ml and the sample volume is 0.1 ml. From the increase in extinction in 1 min, the activity on GPT is calculated.
PRI mme R 2. Kinetic definition of GOT. In the initial mixture, similar to example 1, which, instead of the Al-thick fibers, is about 2 microns, so that the free end of the film and the impregnated paper protrude another 6 mm
50 over the bat (vatochnym canvas). Then everything is cut into test strips 6 mm wide. If 15 µl of whole blood is applied to the sample site, then within 45 seconds of plasma
 penetrates the glass fiber batt under the transparent film, while the red blood cells are held in place. After pressing the film enzyme and indicator paper
The nina contains .200 mmol / l alanine sulfinic acid, under the same conditions as in Example 1, a GOT-containing sample is added. From the extinction change, the GOT content is calculated for 1 min.
EXAMPLE 3. Test strips for GPT.
To prepare the GPT test strips, two papers are impregnated.
Enzyme paper: 0.5 mol / l morpholynes tansulfonic acid (potassium hydroxide, pH 6.5 (MORS buffer), 800 mmol / l alanine, 1 mmol / l potassium hydrogen phosphate; 20,000 units / l P0D; 200000 units / l pyruvate oxidase.
With this solution, absorbing paper 50 microns thick is used, with a surface density of
12 g / m, absorption capacity
 the paper is dried for 30 minutes and cut into strips
1 cm wide. A transparent polycarbonate film with a thickness of 10 mmc together with indicator paper and enzyme paper is applied on one side to plastic strips (ribbons), fixed so that the film is on the outside, the indicator paper in the middle, the enzyme paper inside. Alone
 they are coated with a 15 mm wide fiberglass web, 1.5 mm thick, and applied with a 15 mm wide fiberglass web, 1.5 mm thick, with a fiber thickness of about 2 microns, so that the free end of the film and the impregnated paper protrude by 6 mm
over the bat (vatochnym canvas). Then everything is cut into test strips 6 mm wide. If 15 µl of whole blood is applied to the sample site, then within 45 seconds of plasma
penetrates the glass fiber batt under the transparent film, while the red blood cells are held in place. After pressing the film enzyme and indicator paper
They come in contact with the released plasma and are evenly moistened. The plasma content of pyruvate (GPT) reacts with blue staining, the intensity of which is proportional to the amount (measurement) of GPT and, under certain conditions, can be measured in a remission photometer. Other tested materials, such as serum, plasma, etc., react in the same way.
PRI me R 4. Test strips on GOT
The COT test strips are prepared in the same way as the ORT strips according to Example 3, only in the enzyme paper, instead of alanine, 200 mmol of apanesulfonic acid is contained. The blue color obtained after pressing the transparent film is a measure of the amount of GOT in the sample,
Example 5: Comparison of measurement results in the application of the proposed method and the reagents used after storage are known.
For comparison, apply:
the reagent according to example 3;
The reagent according to Example 3, but instead of 200,000 units / l of the proposed pyruvate oxidase, the paper is impregnated with 200000 units / l of known pyruvate oxidase, as well as an additional 1 mmol / l pyaminepiphosphate (TPP) and 0.5 mmol / l
Using these reagents, 3 samples with pyruvate are examined before and after storage, which show different ORT activity. The measurement is carried out according to example 3,
The test results are presented in table 4.
From Table 4 it follows that, according to the proposed method, even after storage of the reagent for determining pyruvate, the value of the measured values does not significantly deviate from the base value, while according to a known method, the measurement results after storage for 3 days at 60 ° C deviate from baseline values up to 60%.
Similar results are obtained if, instead of the reagent for determining pyruvate according to example 3, the reagent according to example 1 is used, which is lyophilized before storage.

29846
Thus, the proposed method for the determination of pyruvate in biological objects ensures the reliability of the results. The method is suitable
and to determine substrates and
which leads to the formation of pyruvate,
ten

权利要求:
Claims (1)
[1]
Invention Formula
0
A method for determining pyruvate in biological objects, which involves the interaction of the test sample with a reagent containing pyruvate-5-dase, buffer, peroxidase, 4-aminoantipyran, and sulphonic acid, a subsequent analysis of its amount by recording the resulting hydrogen peroxide, characterized by
That is to increase the stability and reliability of the results of the determination by preventing the formation of precipitation and cloudiness, pyruvate oxidase is used, isolated from the lactic acid bacteria of Lactoba-cillua plantarum DSM 2571, or Lbc strains. saliyarius DSM 2573, or Leuconosboc nleeenteroides DSM 2572, or Streptococcus secretion 18 DSM 2574, characterized by optimum activity at pH 5.7, optimum stability at pH 5.6-5.8, Michaelis constant for pyruvate at 25 ° C 0.4 mm / year l, dp phosphate 2.3 mmol / l, mol.m., 146000
5 daltons when determining in a centrifuge
another
Spreadsheets
by
one
40
Specificity to the substrate,%
Inhibitor
Without inhibitor
KCN
NaN, EDTA
Dissolution - Supernatant
Ammonium sulfate fractionation
dextranblau-sepharose
Ammonium sulfate fractionation
ACA E4-mol-sieve
Enzyme activity,%
proposed
ABOUT
ten
100
100
, 01
,one
one
ten
100
85
ABOUT
75
78
thirty
7
ABOUT
100
75
five
62
100
100
98
85
Table 3
67000,37100
55400,4497
25600,8991
7531.030
1025,823,5
27 27.5 29 12
60 62 59 28
212 209 211 81
Editor N. Tupitsa
Compiled by I.Privalova
Tehred M.Morgental Proofreader A.T.
Order 2883/59 Circulation 499 Subscription
BH№fflH USSR State Committee
Inventions n discoveries 113035, Moscow, Zh-35, Raushsk nab., d.4 / 5
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Production and printing company, Uzhgorod, Projecto st., A

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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DE19833306719|DE3306719A1|1983-02-25|1983-02-25|PYRUVATOXIDASE|

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