Method of determining hexoses in supramolecular structures of cells in escherichia coli. RU patent 2510846.

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and molecular biology. Conservation of cells of Escherichia coli in presence of buffered 80-90% glycerol is performed. Cell envelopes are removed with 3% triton X-100. Cell supramolecular structures are successively extracted with increasing concentrations of salts: 0.14 M (bacterioplasm), 0.35 M (loosely linked with cell residue), 2 M NaCl (strongly linked with cell residue), and 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol (cell residue). Acid hydrolysis is carried out in said fractions. Anthrone method is carried out, with preliminary purification of anthrone preparation. Calibration graph is built and quantity of hexoses is determined by means of calculation formula.

EFFECT: invention makes it possible to determine quantity of hexoses in supramolecular structures of bacterial cell of Escherichia coli.

3 dwg, 3 tbl, 1 ex

The invention relates to the biochemistry and molecular biology prokaryotic cells and can be applied to the analysis of molecular-genetic mechanisms of formation of structure of the prokaryotic cells and the role of carbohydrate components in their organization, which is necessary for the disclosure ways of regulating mechanisms of interaction between macro - and microorganisms, and also to find new targets for drugs and the development of environmentally safe treatment and drugs.

There is a method of definition of hexoses [1], in which the acid hydrolysis and color reaction with astronom the products are colored. The drawback of this method is rather low sensitivity (100 g) at the specified ratio acid-thiourea Andron.

There is a method of definition of hexoses [2], based on the ability in the presence of concentrated sulfuric acid to give with astronom painted products. The drawback of this method is that the objects containing hexose, are chemically synthesized reagents - hexose with relatively high concentration of 100-200 mcg/ml.

There is a method to determine the total amount of carbohydrates in bacterial cells [3]. The disadvantage of this method is that bacterial cells did not fraktsionirovanie molecular structure to determine preferential localization levadnyi components inside bacterial cells, and also that this method with a low threshold of sensitivity, as it allows to determine the carbohydrates in the concentration of 10-100 mg/ml

The known method of determination of carbohydrates [4] in cell nuclei. The drawback of this method is that obtaining supramolecular structures is carried out from the cell nuclei of plants, and not from a nuclear-free prokaryotic cells, skin which cannot be charged 0.5% Triton X-100.

The above method of determining carbohydrate components was adopted for the basis in which Uglevodorody components extracted from the cell nuclei, previously frozen in the tissues of wheat germ glycerin environment, then isolated and purified in the density gradient of glycerin with subsequent consecutive extraction nuclear fractions of 0.14 M (nucleoplasm), 0.35 M (chromatin I) and 2 M (chromatin II) chloride, 6 M guanidine hydrochloride in the presence of 0.1% b-mercaptoethanol (nuclear matrix); 0,5 N. NaOH (nuclear-cytoplasmic balance) and the definition of contents of carbohydrates.

The disadvantage of this method is that the homogenization unacceptable for prokaryotes, because culture is composed of separate cells, not integrated into the fabric, 0.5% Triton X-100 does not remove the cell wall prokaryotic cells, extraction of fractions of 0.5-called sodium hydroxide is impossible, because the cell the rest entirely dissolved in 6 M guanidine the hydrochloride from 0.1% beta mercaptoethanol.

The aim of the invention is expanding the range of comparative methods to determine the intracellular localization ecosostenibile component with the method sensitivity of 0.4-1 mg/ml.

This goal is achieved by the fact that in the way of determining the hexoses at supramolecular structures of Escherichia coli cells originally got canned in the presence buffered 80-90% of glycerin cells removed cell membrane 3% Triton X-100, followed consecutive extraction cell supramolecular structures increasing concentration of salts: to 0.14 M (bacteriology); 0,35 M (loosely associated with cellular balance); 2 M NaCl (strongly associated with cellular balance), 6 M guanidine hydrochloride from 0.1% beta mercaptoethanol (cellular balance) and the definition of contents of hexoses.

The invention is illustrated by the following example.

Example. Experiments were performed during the growth of a population of cells of E. coli HB101 (F - ara14 galK2 hsdS20(r B - m B- ) lacY1 leuB6 mtl-1 proA2 recA13 rpsL20(Str R ) supE44,9,14 thi-1 xyl-5 Δ(mcrC-mrr)obtained from the Union of a collection of microorganisms (Puschino) and provided Markusevac T.V. (Institute of biology center of RAS, the group genetics of microorganisms), the strain of E. coli JC-158 (Hfr PO1, thi1, serA6, lacI22, relA1), provided Stupak IV and Stupak EA (Institute of biology of Ufa branch of RAS, laboratory of mathematical and molecular genetics). Cultivation strain of E. coli HB101 was held in liquid nutrient mediums LB and M9. Bacterial cells, used in the experiment was originally located in the bars with agar medium LB (Luria-Bertani): in 1 liter of distilled water dissolve at stirring (magnetic mixer type MM 2A) bacto-triptan (Difco, USA) - 10 g, yeast extract (Difco, USA) - 5 g, sodium chloride (CHP, "Reahim") - 10, Bringing to 7.5 pH was carried out using a 1 M of sodium hydroxide (CHP, "Reahim"). Environment-sterilized when 120-123°at a steam pressure of 1 ATM in the standard autoclave. On 1 litre of environment was added 1.5 g agar-agar (Difco, USA). Culture was kept at a temperature of 4 C. Of the agar column in sterile conditions cells transferred using loops in a liquid LB in the volume of 5 ml, in a chemical test tube with a volume of 20 ml, covered with a cotton-gauze tube and incubated at 37 C, 120 rpm on thermally installation (UFMT-12-250) for 16 hours. From a liquid to a 16 hour culture strain HB101 was rassapalsa "dash" to separate colonies on agar medium LB with the addition of the antibiotic streptomycin (Str) at a concentration of 25 mg/ml in Petri dishes and were incubated at 37 C for 16 hours.

Grown well formed a separate colony of bacteria by using bacteriological loop was one case replanted in a liquid LB with the addition of antibiotic Str at a concentration of 25 mg/ml in volume of 5 ml and were incubated at 37 C, 120 rpm 16 hours. 100 ul of culture was deposited in liquid minimal environment M9 in the amount of 5 ml, and were incubated at 25 C, 120 rpm 16 hours. The composition of the medium M9: Na 2 HPO 4 (CHP, "Reahim") - 6 g, KH 2 PO 4 (CHP, "Reahim") - 3 g, Sodium chloride (CHP, "Reahim") - 0.5 g, NH 4 Cl (CHP, "Reahim") - 1 g on 1 l of distilled water. Environment-sterilized when 120-123°C, at steam pressure 1 ATM in the standard autoclave. After cooling in 1 l of environment was added solutions, sterilized separately: 0.01 M CaCl 2 to 10 ml, 1M MgSO 4 ·7H 2 O - 1 ml of 20% glucose solution ("Biorad"), vitamins to a final concentration of 1 mg/ml, amino acids to a final concentration of 40 mg/ml and antibiotic Str at a concentration of 25 mcg/ml.

In fresh liquid environment M9 in the volume of 50 ml 300 ml flask was sown 600 MKL 16 hour culture and conducted incubation at 25 C, 120 R/min for 6 days.

The first sample was taken in a day in the amount of 1.5 ml Subsequent samples were taken on the second, fourth and sixth day. Controlling the growth of crops was conducted using photocolorimeter KFK-2-change of optical density of cell suspension OD 590 wavelength at 590 nm and sensitivity to 2. The supernatant was removed, the sludge dry a little. To rain was added to 50 ml of medium of the following composition: 80-90% glycerin in 0.01 M Tris-HCl buffer pH 6.8 adding 0.005 M MgCl 2 ; 0.025 M KCl; 0.003 M CaCl2 ; 0.005 M NaCl for preservation of cells at minus 25 degrees C).

Next precipitation cells were washed 3% Triton X-100 in the medium of the following composition: 0.02M triethanolamine (tea)·HCl pH 6.8; 0.005 M MgCl 2 ; 0.025 M KCl; 0.003 M CaCl2 ; 0.005 M NaCl, pH 6,8; shook for 30 min on microsecure (Micro-shaker type 326 m, Poland), followed by centrifugation at 4000 rpm (K-23, GDR) for 20 min for removal of the cell membrane, after what sediment washed twice in the medium of the following composition: 0.005 M MgCl 2 ; 0.025 M KCl; 0.003 M CaCl2 ; 0.005 M NaCl; 0.01 M Tris-HCl pH 6.8, followed by centrifugation at the above conditions. Lower concentration of Triton X-100 was not removed membrane of E. coli cells.

Cytoplasmic proteins were extracted to 0.14 M NaCl, 0.01 M Tris-HCl pH 6.8 buffer. The faction of loosely associated with cellular balance, were isolated by extraction of sludge 0.35 M NaCl, 0.01 M Tris-HCl pH 6.8 buffer. Then the precipitate was fraktsionirovanie suspendirovanie in Tris-HCl buffer with 2 M NaCl. In the sediment remained a fraction containing cellular balance with the cell membrane. Subsequent extraction was performed 6 M guanidine hydrochloride from 0.1% beta mercaptoethanol on Tris-HCl buffer. In the above buffer residue was dissolved completely. Cell fraction was stored at -196 degrees and in nitrogen.

In the above factions spent acid hydrolysis and color reaction with astronom. Method [1] is one of the most common. The essence of the method consists in that after boiling in the presence of concentrated sulfuric acid from peptides otdalyayutsya glucosamine groups, easily splits into monosaccharides. Under the action monosaccharides compounds containing H-groups in the acidic environment formed thioacetate that when interaction with astronom give the products are painted blue, with optimal absorption (especially for hexoses) when And 620 . Direct application of the original method [1] in our conditions no results. Therefore they had done experiments revealing the best conditions of the reaction. According to literary data the net Andron has a melting point 163 degrees C (colorless needles), 154°C [5,6].

The reaction course

For the 0.2 ml of the analyzed solution add 1 ml antropologo reagent, quickly mixed and cooled in an ice bath to 0 C. Then put in boiling water bath for 15 minutes Quickly cooled to room temperature t-20oC. Developing a blue color colorimetrate on SF-26 (LOMO, USSR) against distilled water, And 620 .

Control 1: 0.2 ml of 0.05 M Tris-HCl, pH 8.0+1 ml antropologo reagent

Control 2: 0.2 ml sample+1 ml (74,2% H 2 SO 4 +2% thiourea; without antrona).

The content of hexoses in the analyzed samples estimated per 1 cell by the formula:

C - the number of hexoses (ICG) in supramolecular structures by 1;

1,2 - the volume of the reaction mixture (ml)containing 0.2 ml of sample taken for analysis;

C 1 - the number of hexoses (ICG), was found in the standard curve (figure 1) in the total volume of the reaction mixture;

V - the total amount of the analyzed extract (ml);

A - the number of cells taken in the analysis.

K 1 control 1; K 2 control 2 - indications optical density translated into evidence was found in the standard curve (figure 1).

The method is stable, does not allow sharp fluctuations. The colour intensity of samples is maintained for 1.5 hours, and then decreases.

Table. 1 illustrates the ways of cleaning the sales of the drug antrona (Rahim).

On table 2 shows the efficacy of concentration of sulphuric acid, thiourea on Andronovo reaction.

Figure 1 shows a standard calibration curve for D-glucose. The sensitivity of the method is 0.4 to 1 mg/ml

Figure 2 presents the curve of growth of E. coli HB101. On the y-axis shows the optical density periodic culture. On the x-axis shows the age periodic culture in the day.

Figure 3 shows the dynamics of the content of protein, carbohydrate components of DNA and RNA during growth of a population of cells of E. coli HB101. The amount of protein were determined by the binding protein with Kumasi bright blue G (Loba, Austria) [7, 8]. To determine DNA and RNA used the method A.S. Spirin [9]. Since this methodological approach was developed by us for cell nuclei of plants and methods of its components, ostentatious to determine nanotechnologist, we found it appropriate to provide information on protein, DNA and RNA for the population growth of E. coli HB101 to give full description the obtained suprastructures. For more accurate interpretation of the chart table 3 we present quantitative indicators of Bioorganic composition of bacterial cells of E. coli in periodic culture.

The study of supramolecular structures on the content of carbohydrate component includes a preliminary conservation cells in glycerol environment, removing the cell membrane and the allocation of these cellular fractions of increasing concentrations of salts. Figure 2 illustrates the dynamics of growth periodic culture, analysis figure 3 (table 3) shows the ratio of protein/hexose during the phase of active growth, the deceleration phase and a stationary phase of growth of the population of bacterial cells in periodic culture. Shows the change in the ratio of protein/hexose at the level of all of suprastructures cell populations of E. coli. Interest is the dynamics of the ratio of protein/hexose in genetic and transcription suprastructures of E. coli cells.

The proposed method is recommended in the study of molecular-genetic mechanisms of formation of prokaryotic cell structure and role of carbohydrate components in their organization. The data obtained can be useful in the structure modeling (3D-folding) and the dynamics of biomacromolecule. Knowledge of biochemical processes at different phases of growth of a population of bacteria, allows to reveal the way of regulation mechanisms of the interaction between macro - and microorganisms, which allows controlling the bacterial communities inhabiting a human body.

Color and optical characteristics of the control samples using different concentrations of sulfuric acid and thiourea

Sulphuric acid (% by volume)

The concentration of thiourea (g, %)

Color reaction

dark green

brown-green

0,091±0,004

yellow-blue

0,032±0,004

yellow-blue

0,029±0,004

Bioorganic composition of bacterial cells of Escherichia coli in periodic culture

Growth (day)

The total content in fractions

Bacteriology

Loosely associated with cellular balance

Strongly associated with cell balance

Cellular balance with the cell membrane

The method of definition of hexoses in supramolecular structures of Escherichia coli cells, including conservation of cells in the presence buffered 80-90% of glycerin, removal of cell membranes 3% Triton X-100, followed consecutive extraction cell supramolecular structures increasing concentration of salts: 0,14 M (bacteriology); 0,35 M (loosely associated with cellular balance); 2 M NaCl (strongly associated with cellular balance), 6 M guanidine hydrochloride from 0.1% beta mercaptoethanol (cellular balance), to be held in the above-mentioned factions acid hydrolysis and definition in them of the number of hexoses using antropologo method with preliminary cleaning of the drug antrona, construction of calibration curve and the use of formulas

where C is the number of hexoses (ICG) in supramolecular structures by 1; 1,2 - the volume of the reaction mixture (ml)containing 0.2 ml of sample taken for analysis; C1 - the amount of hexoses (ICG), was found in the standard curve (figure 1) in the total volume of the reaction mixture; V - the total amount of the analyzed extract (ml); And - the number of cells taken in the analysis.