SU1359298A1 - Method of obtaining molecular forms of carboanhydrase - Google Patents

Abstract

This invention relates to a technique for producing purified enzyme preparations. The purpose of the invention is to increase the yield, as well as simplify and accelerate the process. The method consists in the fact that to obtain an extract having carbonic anhydrase activity from the homogenized leaves of the beans, the homogenate is acidified to a pH of 6.0-6.5, followed by dissolving proteins precipitated by ammonium sulfate in a citrate-phosphate buffer 4.5-5.5, gel filtration on sepharose 6B at pH 6.5-7.0, separation and purification of forms 1 and 2 of carbonic anhydrase by ion-exchange chromatography on DEAE-cellulose, and the column is washed with trisulfate buffer pH 8.0-8.5, containing 20-40 mM sodium sulfate, then eluir successively dissolved carbonic anhydrase carbonic anhydrase 1 and 2 trissulfatnym buffer containing 60-70 mM first, then 40-50 mM sodium sulfate at pH 8.0-8.5 and 5.5-6.5 respectively. The method allows to obtain two highly purified forms of carbonic anhydrase, to increase their activity by 2 times, to increase the yield of the target products by 4 times and to ensure high stability of enzymes during storage. 1 tab. 4 il. (cl w sd; o y so oo

Description

The invention relates to biochemistry, in particular, to a technique for the production of purified enzyme preparations from plants, in particular to the preparation of carbonic anhydrase, and can be used in scientific research of various fields of biochemistry, biotechnology, genetics, as well as in medi-physiological research.

Carboanhydrase (EC 4.2.1.1, carbonate hydroliasis - CA) is an enzyme that catalyzes the reversible hydration reaction of utero dioxide. This enzyme is widely distributed in the plant world and

It is important for a wide variety of cell vital processes. Carboanhydrase is a highly sensitive enzyme that is sensitive to zinc deficiency in the mineral nutrition of plants, animals and humans and is used as a diagnostic test for diseases associated with zinc deficiency.

The purpose of the invention is to increase the yield, activity and stability during storage of the target products, simplifying and accelerating the process.

The method is carried out as follows.

The source of raw materials used to pour beans varieties Black Russian, with a high content of SV v beans).

After the leaves have been homogenized, acidification of the homogenate is made by adding 0.1 n. drop by drop of pH 6.0-6.5, followed by centrifugation to obtain a supernatant extract, which possesses space activity, and separation (.) from the precipitated ballast proteins. This makes it possible to completely extract the target products, better to separate them from the impurity Proteins. Acidification of the homogenate to a pI of less than 6 results in a decrease in the amount of ballast protein in the supernatant, however, there is a significant inactivation of both forms of CA, especially Form 1. With an increase in pH of more than 6.5, the amount of impurity protein in the extract (P At pH 8.0, the protein in the extract is 2 times greater, whose pH is 6.0), which reduces the quality of the subsequent exclusion, forms 1 and 2 from this protein.

The extract proteins with CA-activity are salted out with ammonium sulphate in a narrow concentration range (5–5%). The precipitate of proteins obtained at 40% saturation, which does not have CA activity, is discarded. Collect the precipitate. Proteins obtained at 55% saturation with ammonium sulfate.

10 When the fractionation interval is expanded to 30–60%, a strong contamination of the KA fraction with impurity protein occurs.

The precipitate of proteins, obtained by precipitation with ammonium sulphate and containing carbonic anhydrase, is dissolved in a minimum volume of citrate-phosphate buffer with a pH of 4.5-5.5 before conducting gel filtration. Carbonic Anhydrase

20 is transferred to the supernatant upon subsequent centrifugation (20,000 d, 20 min), and the insoluble protein, which does not have CA activity, is discarded. With increasing pH

25, a larger amount of impurity protein passes from the precipitate into the solution, which significantly impairs the efficiency of the separation and purification of the spacecraft at this stage. With a decrease in the value of 30 RP, there is a sharp decrease in the yield of spacecraft in the soluble fraction.

Gel filtration is carried out on a 2.5-70 cm column filled with Separate 6B; proteins elute

from the total leaf protein 35 phosphate buffer with a pH of 6.5-7.0.

Such gel filtration increases the elution rate and speeds up the process of isolating forms 1 and 2 at this stage compared to the prototype type by 2 times. The indicated pH values of the buffer are optimal, since they ensure the stable state of both forms of KA during gel filtration. Reduction

4-5 or an increase in pH value leads to a corresponding decrease in the stability of form 1 or form 2 in solution, until the activity disappears completely during the gel filtration (5 hours), and to the loss of these enzymes during subsequent isolation and purification by other methods. .

55 The carboanhydride fraction obtained after gel filtration is subjected to ion-exchange chromatography on DEAE-cellulose, which is carried out on a column 3–16 cm in size, providing extract proteins with KA activity, salting out ammonium sulphate in a narrow concentration range (40-55 %). The precipitate of proteins obtained at 40% saturation, which does not have CA activity, is discarded. Collect the precipitate. Proteins obtained at 55% saturation with ammonium sulfate.

At -expansion of the fractionation interval to 30-60%, a strong contamination of the KA fraction with impurity protein occurs.

The precipitate of proteins, obtained by salting out with ammonium sulphate and containing carbonic anhydrase, is dissolved in a minimum volume of citrate-phosphate buffer with a pH of 4.5-5.5 before conducting gel filtration. Carbonic Anhydrase

enters the supernatant upon subsequent centrifugation (20,000 d, 20 min), and the insoluble protein, which does not possess CA-activity, is discarded. With increasing pH

a larger amount of impurity protein passes from the precipitate into the solution, which significantly impairs the efficiency of the separation and purification of the spacecraft at this stage. When the RP value decreases, a sharp decrease in the yield of CA into the soluble fraction occurs.

Gel filtration is carried out on a 2.5-70 cm column, which fills a good elution rate and allows the entire process of separating the QA forms to be carried out in 4-5 hours instead of 72 hours, which is necessary for conducting isoelectric focusing, i.e. reducing the time spent on this stage. The use of 0.01 M trisulfate buffer pH 8.0-8.5, containing 20 mm of sodium sulfate as an equilibration buffer, provides an optimal sorption of the initial spacecraft and complete binding to DEAE cellulose. During subsequent washing of DEAE-cellulose with the indicated buffer, KA activity in the eluate is not detected. Using the pH of the equilibration buffer below 8.0 leads to incomplete adsorption of forms 1 and 2 KA on DEAE-cellulose, to partial desorption of the CA and detection of its activity in the eluate. At pH values above 8.5 K / -activity is not detected in the eluate, forms 1 and 2 are fully bound to DEAE-cellulose, but washing with these solutions inactivates form 2.

The picture of eluted forms 1 and 2 KA, in addition to pH, also determines the ionic strength of the buffer, which is created by the addition of sodium sulfate to the buffer solution. When the pH of the buffer is 8.0 - 8.5 and the concentration of sodium sulfate is 60-70 mM, only Form 1 is eluted from DEAE-cellulose. At concentrations of sodium sulfate in a buffer with a pH of 8.0-8.5, less than 60- 70 mM, K.A. activity is not detected in the eluate, and at high concentrations partial forms inactivation of forms 1 and 2 are observed. Obtaining form 2 under these conditions is undesirable due to its strong inactivation in solution with pH 8.0-8.5. Optimal desorption of form-4y from DEAE-cellulose is carried out with 0.01 M nitrate-phosphate buffer, -FN 5.5-6.5, containing 40-50 mM sodium sulfate. Before removing this form 2 with this solution, the DEAE-cellulose column is carefully freed from form 1 and foreign protein first by the above washing with 0.01 M trisulfate buffer, pH 8.0-8.5, containing 60-70 mM sodium sulfate (500 ml) , then with a solution of 0.01 M citrate-phosphate buffer, pH 4.5-5.5 (500 ml), containing 40-50 mM sodium sulfate. In

In all cases, the washing of the column is continued until complete disappearance of the protein in the eluate, which is controlled by measuring the absorbance at 280 nm.

Example 1. The object is the leaves of the Black Russian bean variety. Plants are grown in wooden boxes with soil.

Stage 1. 300 g of freshly picked leaves (28 days old) are fixed with liquid nitrogen and triturated in a porcelain mortar with 300 ml O, 1 M trisulfate buffer, pH 7.8, containing

0.1 M ME and 0.002 EDTA. The grinded mass is slowly thawed by infusion at 4 ° C for 5 hours with occasional stirring. The resulting homogenate, with continuous stirring, is acidified to pH 6.3 by adding dropwise 0.1N H2SO4 and immediately filtered through a double layer of nylon. The filtrate is centrifuged at 22,000 g for 20 minutes. Insufficient fluid containing 1380 mg of protein - leaf extract.

Stage 2. Solid ammonium sulphate is added to the extract up to 40% full;

at this saturation, separated by centrifuging at 10,000 g, 20 min and discarded. Ammonium sulfate is added to the supernatant to 55% saturation. The precipitate with CA activity is collected by centrifugation (lOOOOg, 20 min) and dissolved in a minimum volume (5-10 ml) of 0.01 M citrate-phosphate buffer, pH 5.0, containing 0.05 M ME, stirring. The undissolved protein, which does not possess CA-activity, is separated by centrifugation at 20,000 d, 20 min.

Stage 3. The resulting supernatant

applied to a glass chromatographic column (2.5–70 cm in size) filled with Separate 6B, equilibrated with phosphate buffer, pH 6.8, containing 0.05 M ME. After exiting

100 ml of empty volume, collection of 5 ml fractions is carried out on the fraction collector at a rate of 2 ml / min.

A typical picture of gel filtration on sepharose 6B is shown in FIG.

In fractions, the protein is measured by absorbance at 280 nm and the activity of the CA by the colorimetric method. The most active fractions with an output volume of 1 20210 ml are combined; the resulting volume is 90 ml.

Step 4. An enzyme solution is layered on a column (3-16 cm in size) with DEAE-cellulose (nominal capacity is 0.60-0.80 meq / g, swelling factor 9.0 ml / g dry anion exchange resin, Reanal, Hungary) equilibrated with 0.01 M trisulfate buffer, pH 8.2, containing 0.05 M ME and 20 mM sodium sulfate (200 ml). Non-adsorbed protein is washed out with the same solution, obtaining an eluate that does not have space activity, which is discarded. The fractionation of proteins with DEAE-cellulose is carried out in 2 stages. Initially, the column was washed with 40 mM solutions for washing (200 ml, fraction O and 60 mM (fraction 11, 200 ml) sodium sulfate in 0.01 M trisulfate buffer, pH 8.2. Then the proteins from DEAE-cellulose were eluted with 20 mM dl solutions washing (200 ml, fraction 111) and 40 mM 200 ml, fraction IV, sodium sulfate in 0.01 M citrate-phosphate buffer, pH 5.8. Washing in solutions in all cases continues until the absorption of the protein in the eluate at 280 nm completely disappears. Fractions 1 and 111, which do not have CA-activity, are discarded. KA activity is detected in fractions 11 and 1. Collection of 10 ml fractions is carried out on the fraction collector at a rate of 10 ml / min.

Figure 2 shows a typical elution pattern.

In fractions, the protein is measured by absorption at 280 pm and the activity of CA. The most active fractions with output volumes of 50-150 ml are combined. The first active fraction 11 corresponds to Form 1, contains 94 mg of protein, and has a specific activity of 20,500 U / mg of protein. The second is active - fraction 1 is Form 2, Contains 12 mg of protein and has a specific activity of 9700 U / mg of protein.

Stage 5. The obtained enzyme solutions of forms 1 and 2 KA are separately concentrated by ammonium sulfate at 70% saturation, centrifuged at 22000 g, 20 minutes and stored as precipitates under ammonium sulfate at –20 ° C. The activity of form 1 is maintained for 6 months without change 5 0 5 About g

0 5

0

five

and forms 2–1 months under these conditions.

The resulting preparations of forms 1 and 2 are homogeneous: when tested in a Spinco analytical centrifuge, model E (USA), each enzyme shows a single peak (Fig. 3) with sedimentation coefficients of 15.8 S and 16.0 S, respectively, for forms 1 and 2 ( rotor speed of 48660 rpm, shot taken after 16 minutes of equilibrium at a protein concentration of 3.5 mg / ml). With an analytical disc electrophoresis in a 7.5% polyacrylamide gel, each form migrates as a single protein band (Figure 4).

The results of the purification and isolation of forms 1 and 2 KA from the leaves of the beans are given in the table, where for comparison the results of the purification of natural varieties were also given.

The results shown in the table show that the proposed method compared to the known method allows to increase the activity of homogeneous forms 1 and 2 KA by 2 times and the yield by 4 times (in% of the total leaf protein).

Example 2. The method is carried out as in Example 1. After homogenizing the leaves of the beans, the homogenate is acidified to pH 6.5 and centrifuged. An extract containing 1600 mg of protein with a specific activity of 585 U / mg of protein is salted out with ammonium sulfate in the range of 40-55% and the tank is dissolved in 5 ml of 0.01 M citrate-phosphate buffer, pH 5.5. The resulting supernatant with a specific KA activity of 800 U / mg protein, containing 350 mg of protein, is subjected to gel filtration using Sepharose 6B 0.01 M phosphate buffer, pH 7.0. The KA fraction, with a specific activity of 4000 U / mg protein (180 mg protein), is chromatographed on DEAE-cellulose, initially eluted with Form 1 with 30 mM solutions (for washing) and 70 mM sodium sulfate in 0.01 M trisulfate

buffer, pH 8.5, then form 2 — solutions of 30 mM and 50 mM sodium sulfate in 0.01 M citrate-phosphate buffer, pH 6.5. Forms 1 and 2 are homogeneous with a yield of 7 and 0.6%, a purity of 22 and D p, and a specific activity of 22000 and 9500 U / mg protein, respectively. Indicators are also significantly better than by a known method ..

7

Example 3. The method is carried out as in Example 1. However, the homogenate is acidified to pH 6.0, the precipitate of proteins salted out with ammonium sulfate is dissolved in 0.01 M citrate-phosphate buffer with pH 4.5, gel - filtration on sepharose 6B is carried out at pH 6.5. Subsequent separation of forms 1 and 2 KA by ion exchange chromatography on DEAE-cellulose is carried out with solutions of 20 mM (for washing) and 60 mM sodium sulfate in 0.01 M trisulfate buffer, pH 8 , 0 and a solution of 40 mM sodium sulfate in 0.01 M citrate-phosphate buffer pH 5.5. Forms 1 and 2 KA are implemented in this method with indicators (specific activity, yield, homogeneity, degree of purification, stability), similar to example 1.

Thus, the use of the proposed method of obtaining forms 1 and 2 of carbonic anhydrase from plants simplifies the technology of obtaining the target products and speeds up the process of isolating enzymes 15 times through the use of ion exchange chromatography on DEAE-cellulose instead of isoelectric focusing, allows to obtain highly purified enzymes through the selection of optimal conditions for the purification and purification of carbonic anhydrase in the stages of homogenization, gel filtration, ion exchange chromatography, to increase their activity in two known ways

Getting the extract from the leaves of the beans

Ammonium Sulfate Fraction

eight

per, yield 4 times and storage stability.

Claims (1)

g. The invention The method of obtaining two molecular forms of carbonic anhydrase, involving the homogenization of ground 0 parts of plants in a buffer solution, separation of the solid phase from the homogenate, precipitation of proteins from the obtained extract with ammonium sulfate at a saturation of 40-55%, diluting the precipitate and its gel filtration, followed by separation of the two forms of farboanhydrase, and that, in order to maintain the yield, activity and stability during storage of the target products, to simplify and speed up the process, the homogenate is acidified to a pH of 6.0-6.5, the dissolution of the precipitate is carried out with a pH citrate-phosphate buffer 5 4.5-5.5, gel filtration is carried out on Sepharose 6B phosphate buffer pH 6.5-7.0, followed by separation and purification of the target products by ion exchange chromatography on a column with 0 DEAE-cellulose, while the column is washed with trisulfate a buffer with a pH of 8.0–8.5, containing 20–40 mM sodium sulfate, then eluted with carbonic anhydrase 1 and carbonic anhydride II with a trisulfate buffer containing first 60–70 mM, then 40–50 mM sodium sulfate with a pH of 8.0- 8.5 and 5.5-6.5 respectively. 1.0 100 981 3.0 33 135929810 Table continuation e 250 4300 17.5 40 11000 44.8 6 5000 20.3 The proposed method Getting extract from the leaves of the beans Ammonium sulfate fractionation Gel filtration on sepharose 6B Ion exchange chromatography on DEAE-cellulose e: fraction with form 1 form 2 fractions 12 4300 17.5 11000 44.8 5000 20.3 th way 1.0 1.7 7.7 100 22, ten, 20500 45.56.9 9700 21, 5 V «7 20. 15 / W WS W zoo 220 Fractions., Ml FIG. 1/2 form 2 the form / Fi.T ipopf a 2 Form 1 FIG L