SU1703688A1 - Method of separating nuclease from barley plantlets - Google Patents

Abstract

The invention relates to biochemistry and biotechnology, and can be used for the enzymatic synthesis of nucleospirated tri-phosphates. labeled with phosphorus -32 (33) in the "position of the phosphate group. The aim of the invention is to increase the yield of the target product, free from impurity at-phase, 5-nucleotide and nonspecific phosphatase activities. The method consists in that seedlings of barley 4-6 days are homogenized, extracted, the extract is fractionated with ammonium sulfate 40-80%, the suspension is heated for 15 minutes at 60 ° C. Chromatograph on DEAE-cellulose and silochrome 50/80 red-brown 2K. The output activity 33%, the degree of purification - 426 times. 1 hp f-ly., 6 ill., 5 tab. yo

Description

The invention relates to biochemistry and biotechnology and can be used for the enzymatic synthesis of nucleoside triphosphates. labeled with phosphorus-32 (33) in the "position of the phosphate group.

A known method for producing nuclease. based on traditional methods of purification salt fractionation, gel filtration, ion exchange chromatography 1,

The method does not allow to obtain preparations that meet the requirements for impurity activities, since they do not contain a reliable stage for their elimination (especially ATP-az).

The closest is the method of purification of nuclease from barley seedlings 2. The method includes four purification stages: sulfate-ammonium precipitation of proteins in the range of 40-80% saturation, ion-exchange chromatography on DEAE-Tris-acrylic M at pH 8.0, gel filtration on G-75, AMP Sepharose Affinity Chromatography.

The disadvantages of this method are the low yield of the purified product - no more than 1%. the inappropriate use of AMP-Sepharose affinity sorbent for large-scale purification of the nucleic acid phase due to the small capacity and instability of the sorbent, the lack of an endoscale characteristic of the preparation of the nuclease.

The aim of the invention is to increase the yield of the target product, free from impurity at-phase, 5-nucleotide and nonspecific phosphatase activities.

According to the method of purification of nuclease from barley seedlings, including homo 4 O CO O CO 00

geniization of the raw material, ammonium sulfate precipitation of proteins, ion exchange chromatography at pH 8.0, 4-6 days old barley sprouts are used as raw materials, the homogenate from the raw material is purified by sulfate ammonium precipitation of proteins in the range of 40-80% saturation followed by heating suspensions up to 60 ± 1 ° C for 15 minutes, followed by chromatography on DEAE-cellulose with elution of enzymes with a solution of 0.1 M NaCI and pseudo-affine chromatography on silochrome 50/80 red-brown 2K at pH 8, 0 with pre-elution of impurity proteins with a solution of 0.1 M NaCl and nuclease elution with a solution of 0.5 M NaCl.

Figure 1 shows the dynamics of changes in the activity of the target and impurity enzymes as a function of age (the maximum 3-nucleotide activity was detected in 4-6-day-old seedlings, and not in 6-7-day-old, as in the known method); 2 shows the nuclease resistance to heating up to 60 ° C for 15 minutes (during heat treatment, the impurity dephosphorylating enzymes of 5 -nucleotidase and ATP-ase are partially inactivated (up to 60-70%), hence the expediency of introducing this step into the cleaning procedure ); in FIG. 3, the elution profile of proteins from DEAE-cellulose (pH 8.0) when performing stepwise elution with a 0.1 M NaCI solution (at this stage, the enzyme is further purified 4.1 times, but impurity dephosphorylation enzymes are not separated from the target enzyme ); figure 4 - the profile of the elution of proteins at pH 8.0 with pseudo-affinity sorbent silochrome 50/80 red-brown 2K in the implementation of the step-by-step elution of impurity proteins with a solution of 0.1 M NaCI and elution of nuclease with 3-nucleotidase activity with a solution of 0.5 M NaCI (under these conditions, non-specific phosphatase and b-nucleotides are not adsorbed, ATP-ase binds weakly and completely elutes with 0.1 M NaCl, while nuclease binds well and elutes with 0.5 M NaCl); 5 shows the optimum pH of the H3-nucleotide activity (pH 8.2); 6 is a diagram explaining the proposed method.

The threshold concentrations of NaCf solutions used in chromatography on DEAE-cellulose and silochrome red-brown 2K, as well as the pH of the buffer in chromatography on silochrome red-brown 2K, were chosen experimentally.

Scheme of the nuclease purification procedure

Homogenization of plant material

Protein extraction buffer

I

The precipitation of proteins with ammonium sulfate (40-80% saturation)

t Heat treatment (60 ° C, 15 min)

romatography on DEAE-cellulose, pH ", 0

0.1 M NaCI Chromatography on silochrome red-brown, pH 8.0

0.5MaS1 oTm NaCI

non-adsorbed proteins

NT

nonspecific phosphatase, 5 / -nucleotidase. part of the ATP-aznoe activity

ATPase nuclease

Z-nucleotide activity

I

Concentrated (dialysis against

50% glycerol) PRI me R 1. All purification steps, except for heat treatment and chromatography on an immobilized silicone dye, are carried out at.

The results of the isolation and purification are summarized in table. 1, which shows the data on the purification of nuclease with H-nucleotide activity from 75 g of barley seedlings.

Step 1. Getting the original extract. Barley kernels (Pirkka variety) are germinated in 20 x 30 cm cuvettes on deionized water in the dark at 20 ° C. 4-6-day-old seedlings are separated from the seed and roots, ground in a mortar or homogenized in a knife homogenizer with 0.1 M Tris-HCl buffer, pH 6.5. The homogenate is placed on a shaker for 1 hour to complete the extraction of the enzyme, then centrifuged for 1 hour at 8,000 d, the precipitate is separated, and the supernatant is filtered through Miracloch. The protein concentration in the original extract is 20-30 mg / ml.

Stage 2. Deposition of proteins with ammonium sulfate.

To the initial enzymological one: crystalline ammonium sulfate is added to the 40% saturation (243 g / l) slowly with continuous stirring. The precipitate formed within 1 hour is separated by centrifugation at 10,000 g for 30 minutes. The precipitate is discarded, the supernzant is saturated with ammonium sulfate up to 80% (561 g / l). Leave for 2 hours to form a precipitate.

Stage 3. Heat treatment.

The suspension of proteins saturated with ammonium sulfate is heated to 60 ° C for 15 minutes, then cooled in an ice bath for 20-30 minutes. The suspension is centrifuged in the same mode. The precipitate is dissolved in 50 mM Tris-HCl buffer. pH 8.0 and dialyzed against 1 l of the same buffer with a threefold replacement of the buffer.

Stage 4. Chromatography on DEAE-cellulose.

The desalted protein solution was applied to a DEAE-cellulose column (2.5 x 18 cm) equilibrated with 50 mM Tris-HCl buffer at a rate of 25 ml / h. After applying the protein and washing the column with buffer (100 ml), a solution of 0.1 M NaCl is supplied to elute the enzyme with 3-nucleotide activity (Fig. 3). Fractions with 3-nucleotidase activity are pooled and dialyzed against 1 liter of 50 mM Tris-HCl buffer, pH 8.0.

Stage 5. Chromatography on silochrome 50/80 red-brown 2K.

The removed protein solution is applied to a small column (2x6 cm) with silochrome red-brown, equilibrated with 50 mM Tris-HCl buffer, pH 8.0, at 20 ° C, after which the column is washed with 20 ml of the same buffer to remove non-specific phosphatase and 5 - nucleotidase. Impurity proteins with ATP-ase activity are eliminated with a solution of 0.1 M NaCI (20 ml), nuclease with C-nucleotide activity is eluted with 20 ml of a 05 M solution of NaCI. The elution rate is 40-50 ml / h.

Stage 6. Concentration.

Combining the fraction with 3-nucleotide activity is dialyzed against a 50% glycerol solution prepared in 50 mM Na-acetate buffer pH 6.0 and stored at-10-20 ° C.

Determination of enzyme activity.

Determination of 3-nucleotidase nuclease activity.

3 -N-nucleotidase activity is determined by a colorimetric method. Per unit of activity, the amount of PH in micromoles released per minute of hydrolysis of 3 AMP at 37 ° C is taken.

The method of determination is as follows: 50 µl of the protein sample under study, 100 µl of solution AMP with a concentration of 7.5 mM, 300 µl of a buffer solution of 50 mM TEA-HCI - GM eON are placed in a microsample tube with a volume of 1.5 cm3. pH 8.0: samples / iubuyut 30 min at 37 C. The reaction with, g; m; n; - poured by the addition of 50 mg. x ;; orion acids (9 n.). From analytical Grobirsk

take 100 μl of solution. ;; CVSE-: 0.4 ml of 1 M sodium acetate, 1 m; S .: ..4t tatnogo buffer, pH 4, 0.4 ml a:.,; - About 2 m ;: 1% ascorbic acid solution. prepared for 1 mm solution of copper sulfate. The mixture is left for 10 minutes after which 0.2 ml of a 1% ammonium molybdate solution prepared in 0.05 N is added. H2S04. After 10 minutes, the absorbed samples are measured on a spectrophotometer.

at a wavelength of 660 nm. The calculation of the activity is made by the formula

20

Debbo 14.6 100 Amd --- Gs1 ---- where D Ebbo is the absorption difference at /. 660 nm between the test sample and the control (sample without protein);

14.6 — coefficient of conversion of absorption units, µg PH (determined by calibration);

g is the incubation time (30 min); 31 - atomic mass of R. Determination of the activities of impurity dephosphorylating enzymes.

The determination of the activities of non-specific: phosphatase, 5-nucleotidase and ATP-ases is carried out in a similar way, using (3-glycerophosphate, 5 AMP, ATP, respectively) as substrates. When detecting phosphatase activity with y5-glycerophosphate, 50 mM imidazole buffer, pH 6.5, is used.

PRI mme R 2. The choice of heat treatment conditions.

It is known that barley nuclease is thermostable at t 60 ° C. It is experimentally shown (figure 2) that at this temperature

Inactivation of the impurity dephosphorylating enzymes occurs: b - nucleotides 60%. ATP-basics 70%. Therefore, it is advisable to conduct heat treatment at 60 ± 1 ° С during the cleaning of the target Enzyme.

(thermometer error). The duration of the warm-up is chosen experimentally. The greatest drop in the activity of 5-nucleotidase and ATP-ase occurs in the first 15 minutes of heating at 60 ° C (Fig. 2). With more

prolonged exposure to impurity activities remain at the same level, while the 3-nucleotide activity decreases. On the basis of the data obtained, the warm-up time is 15 minutes.

PRI me R 3. The selection of the concentration of NaCI for stepwise desorption of nucleases from DEAE-cellulose.

The procedure to perform up to step 4 is similar to Example 1. After which, in an analytical version, the target enzyme is desorbed in a NaCI gradient of 0-0.2 M (150 + 150 ml) and the arithmetic average with the root-mean-square error of NaCI is determined (Fig.6, midpoint front elution).

In tab. 2 shows the midpoints of the front elution front of nuclease in M NaCI with chromatography on DEAE-cellulose in a NaCI gradient (0-0.2) M.

As can be seen from the table. 2, NaCl concentration, necessary for desorption of nuclease, 0.1 tO.01 M.

Example 4: Selection of the pH of the buffer solution and the concentration of NaCI for chromatography on silochrome red-brown.

The procedure to perform up to step 5 is similar to Example 1, after which the pH range of the buffer is established for effective sorption of the nuclease at the silochrome with weak sorption of the dephosphorylating enzymes.

In tab. 3 presents sorption data for nuclease with 3-nucleotide activity and impurity enzymes on silochrome red-brown at different pH.

As can be seen from the table. 3, the optimum pH value is 8.0, since despite a slight loss of activity during sorption (on the order of 10%), the pH has drastically reduced the binding of impurity enzymes compared to pH 6.0. At pH 9.0, the proteins in question are not adsorbed.

The choice of NaCI concentration for the stepwise adsorption of the impurity and target enzymes is determined by the desorption efficiency at pH8.0.

In tab. Figure 4 shows the desorption of nuclease and impurity enzymes from silochrome red-brown at various concentrations of NaCl (pH 8.0).

As can be seen from the table. 4. At a concentration of 0.1 M NaCl, desorption of the remaining ATPase and other proteins occurs, while nuclease remains bound to the sorbent. Desorption of the target enzyme begins at a concentration of 0.3 M NaCI and reaches maximum efficiency with 0.5 M NaCI.

Methods of testing the drug.

Nuclease from barley seedlings is used in the synthesis of (5 - P) dAMO and (5 33 P) AMP at the stage of transition of the label from (y-3) RTP ATP to

(a-33) (d) AMP: (y -33 P) ATP + 3 (d) AMP polynucleotide / kinase 3x - (5-33 P) (b) ADP; (3, 5 -33PXd) ADP nuclease / barley (33RHy) AMP + fnl.

The hydrolysis of the 3 -phosphate bond is carried out as with a labeled product (diphosphate) purified after the first reaction, i.e., in the pass-through synthesis without prior purification of the diphosphate. It is significant that in both variants of the synthesis (a -33PXd) AMP, the pH of the reaction medium is 7.0-7.5.

The reaction is carried out with 0.2 E nuclease for 30 min at 37 ° C; the radioactivity loading is 45 mCi (y-P) ATP.

The transition of a label from (3.5 -33P) -phosphate to (5 -33P) -monophosphate is more than 90%, which is calculated from the ratio of peak areas on the HPLC chromatogram. The specificity of nuclease from 3 to 3

to substrates

The substrate specificity of the nuclease with respect to the 3-nucleoside mono- and diphosphates ribo- and deoxyriboriba was studied.

In tab. 5 shows the relative activities (with 3 AMF-100%), shown in the range of pH 7.0-8.5 (substrate specificity of nuclease from barley seedlings). From tab. 5 it is clear that barley nuclease

shows a 3H-nucleotidase activity in relation to 3-substrates of deoxy-p - yes. including Z TMF and 3, 5 TDF, however, the rate of hydrolysis is approximately 100 times less than with Z-ribonucleotides. The optimum pH for ribonucleotides is in the range of pH 8.0-8.5, for deoxyribonucleotides - about 7.0.

Claims (2)

1. The method of purification of nuclease from barley seedlings, including homogenizing the raw material, extracting the homogenate, fractioning the extract with ammonium sulfate in the concentration range of 40-80% unsaturated, ion-exchange chromatography on a carrier containing diethylaminoethyl groups, and chromatographic purification, which is different , in order to increase the yield of the target product, free from impurity at-phase, 5-nucleotide and nonspecific phosphatase activities, the protein suspension after fractionation with ammonium sulfate is emphasized ermoobrabotke at 60 ± 1 ° C for 15 min, ion-exchange chromatography was performed on DEAE-cellulose at pH 8.0 with the elution of the enzyme 0.1 M NaCI, after which pseudo-affinity chromatography was performed on silochrome 50/80 red-brown 2K at pH 8.0 with preliminary elution of the impurity proteins with a solution of 0.1 M NaCI and elution of the target product with 0.5 M NaCI. 2. The method according to claim 1, characterized in that, 4-6 days old barley seedlings are used as the source of the enzyme. Table 1 table 2 ten Table 3 A, E / g weight 14 12 ten eight 6 four 2 ABOUT 34 5 6 7 8 9 10 II 12 13 14 SU Table 4 Table 5 З -ххлзот дзза АТФ-ааа 5-tsuh-eoti, h & for kespe:; and Јgchna phos | anatase Set. i I--. YU eo, 40 .-,.- .five ten v-ig. 2 on, 280 2 I.  Neadsor (3 Drov. Bulk) J t- 60 -I- 120 ......... 3-hl e o TUL h a , behind "- well, about o: 7j; a z a h Nes.G: HCH: -: And OS Ataz 15 20 Detail of heating at 60 ° C, vcffl Z -nuklvotidaza AW-aza 5 -nukh80tidaea nesviiftchnal phosphataea -t- 240 -i- 300 J Ofbtfl EVIL & TA, M.L. oo CO UD O ABOUT L. About B- CT - 03 U t (-, S U U) s oh oh D R Pi C-rJ 2 G nogaso; yi 25 Whitefish. 6 ., „.. Z-nuuleotid-a Olev elvat "to