SU1255640A1 - Method of separating ferredoxine - Google Patents

Description

one

The invention relates to biochemistry, in particular to a method for producing highly purified enzyme preparations, and can be used in the production of enzyme preparations, as well as in scientific research on the biochemistry and physiology of plants, molecular biology,

The aim of the invention is to increase the photoreductive activity, increase the yield of the target product and reduce the time of its release,

12

ammonium sulfate and after 0 min prices trifugiruyut under the specified mode for 10 min. The precipitate containing ferredoxin is dissolved in 40 ml of 5 0.05 M Tris-HC buffer, 0.01 M ME pH 9 and centrifuged at 6 thousand rpm for 5 minutes to remove ballast proteins. The supernatant is desalted and purification of 10 high and low molecular weight substances by filtration through a column (40 X 4.0 cm) with Sephadex G-50, equilibrated with 0.05 M tris-HCl pH 8.2, 0.01 M ME, Protein is eluted

The goal is that according to the method of ferringing fredoxin fs with the same buffer,

from higher plants, including Homo-Desalted protein solution with raw material genification and purification stages: fractionation with ammonium sulfate, gel filtration on Sephadex, and ion-ferredoxin activity with tons per column (3 x 1.5 s with DEAE-cellulose, balanced

exchange chromatography on DEAE-Cellulo-20 0.05 M Tris-HC1 buffer pH 8.2,

4-5-week-old leaves are used in the vine, the homogenization of the raw material is carried out in a buffer in a ratio of 1: 5 with the addition of 0.35 - 0.50 M NaC and 0.01 - 0.02 M mercaptoethanol,

Example 1, Fresh leaves of 4- 5-week-old plants are homogenized in a buffer in a ratio of 1: 5 with the addition of 0.35-0.50 M NaC and 0.01-0.02 M mercaptoethanol. The homogenate is squeezed and fractional salting out is carried out followed by by centrifugation. The first precipitate is discarded. The second precipitate is used to obtain ferredoxin, which is dissolved in Tris-HCl buffer and desalted on Sephadex G-50. Further purification of ferredoxin is carried out using two-fold chromatography on DEAE-whole ylose,

Example 2, Moskovsk 35 variety wheat plants are grown in open ground. A portion of fresh leaves (four-week plants)

0.3 M NaC, 0.01 M ME, The column is washed successively with 0.05 M three HC with pH 8.2 buffer with the addition of 0, 0.15 M NaC. Ferredoxin zlyuyut

25 0.1 M Tris-HC1 buffer supplemented with 0.17 M NaCl, Non-exchange / chromium graphs are re-applied to a column (1x1 cm) with DEAE-cellulose, Ferre doxin stored at 0 ° C under nitrogen

30 or in a frozen state at 20 ° C for a long time without loss of activity.

In tab. 1 The results of obtaining a Christmas tree from 100 g of leaves are given.

35 wheat varieties Moskovska 35,

The homogeneity of the proteins in question is proved by sedimentation equilibrium in the Fugue analytical ultracentre, model E, during electrophoresis

40 in a 10% polyacrylamide gel.

Ferredoxin activity is determined by the rate of photoreduced NADPH wheat chloroplasts in a reaction mixture of the following composition

100 g are homogenized in 500 g of buffer - 45 μmol MgCf; NADP 0.6;

M

Noah mixture of the following composition, M: Tris-HC 0.05 (pH; 8.2); 0.35 NaCl; 0.01 Yu (t, e, in the ratio 1: 5), in a homogenizer for 2 minutes. The resulting dark green mass is filtered through a cloth.

Fractional deposition of ballast: proteins is carried out from the leaf homogenate, adding salt at the rate of 25 g of ammonium sulfate per 100 ml of solution. After centrifugation at 6000 rpm for 10 minutes, the pellet is discarded and 36 g of sugar are added to the supernatant.

fer, ye ,, oxina; 0.05 M Tris-HC C bu (pH 8.2), 10 y-chlorophyll.

Example 3, the allocation of ferr doxin from wheat leaves lead

50 similarly to the example, but with the use of mechanical grinding of frozen leaves in the ratio of leaves: buffer: 2 and without additives, the yield of protein is reduced by 1.5 - 2 times and

55 activity 1.5 times.

PR and ME 4, Isolation of ferr doxin from alfalfa leaves grown in open ground is carried out by anap

56402

ammonium sulfate and after 0 min, centrifuged at the specified mode for 10 min. The precipitate containing ferredoxin is dissolved in 40 ml of 5 0.05 M Tris-HC buffer, 0.01 M ME, pH 9 and centrifuged at 6 thousand rpm for 5 minutes to remove ballast proteins. The supernatant is desalted and purification of 10 high and low molecular weight substances by filtration through a column (40 X 4.0 cm) with Sephadex G-50, equilibrated with 0.05 M Tris-HCl pH 8.2, 0.01 M ME, Protein is eluted

fs with the same buffer

 Desalted protein solution with

ferredoxin activity with t per column size (3 x 1.5 cm) with DEAE-cellulose balanced

0.3 M NaC, 0.01 M ME, The column is successively washed with 0.05 M Tris-HC buffer pH 8.2 with the addition of 0.1 M, 0.15 M NaC. Ferredoxin zlyuyut

0.1 M Tris-HC1 buffer supplemented with 0.17 M NaCl, Non-exchange / chromium trographing is repeated on a column (1x1 cm) with DEAE cellulose, Ferredoxin is stored at 0 ° C under nitrogen

or frozen at 20 ° C for a long time without loss of activity.

In tab. 1 The results of obtaining a Christmas tree from 100 g of leaves are given.

Wheat variety Moscow 35,

The homogeneity of the proteins in question has been proved by sedimentation equilibrium on an analytical ultracentrifuge, model E, during electrophoresis

in a 10% polyacrylamide gel.

Ferredoxin activity is determined by the photoreduction rate of NADP in wheat chloroplasts in the reaction mixture of the following composition:

M

fer, ye ,, oxina; 0.05 M Tris-HC C buffer (pH 8.2), 10 y-chlorophyll.

Example 3, the allocation of ferredoxin from wheat leaves lead

similarly to the example, but using mechanical grinding of frozen leaves in the ratio of leaves: buffer: 2 and without additives, they reduce the yield of protein by 1.5 - 2 times and

activity 1.5 times.

PR and M 4, the Selection of ferredoxin from the leaves of alfalfa grown in open ground, lead anapo220

3

Guided by example 1, Ferredoxin yield 3.5-4 mg per 100 g of leaves with photoreductive activity μmol NADPH

mg.hfh

Example 5, The allocation of ferredoxin from the leaves of peas grown in open ground, is carried out analogously to example 1. The yield of ferredoxin is 3-3.5 mg per 100 g of leaves with photoreductive activity 120

µmol NADPH mgph h

Example 6. The selection of ferredoxin from the leaves of cucumbers grown in open ground, are carried out analogously to example 1. The yield of ferredoxin is 3.0-3.5 mg per 100 g of leaves with photoreductive activity μmol NADPH

180

mg-hf-h

in tab. 2 given the activity of the proposed ferredoxin and the yield of protein from 100 g of wheat leaves varieties Moskovsk 35, alfalfa, cucumbers, peas in comparison with the known.

Example 7. Boundary and optimal values of NaCi 0.35-0.5 M and mercaptoethanol 0.01-0.02 M.

The results of the experiments are given in table. 3

Example 8. Paramount values of parameters.

Leaf homogenate

The second sediment Sephadex G-50 DEAE-cellulose DEAE-cellulose

ten

five

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When changing each of the parameters, all the others are optimal (as in example 1).

The results are given in table. 4. 5 Example 9. The effect of the ratio of green material: buffer on the yield of the isolated protein is shown in t abl. five.;

Example 10. The output of ferredoxin depending on the age of the plants is given in table. 6

Example 11. Reducing the time required to obtain the proposed ferredoxin by eliminating and reducing a number of operations allows the entire process to be carried out in 4.5-5 hours, while it is known in 30-48 hours.

The results of the experiments are given in table. 7

The advantage of the proposed method in comparison with the known method is the use of a combination of purification stages — homogenization of the raw material in a buffer at a weight ratio of leaves: buffer 1: 5 with the addition of 0.35 M - 0.5 M and 0.01 - 0.02 M mercaptoethanol in combination with fractional precipitation with ammonium sulfate, which makes it possible to increase the yield of the target product by a factor of 1.5 to 2 to obtain a more highly active ferredoxin with a high degree of purity and reduce its production time to 4.5-5 hours.

0

five

0

T a

faces

Alfalfa Grade Wheat

Specifications

Concentration, M NaCl Mercaptoethanol

t, MG

Output, 7pl

 100 g of wheat leaves

. μmol NADPH Activity, t--

MG HFCCH

NaCl

Mercaptoethanol

table 2

BY

220

3.5-4.0 2.0-2.5

Indicators

0.35 0.42 0.50 0.0 0.015 0.02

44.5

200 200

180

Table 4

3.1 3.0

3.5 3.9

200 190

150 160

Leaf protein yield ratio: buffer

1: 6 Very strong dilution of proteins extracted from leaves, which entails a loss of up to 10% protein.

It is difficult to work on a centrifuge with large volumes of protein solution.

1: 2 ferredoxin is not completely removed from the leaves

These values are optimal for a 100% yield of ferredoxin.

Table 6

asthenia Ferredoxin yield

mg

--5 --- by the way

100 g leaves

. ,

known proposed

Alfalfa

two week seedlings

4-5 week plants

Peas

two week seedlings

4-5 week plants

Cucumbers

two week seedlings

4-5 week plants

jf.

These definitions have not been carried out by the authors of I (with plants of 4-5 weeks of age).

12556408

Table 5

2.2-2.5 3.5-4

2.2-2.6 3.0-3.5

2.2-2.4 3.0-3.5

Grinding material

Extraction (defrosting)

First salting out Centrifugation

Second defrost

Centrifuging

Desalting at sep dex

G-50 fine

G-50 average

Editor N. Gulko

Compiled by, Soina

Tehred L.Oleynik Proof-reader I. Erdeyi

Order 4787/29 Circulation 490 Subscription

VNIIPI USSR State Committee

for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5

Production and printing company, Uzhgorod, st. Project, 4

1255640 10

Table 7

0-10

1-00

Claims (1)

METHOD FOR ISOLATING FERREDOXIN from higher plants, including homogenization of raw materials and purification stages: ammonium sulfate fractionation, gel filtration on Sephadex and ion-exchange chromatography on DEAE-cellulose, characterized in that in order to increase the yield of the target product and reduce the time of its isolation from higher plants, use the leaves': · ¹ 4-5-week-old plants of the family of cereals, legumes or pumpkin, and the homogenization of raw materials is carried out in the buffer at a mass ratio _with leaves: buffer 1: 5 with the addition of 0.35-F 0.50 M NaCl and 0.01 - 0.02 mercaptoethanol.