SU857145A1 - Method of preparing sorbent for purification of yeast hexokinase - Google Patents

Description

- one .

This invention relates to enzymology and. It is intended to create an easily accessible method for the preparation of a sorbent for the purification of yeast hexoknase used as an important analytical reagent for determining the content of adeioeintrythrin, glucose and other hexoses in biological fluids in medical and scientific practice.

To isolate and purify yeast hexokinase, a number of ion exchange sorbents are known, among which ion exchange celluloses (KM, DEAE, TEAE and others) are most commonly used, which are obtained after its corresponding chemical modification fll.

However, sorbents of this type have a number of significant drawbacks: a low degree of selectivity with respect to a certain class of enzymes, a low degree of purification of the enzyme, as a result of which they are generally accepted, as a rule, only as part of the general schemes of purification of the enzyme.

A more efficient method for the isolation and purification of enzymes is affinity chromatography using biospecific sorbents. Isolated cases of biospecific sorbents for isolating and purifying yeast hexokinase 12) and H. are known.

However, sorbents of this type also provide only partial purification of the enzyme and are of a purely scientific nature. Possibilities of practical application of sorbents of this type are limited or completely eliminated due to the high cost of products used for the synthesis of sorbents, the complexity of synthesis and the low working stability of the resulting sorbents.

The closest in technical essence to the present invention is a method for obtaining a sorbent for purifying yeast hexokinase, which consists in attaching a ligand (-32 Q-aminopyrndine) adenine dinucleotide to the polysaccharide matrix, which is used as a 6-aminocaproyl sepharose, a wire, a wire, a wire, a wire, a wire, a wire, a wire, and a conductor; 1-ethyl-3- (3-di-methylaminopropyl) -carbodiimide hydrochloride with a pL of 4.75 for 24 hours 43.

However, a known method for preparing 30 sorbents for chromatographic purification of yeast hexokinase has a number of significant drawbacks.

First, the initial matrix used for the preparation of the sorbent is expensive, low-stability to the action of mechanical and chemical factors, as a result of which the possibility of using the sorbents obtained on its basis for the preparative isolation and purification of enzymes is limited or completely eliminated.

Secondly, as a ligand of the sorbent (3-aminopyridine), adenine dicucleotide is difficult to use and expensive to have, 2; (it lacks chemical stability, its synthesis is complex and multistage, which ultimately leads to expensive sorbent based on it as a whole and excludes the possibility of using the sorbent for practical purposes with

In other words, the sorbent selectivity for OTHODjeHHK) to yeast hexokinase is low, because the reducible total purification factor for ferments (even in the peak fraction is 20 times and results in the preparation of enzyme with purity of 6%; it is 20% however, there is no information on the extent to which the susceptibility of the desorbed enzyme is active.

The purpose of the invention is to simplify the process of obtaining a sorbent for the purification of yeast hexokinae and increase the selective sorption capacity of the sorbent.

The goal is achieved according to the method of obtaining a sorbent for the purification of yeast hyxocnnase, the end of which is the connection of the ligand to the polysaccharide phosphate cellulose matrix by treating the latter with a solution containing chromium ions (i and) with a gleam of chromium ions (if) and phosphate residues with imitation chromium ions (i and I) and with phosphate residues (if) and phosphate residues (0) containing 0 1 in 0.08-0.1 M acetate buffer solution pH 5.4-5.8.

Treatment of phosphate-cellulose With g-containing solution is usually carried out with constant stirring for 20-24 hours.

The concentration of chromium ions (Mi) in the sorbent obtained by the proposed method is 200-800 µM / g of sorbent.

It should be noted that with a decrease in the concentration of chromium ions () {below 200 µM / g of sorbent), until it is absent in the phosphate cellulose sorption of yeast hexokinase, it decreases to a complete absence of sorption on uncharged Cr phosphate cellulose. On the other hand, when the concentration of chromium ions is higher than 800 μM / g of sorbent, the sorption of the enzyme is irreversible.

The advantages of the proposed method are that which eliminates the need to use complex chemical transformations, expensive and difficult to access substances, simplified. In general, the sorbent preparation method is used. At the same time, by adding chromium ions ((M) to readily available phosphate cellulose, the required amount of metal ion is introduced into the matrix, which is an effective sorption

center of binding of this enzyme, which leads to an increase in the selective sorption capacity of the sorbent.

To obtain the sorbent and its preparation, the following basic equipment is used to purify the yeast hexoin5 kinae: laboratory apparatus for the preparation of the sorbent, chromatographic columns, and an SF-16 spectrophotometer.

Q Phosphate cellulose materials (produced by the Olaine Chemicals Plant) with an analytical capacity of 0.71, 1 mEq / g, C g, (SOj,), j 6H, Q, succinic acid, acetic acid, sodium acetic acid, yeast hexokinase.

Example 1. Obtaining a sorbent.

In a round bottom flask equipped with a mechanical stirrer, 5.0 g of chromium sulfate (M) C yr (SOi,) 3,

800 ml of OD M acetate buffer pH 5.6 and 10.0 g of phosphate cellulose (OPR). The contents of the flask are stirred for 20-24 hours. At the end of the reaction, the sorbent is filtered off, washed with water with a 0.1 M solution of succinic acid to remove excess chromium (I).

The concentrations of chromium ions (I M) in the sorbent are determined from the difference

The concentrations in the initial solution and the solution after phosphate cellulose charging are spectrophotometrically with ethylene diamine tetraacetic acid (EDTA) at 525 nm.

PR and M and p 2. Purification of yeast

Hexocynase.

a) 2.0 g of phosphate cellulose complex with chromium ions (Ml) is loaded into

the chromatographic column (1 to 20 cm) is equilibrated with 5 mM of succinate buffer solution pH 6.0, containing 0.5 EDTA and 4.0 mm of the enzyme solution of yeast hexokinase is applied

Claims (2)

Firm Fluka (Iveydari). Protein concentration 1.45 mg / ml, specific activity 28.0 units / mg of protein. Upon elution with 330 ml of the initial buffer solution pH 6.0, ballast proteins are removed. The elution of active hexokinase was carried out with a gradient of sodium chloride from 0.005 M to 0.25 M in a succinate buffer solution pH 6.0. The specific activity of hexokinase after desorption is 300 units / mg of protein. The yield on the activity of 90%. b) 2.0 g of the sorbent is loaded into an xp matrix column (1 x 20 cm) equilibrated with 5 mM succinate buffer solution pH 6.0, containing 0.5 mM EDTA, and 5, O ml of enzyme preparation obtained after two hours is applied autolysis at 3-7 tpPH / vu HPM / GO aa 3 suspensions 1.8 g of baker's yeast in 5 ml of 0.2 M, with 0.5 mM EDTA and subsequent dialysis against the initial buffer solution pH 6.0. Protein concentration is 9.6 mg / mp, specific activity 4, units / mg of protein. Upon elution with 340 ml of a succinate buffered solution of pH 6.0 v. Silvagates ballast proteins. The elution of hexoxynes was carried out with a gradient of chloride from 0.005 to 0.5 M in the initial buffer solution pH 6.0. The specific activity of hexokinase after desorption is 42 units / mg of protein. The yield on the activity of 61%. c) a chromatographic column filled with sorbent — as in Example 2 (a and b), equilibrated with 50 mM borate buffer solution pH 7.6, containing 0.5 mM EDTA — was applied 3.5 ml of the basic preparation of yeast hexokinase obtained after autolysis 1.6 g of dried baker's yeast for 2 hours at 37 ° C in 5 ml of 042 M Na, HPOiH chromatographic purification on a column of DEDE-cellulose Protein concentration 0.98 mg / mp, specific activity 33 YDP / mg squirrel. Upon elution with 200 ml of borate buffer solution pH 7.6, the ballast proteins are washed out. The active hexokinase eluted with an increase in the concentration of succinic acid from 0.001 to 0.05 M in the initial borate buffer solution pH 7.6. The specific activity of hexokinase 300 units / m1 protein. Bbaxi, d in activity not lower than 50%. d) in a chromatographic column as in Example 2, equilibrated with 50 borate buffer solution pH 7.6, 3.5 ml of Fluka (Switzerland) hexocinase enzyme preparation was applied. The protein concentration is 2.0 mg / ml, the specific activity is 31 units / mg protein. Upon elution with 50 ml of initial buffer solution pH 7.6, the ballast proteins are washed out. Active hexokinase is desorbed with a succinic acid of succinic acid from 1 mM to 50 mM in an initial buffer solution of 7.6. The specific activity of the desorbing enzyme is 990 U / mg protein with a yield of 75% activity. Example 2 g of sorbent (the concentration of chromium (III) ions 200 µM / g of sorbent) is loaded into the chromatographic column (1 x 20 cm) equilibrated with 50 mM borate buffer pH 7.6 and 0.5 ml of EDTA is applied, and 3 ml of farm solution are applied yeast hecookinae. 5 obtained after autolysis of 1.5 g of dried Baker's yeast for 2 h at 37 ° C in 5 ml of 0.2 M, and desalting on Sephadex U-25. The protein concentration is 0.98 mg / mp, the specific activity is 33 units / mg. When elution 170ml iv.-.... l.pl. t ,, jL--: ballast proteins are washed out of the original buffer solution. Active hexokinae-alu- mination increases with an increase in the concentration of succinic acid from 0.001 to 0.05 M in a borate buffer solution pH 7.6 with 0.5 mM EDTA. The specific activity of hexokinase is 211 units / mg. The yield on the activity of 45%. Example. 2 g of sorbent (concentration of chromium (III) ions 800 µM / g of sorbent) is loaded onto a chromatographic column (1 x 20 cm), equilibrated with 50 mM borate buffer solution pH 7.6, containing 0.5 mM EDGHA, and 8 ml of enzyme a solution obtained after autolysis of 2.8 g of baker's yeast for 2 hours with wl ml of 0.2 M, chromatographic purification on a column with DEAE-cellulose and desalting on Sephadex U-25. The protein concentration of the enzyme preparation is 1.3 mg / MP, the specific activity is 10.4 units / mg. When elution of 850 ml of the initial buffer solution, ballast proteins are washed out. Active hexokinase is eluted at a turn & amphibian concentration of succinic acid from 0.001 to 0.1 M in borate buffer pH 7.6 with 0.5 mM EDTA. The specific activity of hexokinase is 90.4 units / mg. The yield on the activity of 32%. The proposed method of preparing a sorbent based on polysaccharide - fosfattsellkiyuey through its charging chromium ions (lit) provides, as compared nsha with izaestmymi sposobgmi preparation of sorbents for purification of yeast hexokinase, cheapness, availability of starting vetsestv for producing an adsorbent, simplicity and availability of the method for producing sorbitol: in an unlimited amount, the high efficiency of the sorbent with respect to yeast hexokinase, comparable to the best1 "oOpaaitaMt foreign counterparts, i.e., an increase in the specific activity of the enzyme in one operation of sorption - desorption on average 10-15 times with preservation of the activity of the desorbed enzyme by 50-90%. Claims 1. Method for preparing sorbent for cleaning burnt hexokinase cores by drawing a ligand to a polysaccharide matrix, which differs from using phosphate cellulose in a process of simplifying the process and increasing the sorption capacity of the sorbent as a polysaccharide matrix; Processing with a solution containing chromium (III) ions at a molar ratio of chromium (III) ions and phosphate residues (0.25-2): 1 in 0.080, 1. M acetate buffer solution pH 5.4 to 5.8. 2. The method according to claim 1, from l and h and Ц and с so that the processing of cellulose phosphate with rt-containing solution is carried out at a constant cross-linking for 20-24 hours. Information sources accepted into account when examining 1. Womach F.S., Welch M.K., MIelsenfi, Colowich SP, Purification and 58 ferrological comparison of the isofergants of the yeast hexonokine R-1 and PP, Arch. Biochem, Btophys. 158, 1973, p. 451-457. 2. Lee C, Y., Lagarus L. N., Kakboff D. S., Russel R.V. , Laver Og.M. Kaplan N. 0. Purification of Kinases by General Law Affiliate Chromatography, Archives of Biochem. Biophys, 178, (197.7) 8 (prototype) h. Wright S.L., Warsy A.S., Ho1rayde I.G., Tgaueg G. R., BJochem G., 175 (1978), 125.. 4. Karenman I.M. Photometric analysis. M., Himi, 1975, p. 260