RU2007423C1 - Method of serum albumin purification - Google Patents

Abstract

FIELD: analytical and preparative biochemistry. SUBSTANCE: initial preparation or albumin is precipitated with trichloroacetic acid from serum blood directly. Precipitate is dissolved in ethanol acidified with hydrochloric acid and subjected for gel-filtration using acidified ethanol as a solvent for elution. Then protein is isolated from eluate obtained by ultrafiltration, salting out with sodium acetate or by precipitation with alkali. Protein is lyophilized, and a fraction of native serum albumin is isolated. EFFECT: simplified process, retained functional activity of the end product.

Description

 The invention relates to analytical biochemistry and molecular biology. It can be used to obtain standard organophilic ligands free of standard serum albumin preparations used in experimental and clinical medicine, in biotechnology, in the work of biochemical, biophysical, hematological, pharmacological and immunological laboratories.

A known method for the purification of blood plasma albumin from associated non-covalent bonds of fatty acids and minor organophilic ligands, which consists in the fact that the initial preparation of serum albumin is first subjected to fractional salting out with ammonium sulfate in the range of pH 7.0-6.0 and pH 6.0 -4.0. The last fraction is desalted and lyophilized. The lyophilized protein was subjected to extraction four times with a mixture of chloroform and ethanol at a volume ratio (5: 1) - (8: 1) for 12-18 minutes at ^about 36-38, wherein the first extraction is carried out with the addition of concentrated HCl to a final concentration in the extracting mixture 10 ^-3 - 10 ^-4 M.

 To isolate the native albumin fraction, the extracted, thoroughly dried in vacuum protein is again subjected to fractional salting out with ammonium sulfate to isolate the fraction which is first salted out in the pH range 7.0-6.0, which is discarded and then salted out at pH 6.0-4.0 , which is desalted, lyophilized.

 The main disadvantage of this method is that the thus purified serum albumin has low functional activity as a transporter of endogenous organic anions, in particular bilirubin. In particular, for human serum albumin purified by this method, camel or cattle, this activity, determined by the method developed in the Institute of Biology of the USSR Academy of Sciences, is 20.9, respectively; 21.4 and 19.1% of the initial concentration of bilirubin bound to membrane particles.

 Apparently this is due to the fact that chloroform has a strong denaturing albumin effect and / or that chloroform, extracting fatty acids and minor organophilic ligands from a molecule of serum albumin, can itself quite strongly bind a protein molecule as a ligand as a halide-containing hydrocarbon.

A disadvantage of the method is furthermore the fact that the manipulation of the extraction mixture containing chloroform at 36-38 ^{° C} unsafe for human, as relating to low-boiling halogenated hydrocarbons is chloroform hepatic strong poison.

 The disadvantage of the method also lies in its complexity and cumbersomeness (repeated repeated extraction, repeated fractional salting out of the protein, etc.).

 The aim of the invention is to preserve the functional activity of purified serum albumin as a conveyor of endogenous organic anions and to simplify the method.

 This goal is achieved by the fact that the proposed method for the purification of serum albumin from associated ligands, namely, that the initial preparation is dissolved in acidified (concentrated hydrochloric acid) ethanol, subjected to gel filtration, using acidified (concentrated hydrochloric acid) ethanol as an eluting solvent, and isolated from the eluate protein, lyophilize and isolate the fraction of native serum albumin.

 Protein from the eluate is isolated by ultrafiltration, or salting out with sodium acetate or alkali precipitation.

 The native serum albumin fraction is isolated from the lyophilized purified protein preparation by gel filtration of a fraction of the drug dissolved in distilled water obtained by salting out with ammonium sulfate at pH-5.0, and lyophilized.

 Ethanol is selected as a solvent in the proposed method from all possible organic solvents. The choice is due to the fact that ethanol as an endogenous metabolite is present in the circulation system, constantly in contact with serum albumin. Ethanol is used in the form of water-alcohol mixtures, in particular for the systematic fractionation of blood plasma proteins. The presence of water in such mixtures makes it necessary to carefully and very difficultly observe different modes of their cooling, changing values of their ionic strength and changing their electrolyte composition, which makes it possible to obtain discrete protein fractions, avoiding the action of ethanol denaturing proteins in water-alcohol mixtures.

It was possible to find a simple condition that allows us to use not water-alcohol mixtures, but 98% ethanol cooled to - (5-7) ^о С as a solvent of a dry whey protein preparation, which completely eliminates the mentioned difficulties that arise when working with water- alcohol mixtures. This condition is a certain acidification of ethanol. For the first time it was found that a dry preparation of serum albumin can be almost completely dissolved within 7-10 min, cooled to - 5-7 ^of ethanol, freshly distilled ethanol if after said cooling acidify kontsetrirovannoy 11-12 N HCl to pH 5-7 with ^about equal 1.85-1.95 (hydrogen index of freshly distilled ethanol was 5,20-5,90 at ²² ° C). It should be emphasized that the concept of "hydrogen indicator" here has a conditional meaning, since we are not talking about water, but about ethanol, and is used only for comparative quantitative characteristics.

 For the most complete dissolution of the protein in acidified ethanol, constant vigorous stirring of the initially occurring suspension during the entire specified time of dissolution is necessary. A necessary and sufficient condition for such a dissolution is the nativeness of the protein, the absence of hemolysis during the isolation of the protein from blood serum and the almost complete absence of other proteins in the plasma (serum) of the albumin preparation. The maximum concentration of albumin dissolved in ethanol (without significant opalescence) is 0.8-1.2 mg / ml.

Acidification of ethanol to pH _{- (5-7)} o _C = 1.95 may not be sufficient to dissolve serum albumin (especially when an extremely large number of organophilic ligands, in particular fatty acids, are associated with the protein, as is the case when working, for example, with yak serum albumin).

Acidification of ethanol to pH _{- (5-7)} o _C 1.85 is impractical, since the insolubility of albumin in ethanol acidified to pH _{- (5-7)} o _C = 1.85, indicates that the albumin preparation is either devoid of nativeness either contains too much gel or its derivatives, or contains an admixture of other proteins in the blood plasma (serum) that counteract the dissolution of albumin. In model experiments, it was first discovered that gel-filtration of an ethanol solution of serum albumin using acidified to pH _{- (5-7)} o _C = 1.85-1.95 ethanol as an eluting solvent allows a clear separation of higher fatty acids, for example palmitic or oleic acid, from protein. Analytical thin-layer chromatography of a chloroform extract of the eluted protein purified by the proposed method shows that such filtration allows to completely remove not only unesterified fatty acids from the protein of the preparation, but also the mentioned minor organophilic ligands. When the total content of non-esterified fatty acids in the initial serum albumin preparation (based on the amount of protein) is 3-7 mol / mol, their total content in the eluted protein is based on the results of Weigel W. colorimetry calibrated by palmitic acid (Die Pharmazie 1956, N 12, S 786-791), does not exceed 0.001-0.002 mol / mol.

 It was also first discovered that the gel filtration described allows trichloroacetic acid to be separated from serum albumin dissolved in acidified ethanol. This circumstance opens up the possibility of using for the proposed purification procedure albumin pre-precipitated with trichloroacetic acid directly from Korner A Debro I. R. blood serum (Nature 1956, V. 178, N 4541, p. 1067-1069), which significantly expands the possibilities of protein purification. Without gel filtration described, precipitation of albumin from blood serum with trichloroacetic acid followed by selective dissolution of precipitated albumin is not feasible from the point of view of further use of albumin, since trichloroacetic acid is energetically bound by precipitated albumin (Scatchard G. Black ES // J Phys Colloid Chem. 1949, V 53, N 1, p. 88-99).

 As established in the experiments, either ultrafiltration, or salting out, or alkalization of ethanol can be used to isolate whey protein from the eluate obtained by gel filtration.

Crystalline sodium acetate was selected to salify the protein from acidified ethanol. The choice is due to the fact that sodium acetate as a metabolite is present in the in vivo circulation system and is constantly in contact with serum albumin, because sodium acetate is soluble in acidified ethanol, and also that the Na ⁺ cation and the CH ₃ COO anion ^are practically not bound by serum molecules albumin.

To isolate serum albumin from acidified ethanol, in addition to salting out the protein by saturating ethanol with crystalline sodium acetate, alkalization of ethanol to pH _{- (5-7)} o _C 7.50-8.00 can be used. This alkalization is achieved by introducing into the albumin, acidified with ethanol, a micro amount of saturated (14.4 g) NaOH on a thin glass rod with constant potentiometric control. With this alkalization, the protein dissolved in ethanol completely precipitates. When ethanol is alkalized to pH _{- (5-7)} o _C 7.50, the precipitation of the protein in the precipitate is, according to the results of spectrophotometry in the UV region of the supernatant, incomplete. Alkalization of ethanol to pH _{- (5-7)} o _C 8.0 is impractical, since protein precipitation at pH _{- (5-7)} o _C = 8.0 is complete.

Both when salting out the protein and when it is precipitated by alkalization of ethanol, it is necessary to thoroughly mix the mixture, followed by 10-20 minutes incubation at - (5-7) ^о С for more compact formation of the protein precipitate, which is separated by filtration at - (5-7) ^about C, and lyophilized.

To isolate native albumin from a lyophilized protein, purified from the ligands associated with it, the resulting preparation is dissolved in distilled water, bringing the pH of the solution to 7.20 - 7.20 (1-2 ^about ). At pHH _1-2 o _C <<7.20, the dissolution of the lyophilized protein occurs very slowly and is incomplete. At p H _1-2 ° _C 7.40, dissolution occurs quickly and is almost complete. Therefore, alkalization to pH _1-2 o _C > 7.40 is impractical. A saturated solution of ammonium sulfate is added to the resulting solution to a final concentration of 2.8-3.0 M necessary for salting out serum albumin. The pH of the protein solution is adjusted to pH _1-2 ° _C 6.0, adding a micro amount of 0.2 N. Hcl under constant potentiometric control and with constant stirring. Then the sample is left to stand for 20-30 min incubation at 1-2 ^{° C,} after which vysolenny protein was separated by centrifugation (10000g for 30 minutes 1-2 ° ^C) and discarded. Hydrogen index of the supernatant was adjusted to 5.0 (1-2 ^C), and after 20-30 minutes incubation at 1-2 ^{° C} the protein precipitate collected by centrifugation (see. Above), was dissolved in a small amount of distilled water, adjusted pH value solution to 7.0 (1-2 ° ^C), filtered through a column of Sephadex G-75, using as the eluting solvent distilled water (pH _1-2 o _C 7,0) and protein containing portions of the eluate lyophilized. Lyophilized protein is serum albumin purified from higher fatty acids and other minor organophilic ligands, since it is albumin that is salted out with neutral salts at the isoelectric point only at a 70-100% salt solution saturation.

All operations with alcoholic solutions of serum albumin is carried out at a constant temperature - (5-7) ^C. At temperatures above -5 ^{° C,} as shown by observation becomes possible loss of certain protein functional activity of its bilirubin transporter as determined in vitro.

Cooling to a temperature below -2-7 ^C. impractical because a sizeable increase in the functional activity of said protein compared with the activity being found at a temperature of -7 ^{° C,} does not.

All operations with aqueous solutions of serum albumin is carried out at a constant temperature of 1-2 ° ^C. Such cooling is necessary to limit as freed from organophilic ligands serum albumin is very sensitive to an increase in environmental temperature. Cooling to a temperature below 1 ^{° C} is undesirable because of the danger of freezing water environments. Cooling to a temperature above 2 ^{° C} is undesirable due to said thermal lability of the purified albumin.

EXAMPLE 1. For gel filtration, a vertical glass column (40x2.5 cm) was used, completely surrounded by a glass casing, in the lower part of which a special "process" is provided to remove accumulated during operation in the space between the column and the melt water casing. This space was completely filled with a mixture of ice and NaCl (8-12%), which cooled the contents of the column to - (4.9-7.5) ^о С. The column was filled with Sephadex LH60 after daily swelling in acidified 98% ethanol at - ( 4.9-7.5) ^about C. The same acidified ethanol was used as an eluting solvent at a flow rate through the column of 1.5 ml / min. The column was considered ready for operation in the complete absence of light absorption at λ 280 nm in the resulting eluate portions.

A portion (15 mg) of the dried human serum albumin (Blood transfusion station, Vitebsk), placed in chilled to - (4,8-7,5) ^C glass weighing bottle with a ferromagnetic agitating element. The weighing bottle measure 10 ml chilled to - (4,9-7,5) ^C. 98% ethanol and 3.5 l of 11 n. Hcl (the final concentration of Hcl was in this case ≈ 3x10 ^-3 M). With constant vigorous stirring of the contents of the box, complete dissolution of the protein occurred within 7 minutes.

The resulting ethanolic albumin solution was applied to a Sephadex LH60 column. Protein was eluted in the above mode using spectrophotometry of the resulting eluate portions (D ₂₈₀ ) to construct an elution profile. Portions of the eluate containing protein, identified by the absorption of light at λ 280 nm, were combined and then subjected to ultrafiltration using an FM 02-10 filter cell. Protein isolated by ultrafiltration was lyophilized.

For the isolation of a purified native protein albumin lyophilized protein was dissolved in 2-3 ml, cooled to 1-2 ^{° C} with distilled water (pH _1-2 o _C 7,4). Crystalline ammonium sulfate was added to the protein solution to a final concentration of 28 M with constant vigorous stirring of the mixture. The addition of 0.1 N. Hcl the pH was adjusted to 6.0. Vysolenny protein was separated by centrifugation (18,000 g, 1-2 ^{° C,} 10 min) and discarded. The addition of 0.1 N. Hcl the pH of the supernatant was adjusted to 5.0. Salted protein was separated by centrifugation in the same mode. The precipitate was dissolved in 1.0-1.5 ml of distilled water (pH _1-2 ° _C 7.0). The protein solution was applied to a Sephadex G-75 column. Distilled water was used as an eluting solvent (pH _1-2 ° _C = 7.0). Portions of the eluate containing protein, identified by their absorption of light at λ 280 nm, were combined and lyophilized. The yield of purified native lyophilized protein was 9.8 mg (65%). Its in vitro functional activity as a transporter of organic anions (bilirubin) was 34.5%, which is 1.7 times higher than the activity of the protein purified by the prototype method.

Example 2. To 1 ml of serum of a camel (experiments were carried out at the Institute of Biotechnology of the Academy of Sciences of the People's Republic of Mongolia. Ulan Bator), 120 μl of a 50% aqueous trichloroacetic acid solution was added. After thorough mixing, the mixture was allowed to stand at 1-2 ^{° C} for 10 minutes for tighter clearance precipitate which was separated by centrifugation (5000 g, 1-2 ^{° C,} 10 min). The supernatant was discarded. The thoroughly drained centrifuge tube was added 5 mL of chilled to - (4,9-7,5) ^C. 98% ethanol and 1 .mu.l of 11 n. Hcl (final concentration of Hcl 2.0x10 ^-3 M). The resulting albumin ethanol solution was filtered through Sephadex LH60 as described in Example 1.

Protein-containing eluate portions were pooled. To isolate the protein, 30 mg of crystalline sodium acetate (CH ₃ COONa ˙ 3H ₂ O) was added to the total volume of these portion of the eluate (6 ml). The mixture was left to stand at 1-2 for ^about 30 minutes for a more compact design vysolennogo protein for which separation using centrifugation (10000g, 1-2 ^{° C,} 10 min). After removal of the supernatant, the protein precipitate was lyophilized. Lyophilized protein was dissolved in 1 ml of distilled water (pH _1-2 ° _C 7.4). Further isolation of native albumin from the purified protein preparation was carried out by fractional salting out of albumin with ammonium sulfate, with desalting of the protein and its lyophilization as described in Example 1. The yield of purified lyophilized protein was 22 mg (≈ 63%).

 Its functional activity as a bilirubin transporter, determined in vitro, was 34.8%, which is 1.6 times higher than the activity of the protein purified by the prototype method.

PRI me R 3. To a weighed portion (20 mg) of dry bovine serum albumin (Reanal, Hungary) was added 10 ml chilled to - (4.9-7.5) ^o With 98% ethanol and 9 μl 11 n. Hcl (final concentration of Hcl 11x10 ^-3 M). The ethanol solution was subjected to gel filtration as described in Example 1. In the total volume of protein-containing portions of the eluate, 1 μl of 14 N was added. NaOH with constant vigorous stirring. The resulting protein precipitate was separated by centrifugation as described in Example 2. The yield of purified lyophilized protein was 14 mg (70%). The resulting protein was lyophilized and a native albumin fraction was isolated from it as described in Example 2.

 Its functional activity as a bilirubin transporter, determined in vitro, was 32.7%, which is 1.7 times higher than the activity of the protein purified by the prototype method.

 Thus, the examples of specific performance show that the functional activity of serum albumin of various species as a transporter of organic anions (bilirubin) purified by the proposed method is 1.6-1.7 times higher than the activity of the protein purified by the prototype method . In addition, the proposed method for the purification of albumin is much simpler than the prototype method, since it does not require repeated extraction with highly toxic chloroform and repeated fractional salting out of the protein, and it also allows purification of not only dry commercial albumin preparations, but also the protein precipitated with trichloroacetic acid from blood serum. (56) Copyright certificate of the USSR N 1312947, cl. S 07 K 3/12, 1989.

Claims (2)

1. METHOD FOR CLEANING SERUM ALBUMINE, including fractionation, desalination, freeze drying, characterized in that, in order to simplify the process and maintain the functional activity of the target product, the original human albumin is dissolved in chilled to 4.9 - 7.5 ^o With 98% - ethanol acidified with concentrated hydrochloric acid is subjected to gel filtration, eluted with acidified ethanol and isolated.  2. The method according to p. 1, characterized in that the target product is isolated by ultrafiltration or precipitation with sodium acetate or sodium hydroxide.