Method of producing biospecific polymer sorbent for extracting proteinase

FIELD: chemistry.

SUBSTANCE: in order to produce a biospecific polymer sorbent for extracting proteinase, an aqueous solution undergoes radical polymerisation, where said solution contains 0.1-1.5 wt % ovomucoid from duck egg albumen, acylated with acyl chloride of acrylic or methacrylic acid, 7.0-20.0 wt % hydrophilic monomer consisting of acrylamide, methacrylamide or N-vinylpyrrolidon, and 0.7-15.0 bifunctional cross-linking agent. The hydrophilic monomer is further mixed with N-ethylbromide dimethylaminoethyl methacrylate, taken in amount of 45-55 wt % with respect to weight of the hydrophilic monomer.

EFFECT: addition of N-ethylbromide dimethylaminoethyl methacrylate to the hydrophilic monomer increases efficiency of sorption of proteinase from biological fluids.

1 tbl, 2 ex

 

The invention relates to the field of polymer chemistry, biochemistry and medicine, namely to a method for biospecific polymer Sobota, which is used to remove proteolyses enzymes from blood and other biological liquids with the purpose of detoxification in pathological conditions involving activation of proteolysis and enzyme intoxication, including sepsis, purulent peritonitis, pancreatitis, asthma, etc.

A method of obtaining biospecific polymer sorbent for separation of proteases by the interaction of trypsin inhibitor from soy activated cross-linked polysaccharide - separate [Gilliam E.V., Kitto G.B., Isolation of starfish trypsin by affinity chromatography, Comparative Biochemistry and discrimination, 1976, V.54, No.1, p.21-26].

The disadvantage of this method is the low efficiency of sorption component of 0.2 mg of enzyme per 1 mg of chemically bound to the polymer inhibitor.

The closest in technical essence and the achieved results is the method of obtaining a biospecific polymer sorbent for separation of proteases by radical polymerization of an aqueous solution containing 0.1 to 1.5 wt.%, ovomucoid of protein duck eggs, acylated with the acid chloride of acrylic or methacrylic acid, a 7.0-20.0 wt.%, hydrophilic monomer and 0.7-15.0 wt.% bifunctional cross-linking agents is [USSR Author's certificate No. 1137388 A, G01N 33/50, bull. No. 4, 1985]. As the hydrophilic monomer used acrylamide, methacrylamide or N-vinyl pyrrolidone. The effectiveness of the sorbent in the sorption of proteinases from the dog's blood or human blood plasma is 0.8-1.0 mg of enzyme per 1 mg associated with polymer ovomucoid.

The disadvantage of this method is the low efficiency of sorption when removing proteases of the blood. The reason for this is that the optimum pH for the action of ovomucoid equal to 8.0, the maximum capacity of the sorbent possesses when removing proteases from a solution with a pH of 8.0.

However, the pH of the blood is different from this value and is approximately 7.4.

The objective of the invention is to increase the efficiency of the sorbent in the sorption of proteinases of the blood and plasma.

The technical result that is achievable with the use of the invention is to increase the efficiency of the sorbent in the sorption of proteinases of the blood and plasma.

The technical result is achieved in that in the method of obtaining a biospecific polymer sorbent for separation of proteases by radical polymerization of an aqueous solution containing 0.1 to 1.5 wt.%, ovomucoid of protein duck eggs, acylated with the acid chloride of acrylic or methacrylic acid, a 7.0-20.0 wt.%, hydrophilic monomer, consisting of acrylamide, methacrylamide or N-vinylpyrrolidone, and 0.7-15.0 wt.% the all linking agent, in the hydrophilic monomer is further added N-ethylbromide dimethylaminoethylmethacrylate, taken in an amount of 45 to 55 wt.%, in relation to the weight of the hydrophilic monomer.

As a cross-linking agent used is N,N-methylenebisacrylamide (BIS) or fluids methacrylic acid and polyoxyethyleneglycol with a degree of polymerization of 3 (TGM-3) or fluids methacrylic acid and polyoxyethyleneglycol with a degree of polymerization of 13 (TGM-13).

Used ovomucoid is a glycoprotein with molecular weight of 31,000 and contains two binding site of trypsin and one binding site of chymotrypsin, i.e. in the limit of 1 mg ovomucoid can bind up to 1.5 mg of trypsin and 0.7 mg of chymotrypsin.

Ovomucoid separated from protein duck eggs by the method of [Shulgin M.N., Valuev T.A., Koester YA, Mosolov CENTURY Properties duck ovomucoid, purified by the method of affinity chromatography on triplinerve, Biochemistry, 1981, vol.46, No. 3, s-480].

N-ethylbromide of dimethylaminoethylmethacrylate (EB-DMA) is produced by adding a 0.035 mol of dimethylaminoethylmethacrylate dropwise with stirring to 0.8 mol of ethylbromide at 0C in the presence of the polymerization inhibitor is hydroquinone in amounts of 1 wt.%. The formed precipitate is filtered off, washed with ether and dried. The melting point of DL-DMA equal to 103C.

The mechanism of action of N-ethylbromide of dimethylaminoethylmethacrylate EB-DMA) is to give crosslinked polymer of a positive charge, that leads to a change in local pH at the active center of ovomucoid and provides the offset of the effective optimum pH for the action of ovomucoid from 8.0 to 7,35-7,45.

Example 1.

In 91 ml of bicarbonate buffer (pH 8.0) dissolve 1.0 g of ovomucoid. To the solution at 0C is added 0.01 ml of the acid chloride of acrylic acid (HAACKE). The reaction mixture was stirred at 0C for 15 minutes. To the solution add of 3.85 g of acrylamide and 3.15 g of DL-DMA, 1.0 g of BIS and the polymerization initiator is 0.01 g of ammonium persulfate and 0.2 ml of 0.4%solution of N,N,N',N'-tetramethylethylenediamine. Through the solution for 10 minutes to let the nitrogen to remove dissolved oxygen. The polymerization carried out by keeping the reaction mixture at 10C for 20 minutes.

The resulting sorbent crushed by pushing through a sieve with a pore diameter of 1.0 mm and washed with water to remove unreacted substances.

The completeness of the removal spectrophotometric control.

The number associated ovomucoid is determined by the difference between the initial concentration of ovomucoid and its concentration in the wash water. It is equal to 4.2 mg per 1 g of sorbent. Then the sorbent is kept in a physiological solution (0.9%NaCl).

To study the properties of the sorbent he is placed in a column of 30 ml and passed through the column with 250 ml of human blood plasma containing trypsin.

Number is the number of adsorbed trypsin is determined by rinsing it with speakers aqueous solution with a pH of 1.5.

The composition of the initial solution and the properties of the sorbent in the table.

Examples 2-12.

The process is conducted according to example 1, using various initial connection.

The composition of the initial mixture and the properties of the sorbents is given in the table.

# exampleOriginal mix*, g/100 g of solution (wt.%)The content of ovomucoid in the sorbent (mg/gThe capacity of the sorbent, mg trypsin/g sorbentThe effectiveness of adsorption, mg trypsin/mg ovomucoid
OvomucoidThe monomerEB-DMAA crosslinking agent
11.0 KHAAK3,85 AA3,151,0 BIS2,63,35***1,28
21.0 KHAAK3,15 AA3,850,7 BIS1,9of 2.26***1,19
31.0 KHAAK3,5 AA3,50,7 BIS1,31,70***1,21
41.0 KHAAK10,0 AA10,01,5 BIS2,83,13**1,12
50.1 KHAAK5,5 AA4,515,0 TGM-130,91,33***1,47
60.8 CHAMAK6,75 AA8,2510,0 TGM-132,33,00**1,30
71.5 KHAAK5,0 MAA5,08,0 TGM-33,23,81***1,19
8 1.5 km CHAMAK4,0 PG4,010,0 BIS2,02,55***1,28
90.5 KHAAK3,5 EAP3,51,5 BIS1,11,63***1,48
100.1 CHAMAK4,5 MAA5,55,0 TGM-30,60,86**1,43
111.5 KHAAK7,5 AA7,51,5 BIS2,42,90***1,21
121,2 KHAAK7,5 AA7,51,5 BIS1,51,85***1,23
* KHAAK - acid chloride of acrylic acid, HAMAC - acid chloride meta is iloveu acid, AA - acrylamide, MAA - methacrylamide, EAP - N-vinyl pyrrolidone.
** Sorption from the blood of dogs.
*** Sorption from human blood plasma.

It is seen that the introduction of the sorbent positively charged monomer increases the efficiency of sorption of proteases from biological fluids by 20-50% compared to the prototype (from 0.8 to 1.0 to 1.19 to 1.47 mg of enzyme/mg of ovomucoid).

Limit the number of monomers for obtaining the sorbent is determined by the fact that when the content of the links of the monomers in the sorbent optimum pH for the action of ovomucoid equal to the pH of the solution, which is sorption.

Thus, the present invention allows to obtain a biospecific sorbents proteases with high sorption efficiency of proteases from biological fluids.

In addition, since the main contribution to the cost of the sorbent contributes ovomucoid, the use of the proposed method will allow more efficient use of ovomucoid and significantly reduce the cost of operation of sorption.

A method of obtaining a biospecific polymer sorbent for separation of proteases by radical polymerization of an aqueous solution containing 0.1 to 1.5 wt.% ovomucoid of protein duck eggs, acelerando what about the acrylic acid chloride or methacrylic acid, a 7.0-20.0 wt.% hydrophilic monomer, consisting of acrylamide, methacrylamide or N-vinylpyrrolidone, and 0.7-15.0 wt.% bifunctional cross-linking agent, characterized in that the hydrophilic monomer is further added N-ethylbromide dimethylaminoethylmethacrylate, taken in an amount of 45 to 55 wt.% in relation to the weight of the hydrophilic monomer.



 

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