RU2016901C1 - Immobilized catalase - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves immobilization of catalase with immunoglobulin and polystyrene. Immobilized enzyme shows stability and can be used repeatedly. EFFECT: increased activity of catalase. 5 tbl

Description

 The invention relates to enzymology and can be used for research purposes and in the production of blood products to improve their quality.

 Known immobilized composition based on catalase, albumin associated with mushroom mycelium using glutaraldehyde and complexing buffer, used for analytical purposes and in the food industry [1].

 When catalase stabilizes blood products (immunoglobulin and albumin), the quality of the drug (physico-chemical properties) improves. The level of fragmentation, malondialdehyde is reduced. However, the degree of opalescence does not differ from the control. In addition, the introduction of catalase into blood preparations sets the task of liberation from the enzyme, since the drugs used in medicine should not contain any impurities.

 The purpose of the invention is the creation of a sorbent KIGS-1 based on immobilized catalase to improve the physico-chemical properties of blood products.

 This is achieved by a composition of compounds based on catalase and immunoglobulin (chemically) associated with polystyrene (physicochemical), used in accordance with the sorbent for purification of blood products from activated oxygen species.

Significant differences of the new biologically active substance (BAS) are
stability of the catalase enzyme in an immobilized state;
increased catalase activity.

 The method is as follows.

An equivalent volume of catalase solution containing 2.8-3.2 mg is added to 5-6 ml of a donor immunoglobulin solution diluted with 0.01 M phosphate buffer pH 7.0-7.4 to a concentration of 5-6 mg protein in 1 ml protein in 1 ml, stirred for 5-7 minutes and 0.5 ml of a 1% aqueous solution of glutaraldehyde are poured dropwise; stirred for 2.5-3 hours at room temperature, centrifuged at 15,000 rpm for 25-30 minutes, the supernatant is an immunoglobulin-catalysis complex. The immobilization of the complex is carried out by adsorption on polystyrene granules with a size of 3x5 mm. To this end, the volume of the resulting supernatant was adjusted with 0.01 M phosphate buffer, pH 7.0-7.2 to 50-60 mL, poured polystyrene plates (30-32 g) and left for 18 hours at 4 ^{° C;} unbound protein is removed by washing with 100-120 ml of physiological saline, pH 7.0-7.2. The resulting KIGS sorbent-filled column 1 and passed therethrough at a liquid blood product flow rate of 120-150 ml / h at 25 ° ^C.

 PRI me R 1. The choice of the optimal pH of the buffer upon receipt of the sorbent KIGS-1.

 Upon receipt of the KIGS-1 sorbent, the following pH values of the complexing buffer were used: 5.0; 7.0; 7.2; 7.4; 8.6; 9.6

 As can be seen from the data in table 1, the optimal pH of the complexing buffer is 7.0-7.4.

 In addition, as a result of the immobilization of catalase and immunoglobulin, the specific activity of the enzyme increases significantly. If before immobilization it amounted to 35,000 conventional units, then after an activity of 42,000 units was observed. activity.

 The use of immobilized catalase as part of the KIGS-1 sorbent for stabilizing blood products.

The resulting sorbent KIGS-1 (based on immobilized catalase) fill the column and passed therethrough at a liquid blood product flow rate of 120-150 ml / h at temperature of 20-25 ^C. The efficacy of the sorbent are shown in Table 2, 3, 4, 5.

 As the data in the tables indicate, the efficiency of purification of blood products - immunoglobulin and albumin with the KIGS-1 sorbent exceeds the efficiency of catalase taken as a prototype. In addition, the use of a sorbent as a stabilizer allows it to be used repeatedly for these purposes, and foreign substances do not enter the blood devices.

According to the data in Table 3, the physicochemical properties of albumin passed through the KIGS-1 sorbent are significantly improved in all respects, while those stabilized by the prototype method are characterized by a high degree of opalescence. The paper presents data in preparation accelerated aging (1 month at ³⁷ ° C).

 Passing albumin through the KIGS-1 sorbent also contributes to an increase in the% of monomer and prevents the polymerization of albumin (Table 4).

 As the data in Table 5 show, the transmission of immunoglobulin through the KIGS-1 sorbent based on immobilized catalase significantly improves the physicochemical properties of immunoglobulin compared to catalase-stabilized immunoglobulin.

 Thus, passing through the KIGS-1 sorbent based on immobilized catalase significantly improves the physicochemical properties of albumin and immunoglobulin.

 Proof of freedom of active centers - immunoglobulin and catalase enzyme in the immobilized immunoglobulin + catalase complex.

 The immunological activity of immunoglobulin and the enzymatic activity of catalase were tested as follows.

 In the wells of a polystyrene tablet (Flow company), rabbit antiserum was titrated to human immunoglobulin class G 0.01 M carbonate-bicarbonate buffer pH 9.6 to titers 20480, incubated. Antiserum is bound to polystyrene, unbound serum is removed by washing three times with tap water and washing three times with saline with Tween-20 detergent at a concentration of 0.05%.

Then, a catalase + immunoglobulin complex of 0.2 ml is added to the wells. Plates were incubated for 1 hour at ³⁷ ° C, unbound complex with antiserum catalase + immunoglobulin washed as above. The wells of the plates is made as indicator systems for catalase enzyme activity, 0.2 ml of 0.025% H ₂ O ₂ (hydrogen peroxide), with incubation for 10 minutes at ³⁷ ° C, then 0.2 ml of a 4% solution of ammonium molybdate with incubation at room temperature for 30 minutes In the wells where catalase reacted with H ₂ O ₂ peroxide, no yellow staining was observed.

 The titer of the drug in the experiments was 10,240. The more immunoglobulin, the more catalase, the less pronounced yellow staining. Persistent yellow staining was observed in the control rows. Therefore, the evidence of the action of the active center of an immunoglobulin is its interaction with antiserum: the safety of the catalase center is interaction with hydrogen peroxide.

 Thus, this experiment proves that the active centers of immunoglobulin and catalase remain free in this immobilized complex.

-R-

=N-Jg,, где К - остаток каталазы;
I_g - остаток иммуноглобулина;
R - остаток глутарового альдегида.Thus, the structural formula of the immobilized complex is as follows
KN =

-R-

= N-Jg ,, where K is the rest of the catalase;
I _g is the remainder of the immunoglobulin;
R is the remainder of glutaraldehyde.

 Catalase and immunoglobulin are linked by glutaraldehyde containing two aldehyde groups at both ends of the chain. These groups react at neutral pH with free amino groups. One end of glutaraldehyde is attached to catalase lysine, the other to immunoglobulin lysine. With polystyrene, binding occurs physically and chemically through hydrophobic addition. The active centers of catalase and immunoglobulin remain free, which is manifested in their specific action - binding of antigens (antiserum) and decomposition of hydrogen peroxide (lack of staining with ammonium molybdate).

 A positive effect is achieved by increasing the activity of the enzyme catalase due to its immobilization (with immunoglobulin and polystyrene), as well as the stability of the enzyme and, as a result, the possibility of using it for repeated use.

Claims (1)

IMMOBILIZED CATALASE of the general formula
KN =

-R-

= N-Jg ,,
where K is the residue of catalase;
Jg - the remainder of the immunoglobulin as a sorbent to improve the physico-chemical properties of blood products from activated forms of oxygen;
R is the remainder of glutaraldehyde.

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