RU2046143C1 - Process for preparing immobilized dismutase superoxide - Google Patents

Abstract

FIELD: biotechnology and chemistry of high molecular biologically active compounds and biological practice for the production of a stable oxygen transfer agent having prolonged antioxidant activity. SUBSTANCE: 1-2% by weight of glutaric aldehyde (GA) is successively added to a 0.4-1.0 $ by weight of dismutase superoxide (DSO) solution at a DSO to GA molar ratio of (95.4-228) and 0.68-5.0% by weight of a hemoglobin (HG) solution at a DSO to HG molar ratio of 1:(1-13.4). The desired DSO derivative is biodestructable in the blood route. It maintains total superoxide activity and has a high level of a gas- transport function, and high MM values promote prolonged activity. The process allows the transfer agent to be covalently added to the polymer, this corresponds to 68-93% by weight of DSO introduced in the reaction. EFFECT: improved properties of the immobilized dismutase superoxide. 2 dwg, 1 tbl

Description

 The invention relates to the chemistry of high molecular weight biologically active compounds and can find application in biochemical practice to obtain a stable oxygen carrier having simultaneously prolonged antioxidant activity.

 A known method for producing covalently immobilized superoxide dismutase (superoxide: superoxide oxidoreductase) (SOD) on polyethylene glycol (PEG). It is implemented using the combination of the following essential features.

в органическом растворителе активировали ПЭГ-5000. К активированному ПЭГ добавляли СОД и вели реакцию при перемешивании при комнатной температуре. Целевой продукт выделяли хроматографией.Cyanurate chloride

PEG-5000 was activated in an organic solvent. SOD was added to the activated PEG and the reaction was carried out with stirring at room temperature. The target product was isolated by chromatography.

 The main disadvantage of this method is the impossibility of biodegradation of the obtained polymer derivative of SOD in the body. In addition, the target product does not have the ability to transfer oxygen.

 The aim of the invention is to give immobilized SOD the ability to biodegradation and gas transport functions.

 The goal is achieved by the method of obtaining immobilized SOD, which is implemented by the following set of essential features.

To an aqueous 0.4-1% (wt.) Solution of SOD with a pH of 6.8-7.4 add a 1-2% (wt.) Aqueous solution of glutaraldehyde (HA) at 5-15 ^about C. The molar ratio of SOD: HA 1: (95.4-228). A 0.68-5% (wt.) Aqueous hemoglobin solution (GB) is added to the reaction mixture with stirring. The ratio of SOD GB 1: (1-13,4). The reaction is completed by the introduction of a reagent that blocks free aldehyde and unsaturated groups that occur during the reaction in immobilized SOD. The target product is isolated by chromatography.

 The target product was characterized by an MM value of 32,000 by the amount of added SOD, the absence of an impurity of SOD that was not covalently bound in it was controlled, the activity of immobilized SOD was determined, and the gas transport function was evaluated.

 An analysis of the known level of science and technology showed that the claimed method for producing immobilized SOD is not known.

 As an illustration of the claimed method are shown in figures 1 and 2 (according to example 1). For the remaining examples, the figures are largely similar.

 In FIG. Figure 1 shows the chromatogram of the reaction products obtained on a column (1.8 x 60 cm) with 6 V sepharose. Optical density along the ordinate axis, and elution volume along the abscissa axis.

Figure 2 shows the oxygen dissociation curves for polymeric GB (PHB) obtained by the interaction of GB isolated from red blood cells with a bifunctional cross-linking agent in aqueous solutions, superoxide dismutase (superoxide: superoxide oxide reductase) М of immobility 1 (curve 2). On the ordinate axis, the saturation of the GB units with oxygen (c) on the abscissa axis P _{O2 is the} partial pressure of oxygen (in mmHg).

It is clearly seen that for curves 1 and 2 P _{50 the} partial pressure of oxygen during the half-saturation of the polymer derivatives with oxygen is practically equal to 24 and 25, respectively. Moreover, the parameter characterizing the cooperativity of the process of oxygen addition to PHB for the compared polymer products is also identical and equal to 1.3.

 SOD activity was determined spectrophotometrically at λ 560 nm by calibrating the recovery of the nitrotetrazolium blue dye in the riboflavin-methionine superoxide radical repair system.

The hemoglobin concentration was determined spectrophotometrically at λ = 540 nm using a cyanomethhemoglobin derivative, the mass-average molecular mass was determined based on MMP on Sepharose 6B using standard calibration proteins in the MM range from 14 ˙ 10 ³ to 900 ˙ 10 ³ .

PRI me R 1. To 1.48 ml of a 0.5% (wt.) Aqueous (0.15 M sodium chloride) solution of SOD slowly add 0.25 ml of 1.9% (wt.) an aqueous solution of HA. The reaction mixture was stirred for 60 min at ¹⁰ ° C and added 4 ml of 5% (wt.) Aqueous (0.15 M sodium chloride) solution Gb. The mixture was again stirred for 60 min at ¹⁰ ° C and added 4 ml of 5% (wt.) Aqueous (0.15 M sodium chloride) solution Gb. The mixture was stirred again for 60 minutes at the same temperature and added dropwise to complete the reaction with 0.26 ml of a 0.7% (w / w) aqueous solution of NaBH ₄ pH 8. The mixture was stirred for 30 minutes and introduced into a chromatographic column with 6 V Sepharose equilibrated 0.04 M phosphate buffered saline pH 7.4. Size exclusion was conducted at 12 ^{° C} at a speed of 13 ml / hr, elution was carried out with 0.04M phosphate buffer pH 7.4. The amount of bound SOD is 6.2 mg, i.e. 84% of the amount of enzyme introduced into the reaction.

 The determination of activity was carried out by the known method of Beyer and Fridovich. The magnitude of activity, the indicator MM PHB and the molar ratio of SOD: GB are presented in the table.

PRI me R 2. To 1.11 ml of a 1% (wt.) Aqueous (0.15 M sodium chloride) solution of SOD pH 7 add 0.2 ml of a 1.9% aqueous solution of HA. The reaction mixture was stirred for 45 min at ¹⁵ ° C and add 3 mL of 5% aqueous solution (0.15 M sodium chloride) solution Gb. The mixture was stirred for 90 minutes at the same temperature and 0.39 ml of a 0.38% (w / w) aqueous solution of NaBH ₄ pH 8.6 was added dropwise to complete the reaction. The mixture is stirred for 30 minutes. The solution was gel chromatographed under the conditions of Example 1. The amount of bound SOD was 10.32 mg, i.e. 93% of the amount of enzyme introduced into the reaction.

PRI me R 3. To 2 ml of a 0.65% (wt.) Aqueous (0.15 M sodium chloride) solution of SOD pH 7.4 slowly add 0.375 ml of a 1% (wt.) Aqueous solution HA at t ^about 12 ° C. The reaction mixture is stirred for 60 minutes at the same temperature and 5 ml of a 1% (wt.) Aqueous (0.15 M sodium chloride) GB solution are added. The mixture was stirred for 120 minutes at the same temperature, after which a 0.55 minute 0.27% (wt.) Aqueous solution of NaBH ₄ pH 9 was added to complete the reaction. After stirring for 30 minutes, the mixture was gel chromatographed under the conditions of Example 1. bound SOD 9.6 mg, i.e. 74% of the amount of enzyme introduced into the reaction.

PRI me R 4. To 0.8 ml of a 0.90% (wt.) Aqueous (0.15 M sodium chloride) solution of SOD pH 7.2 slowly add 0.126 ml of 1.9% (wt. .) aqueous solution of GA at 5 ^C. the reaction mixture was stirred for 60 minutes at the same temperature and add 2 ml 0.68% solution (wt.) aqueous (0.15 M sodium chloride) hemoglobin solution. The mixture was stirred for 240 minutes at the same temperature. To complete the reaction add 0.29 ml of a 0.3% (wt.) Aqueous solution of NaBH ₄ with a pH of 8.2. After stirring for 30 minutes, the mixture was gel chromatographed under the conditions of Example 1. The amount of covalently bound SOD was 4.9 mg, i.e. 68% of the enzyme introduced into the reaction.

 Examples 5-12 are made under the conditions of example 1. All data are summarized in table.

Biological tests were carried out at the Leningrad Research Institute of Hematology and Blood Transfusion of the Ministry of Health of the RSFSR. The drug with MM (130-220) × 10 ³ was administered to five dogs (weighing 16-17 kg), 25 rats (weighing 0.20-0.25 kg), 10 rabbits (weighing 2-2.5 kg). The drug was administered six times within 2 weeks intravenously (rabbits in the ear vein, rats in the tail vein, dogs in the femoral vein). 3 days after completion of the course of administration (1% solution at a dose of 1 to 4 g / kg of animal weight), insignificant hemosiderin granules were observed in the kidneys and spleen. After 14 days, only traces of granules were observed. After 30-45 days, no traces were recorded.

 Analysis of the experimental material and these figures allows us to draw the following conclusions. The objective of the invention is achieved, firstly, due to the fact that the derivative is biodegradable in the bloodstream, and secondly, it fully retains superoxide activity and at the same time is characterized by a high level of gas transport function, i.e. ability to reversible oxygenation deoxygenation. High MM conjugate provide a prolonged action, (the half-life of immobilized SOD is 10-12 hours, the period of complete elimination of the initial SOD is about 10 minutes), thus the prolongation in time is 60-72 times longer.

 The claimed method provides covalent attachment of 68-93% by weight of the SOD introduced into the reaction, which ensures high efficiency of the method.

Claims (1)

METHOD FOR PRODUCING AN IMMOBILIZED SUPEROXIDE DISMUTASE, which involves the interaction of aqueous hemoglobin solutions, an enzyme and glutaraldehyde, the completion of the reaction by the introduction of a reagent blocking free aldehyde and unsaturated groups, followed by isolation of the target product by chromatography, characterized in that 0.1 is added to the reaction by adding 0 water to 4% solution of superoxide dismutase (SOD) pH 6.8 7.4 1.0 2.0% aqueous solution of glutaraldehyde (HA) at a molar ratio of 1: (95.4 228) respectively, and 0.68 of a 5.0% aqueous hemoglobin solution (GB) with a ratio of SOD: GB. 1: (1 13.4), respectively, and a temperature of 5 15 ^o C.

Images