DD210693A1 - PROCESS FOR IMMOBILIZING BIOLOGICALLY ACTIVE COMPOUNDS - Google Patents

Abstract

A new method is described for the immobilization of biologically active compounds of low or high molecular weight structure, in which haemoglobin or its prosthetic group Haemin are first bound coordinately to imidazole- or pyridine-containing carrier materials. Subsequent activation of these modified carriers with bifunctional reagents leads to reactive carriers can be used for the immobilization of biologically active compounds. In this way, biologically active immobilizates can be produced with a wide range of applications in medicine, pharmacy, biotechnology, chemistry, agriculture and environmental protection.

Description

-A-

"Method for -immobilization" of biologically active compounds

Field of application of the invention

The invention relates to a method for immobilizing biologically active compounds. The Imraobilisate formed can be used in the transformation of materials or find application in medicine, biotechnology and environmental protection.

Characteristic of the Known Technical Solutions In recent years, a whole series of methods for the immobilization of biologically active compounds have been developed "which have been described in detail in various reviews articles / for example in Mosbach, K., edi. Methods in Enzymology, Vol. 44, Academic Press, New York (1976); Chang, TMS, ed., Bioraedical Application of Immobilized Enzymes and Proteins, Vol. I, Plenum Press, New York (1977). The immobilization of the biologically active compounds can then be achieved by adsorption, gel entrapment, microencapsulation, and covalent bonding. * All these methods are characterized by a whole series of advantages as well as drawbacks, which limit their wide range of applications again and again. to develop simple and inexpensive immobilization methods that yield immobilization products with improved activity and stability.

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From the practical point of view, adsorption is the simplest and most economical method of immobilization. However, biologically active compounds immobilized in this way are also readily desorbed again in the current state of the art, for example by changing the temperature * of the pH or ionic strength, However, this disadvantage is often compensated by the fact that the carriers used for the adsorption can be used repeatedly for adsorptive binding of the biologically active compounds, The disadvantage of easy desorption in the non-covalent bond can be eliminated by covalent binding of the biologically active compounds to insoluble carriers, but this often represents the economically less favorable variant, because reuse of the carrier materials after loss of biological activity in most cases not possible ch is.

Object of the invention

The aim of the invention is to develop a generally applicable immobilization method for biologically active compounds of low and high molecular structure by coordinative bonding * which is characterized by a wide range of applications and high binding stability.

Presentation of the nature of. invention

According to the invention, immobilizates of biologically active compounds are prepared by coordinating hemoglobin or its prosthetic group hemin with heterocyclic-containing, insoluble support materials, activating the formed modified support materials with a chemical reagent, and then covalently bonding compounds having biological activity to the activated support material or by first dissolving soluble conjugates from biologically active

bonds and hemoglobin or hemin, which are coordinatively fixed to heterocyclic, insoluble support materials.

Immobilizates of hemoglobin or hemin (iron (III) -protoporphyrin IX) and heterocyclic-containing supports with covalently bonded imidazole or pyridine can be prepared in a simple manner by combining the components in a suitable solvent and are characterized by a high stability. Coordinative binding of the hemin to the abovementioned carrier materials takes place in organic solvents such as dimethylformamide or hexamethylphosphoric acid or strongly alkaline aqueous solutions, while the hemoglobin is bound in aqueous buffer systems of pH 6 to 12. As insoluble support materials with covalently bonded imidazole and pyridine are those inorganic, organic or bioorganic nature with macroporous structure in question, for example those of porous glass or silica gel, polymers of styrene, acrylate or methacrylate type or those based on polysaccharides such as cellulose or networked extraterrestrials.

The further activation of the modified carrier materials with coordinatively bound hemin or hemoglobin for the immobilization of biologically active compounds is carried out in various variants. The immobilized hemin, for example, is advantageously activated by carbodiimides or by the mixed anhydride method with chloroformates. Much easier immobilized hemin with activated carboxyl groups can be obtained if the coordinate binding with activated esters of hemin, for example the p-nitrophenyl or N-hydroxysuccinimide esters, is carried out with imidazole- or pyridine-containing carrier materials in chlorinated hydrocarbons.

The insoluble support materials with coordinating bound hemoglobin are activated according to the invention preferably with bifunctional reagents such as dialdehydes, quinones, diisocyanates, diisothiocyanates, bisdiazonium compounds, diimido esters, cyanuric chloride and others. Since the hemoglobin with its functional groups serves as the immobilization matrix Depending on the solubility of the bifunctional Aktiierungsreagentien the activation reactions can not necessarily be retained, the activation reactions can be carried out both in aqueous systems and in organic solvents - in the latter often also under denaturation of hemoglobin. ,

High molecular biologically active compounds such as proteins with and without enzymatic activity or else low molecular weight biologically active compounds can be covalently bound to the carrier materials activated by the abovementioned methods, the biologically active compounds suitable for this purpose, for example proteins, coenzymes, enzymes , Antibodies, antigens, haptens, vitamins or hormones must contain nucleophilic functional groups, such as amino-, hydroxyl- or mercapto groups, which are not essential for biological activity. The immobilization of the water-soluble proteins is preferably carried out in aqueous buffer systems at temperatures of 0 40 C ₉ wherein the nature and the pH of the buffer is determined primarily by the stability of the Biomakroraoleküle used haptens, vitamins and hormones, however, depending on the solubility in aqueous systems or organic solvents or their mixtures with the activated support materials are reacted, in which case higher working temperatures than 40 ° C are possible «

On the other hand, it is also possible to produce soluble conjugates of hemoglobin or hemin with biologically active compounds by known methods and to immobilize them. As a rule, conjugates which are capable of coordinating with imidazole- or pyridine-containing support materials can also be used for the processes described above using bifunctional activating reagents, although the same reaction conditions can be used here, in contrast to the heterogeneous reaction described therein homogeneous phases is worked. As a result, when obtaining the hemoglobin or heme-containing conjugates of biologically active compounds, an additional but important step for their isolation often has to be carried out in as uniform a form as possible, for which the methods of gel chromatography or affinity chromatography can be used. The process for immobilizing biologically active compounds by immobilizing their conjugates is indeed more complicated by the additional chromatography steps, but in some cases immobilizates are obtained with substantially higher biological activities. The immobilizates of biologically active compounds which can be prepared by the two types described are distinguished by high binding stability. Desorption by changing the temperature, the pH or the ionic strength does not occur when using these immobilizates. Their application is therefore particularly indicated where changes in the reaction conditions of this type occur and, as a result of the desorption caused by them, losses of the biological activity of the immobilizates occur.

The invention will be explained by examples which are not intended to limit the content.

O u rd e ssuccessful

1 gram of a manufactured according to the DOR-WP 124405 imidazolha It ig en polymers with 250 ml borate buffer (0.1 mol / L; pH-word 9 _a 0) are washed ", 25 mg lyophilized hemoglobin in 5 ml of the same Borate buffer dissolved and the solution is added to the buffered carrier. The suspension is shaken for 3 hours at ambient temperature and then filtered. The filtered-off polymer is washed with 500 ml of sodium chloride solution (1 mol / l) and 500 ml of distilled water, so that the eluate is colorless and then recirculated with 250 ml of phosphate buffer (O ₃ l sal / L. · pH 7.5) Add 10 ml of a 1 % solution of glutaraldehyde in the above phosphate buffer to the polymer and shake the suspension again for 3 hours at ambient temperature. After filtering off the activated carrier, dilute with 250 ml of distilled water and 250 ml of phosphate buffer ( 0 _s 1 mol / L; pH 7.5; + 1 x 10 -5 mol / L EDTA) 500 urease units in 10 ml of the above phosphate buffer are added to the carrier and the suspension is shaken at 5 ° C. for 12 hours. The ureaeira immobilizate is separated off, with 500 ml of Na

- 3 triumchloridlösung (1 mol / L + 1 χ 10 mol / L EDTA) washed * and in the eluate, the unbound urease activity is determined by the pH-state method Thereafter, 70 % of urease · »activity bound to the carrier»

1 g of a pyridine-containing polymer prepared according to DDR-WP 136 269 is loaded with hemoglobin and activated with a 1% glutaraldehyde solution as described in Example 1. The activated polymer is mixed with 250 ml of phosphate buffer (O ₁ , 1Hal / L, pH washed 7 _S 5) _β 50 iag NAD (-SS- Nicotinäinidadenindinucleofcid) are dissolved -in the same phosphate buffer _given} ZUiIt polymers ,, and the suspension is 12 stun "-

shaken at 5 ° C. The carrier is separated and washed with 100 ml of Phoophatpuff (0.1 mol / L, pH 7.0). NAD binding was complete. The activity yield was 10 % when the activity of 50 mg of soluble NAD was 100% (reaction with alcohol dehydrogenase and ethanol).

Example 5

1 Grasira one after Collraan et. al. (D, Amer. Chem. Soc, 96 (1974), 6800) imidazole-containing Polystyrenderivateo is loaded according to Example 1 with hemoglobin. The support is washed with distilled water, a mixture of water and acetone (1/1, v / v) and acetone. 75 mg of 2,4,6-Trichlorosyl-triazine are dissolved in 10 ml of acetone, at 0 - 5 ⁰ C, the hemoglobin carrier is added to the solution, and the suspension is shaken at 5 ° C for 6 hours. The activated carrier is separated and washed with 100 ml of acetone, 250 ml of distilled water and 250 ml of borate buffer (0.1 mol / L, pH 9.0). 50 milligrams of glucose oxidase with an activity of 40 units per milligram dissolved in 5 ml of borate buffer (0.1 mol / L; pH 9.0) was dissolved was added to the activated carrier, and the suspension is shaken for 12 hours at 5 ⁰ C. The support is filtered off, washed with 250 ml of ice-cold sodium chloride solution (1 mol / L), and in the eluate the glucose oxidase activity is determined (according to the o-dianisidine method). No enzyme activity is found in the eluate. One gram of carrier after immobilization has a glucose oxidase activity corresponding to 10 μg of soluble glucose oxidase with an activity of 40 units per milligram.

Example 4 . . ' , , ; 10 'of an imidazole-containing carrier based on the macroporous perl cellulose are loaded according to Example 1 with hemoglobin. The carrier is as described in Example 3, washed with acetone and Ό - 5 ⁰ C and 10 ml of a solution comparison

sets "which is prepared from 100 mg hexamethylene-di-iso · = · cyanate in 10 ral getrocknetera acetone" The suspension is 1 hour at 0 - 5 ⁰ C stored and then agitated for 3 hours at ambient temperature "After filtration, the activated carrier washed with 250 ml of acetone, in vacuo, the residual acetone is filtered off with suction ^ and the support is in 10 ml of an aqueous peroxidase solution with an activity of 310 units per 10 ml registered The suspension is shaken for 12 hours at 5 ⁰ C ο After this time is a complete peroxidase binding occurred because after washing the filtered carrier with sodium chloride solution, neither peroxidase activity nor protein could be detected in the starting material. The immobilizate had a peroxidase reactivity of 110 units per gram of carrier (with guaiacol as substrate) O

1 gram of an imidazole-containing, porous silica-based support prepared according to Leal et al. (CJ * Araer _s Chem. Soc. 97 (1975) 5125) is washed with 250 ml of chloroform, 50 milligrams of hemin-bis-p-nitrophenyl ester Dissolved in 10 ml of chloroform and added to the imidazole-containing silica gel. "The suspension is 8 hours at ambient temperature. The support is washed first with chloroform until the eluate is colorless and then further with acetone and distilled water. 100 milligrams of glucose dehydrogenase are dissolved in 10 ml of borate buffer (O ₄ I mol / L; pH 9 Q). The modified silica gel is added to this solution. The immobilization is carried out at 5 ° C. for 12 hours. The support is then filtered off and washed with sodium chloride solution (1 well / L) and phosphate buffer (O ₈ I mol / L; pH 7 , 5) ₀ to washed in the eluate no longer detectable protein is "1 Gramra this Glucosedehydrogenaseträgers has a © turnover rate of 0.3 rag NADP to NADPH per minute (determined spectrophotometrically) ·

Example 6

1 gram of a carrier prepared by the '$ £ 160 192 of an imidazole-containing polymers, and hemin is kept for 10 hours in 10 ml of absolute acetone and then degassed by applying a vacuum. 250 milligrams of ethyl chloroformate in 10 ml of absolute acetone are added to the suspension while stirring at 0-5.degree. The suspension is stirred for a further 3 hours at 0-5 ° C, and the carrier is separated by filtration and washed with 250 ml of acetone. 100 milligrams of albumin or IgG are dissolved in 25 ml of phosphate buffer (0.1 mol / L, pH 7, 5) and stirred with the activated carrier at 5 ^° C. for 12 hours. After filtration, the product is washed with 250 ml of sodium chloride solution (1 mol / L). To 1 gram of the carrier, 84 milligrams of albumin or 60 mg of IgG are bound.

Example 7

20 milligrams of lyophilized hemoglobin and 300 milligrams of urease with an activity of 3 units per milligram are dissolved in 50 ml of phosphate buffer (0.1 mol / L, pH 7.5) and added with 2 ml of a 25 % glutaraldehyde solution. The solution is stored for 4 hours at 5 ⁰ C and then treated with 1 gram of an imidazole-containing polymer prepared according to the DDR-VVP 124 405. The suspension is stirred for 12 hours at 5 ⁰ C and then filtered. The polymer is washed with sodium chloride solution (1 mol / L) and its activity is determined by the pH-state method with urea as substrate. One gram of the carrier has an activity of 255 units. However, after gel chromatographic purification of the conjugate, one gram of carrier has an activity of 450 units.

Claims (2)

1, Process for the immobilization of compounds with nucleophilic properties possessing functional groups of low and high molecular weight structure, characterized in that. Hemoglobin or its prosthetic group hemin to insoluble carrier materials having covalently bonded 3-nitro heterocycles having at least one endocyclic nitrogen atom having free electron pair coordinate activated the modified carrier drug oils formed with a chemical reagent and functionalizing the compounds having nucieophilic properties on the activated carrier material Covalently bound to groups of high molecular weight structure, or by first preparing soluble conjugates of the compounds with nucleophilic properties of functionally low molecular weight and high molecular weight structures and hemoglobin or harain attached to the insoluble support materials with covalently bonded nitrogen c yc1eη _e the eb0η fa11s miηdes ten & have an endocyclic nitrogen atom with a free electron pair, coordinated ati ν geb ι? η e e η we rde η _β 2. Method according to point ^ ->, characterized in that the compounds with nucleophilic properties possessing functional groups of low and high molecular structure biologically active substances such as plasma proteins, Enz / me, antibodies, antigons, hemagglutinins, compounds with protein character quite, in general or heptene, coenzymes, vitamins, hormones "peptides or amino acids and naturally or synthetically produced activators or inhibitors"