CS269750B1 - Method of proteins immobilization - Google Patents

Abstract

A method of immobilizing proteins, for example; enzymes and antibodies for the preparation of hetsrogen-j biocatalysts, immunochemical stands and affinity chromatography on organic! inorganic carriers containing hydroxylo- amino, or sulfhydryl groups, characterized in that the carrier is activated By the action of perfluorinated olefin, e.g. perfluoropropene or perfluorocyclobutene or a fluorinated diene or polyene e.g., perfluoro-1,3-butadiene or perl fluorohexatriene or 3-chloro-nonafluoro- -1,5-hexadiene, optionally in the presence of potassium fluoride and thus activated the carrier binds the proteins with covalent bond generated by the nucleophilic addition of its groups for example sulfhydryl or hydroxyl or amino groups and perfluorinated a double bond present on the surfaces activated carriers.

Description

EN 269 750 B1 1

The present invention relates to a method for immobilizing proteins, for example enzymes and antibodies, for the preparation of heterogeneous biocatalysts, solid phase immunochemical assays and affinity chromatography.

In order to prepare the immobilized proteins for this purpose, a number of methods have been developed, predominantly based on various forms of protein fixation on suitable carriers. The problem of immobilization of enzymes generally applicable to other proteins is described in detail in many monographs and review articles, for example Chibata 1: Imobilized Enzymes.

Research and Development, Kodansha, Tokyo (1978), Goldatein L., Manecke G .: The chemistry

of Enzyme Immobilization, Applied Biochemistry and Bioengineering, Academic Press, New I

York (1976), Wiseman, A. Handbock of enzyme biotechnology. Ellis Horwood, Chichester (1975), Kas 3., Marek M., Valentova O.s Immobilized Enzymes. (Principles of immobilization, properties and use of immobilized enzymes), Microbial technology, Academia Praha * (1988), Marek M., Káš 3 .; Techniques of Enzyme Immobilization by Covalent Binding, Chem. Industry 34, 337 (1984).

The easiest way to immobilize proteins is to adsorb them to different types of carriers.

However, the sorption forces that hold the protein on the carrier surface are in many cases relatively weak and immobilization is therefore often inadequate. For these reasons, sorption is very often combined with subsequent cross-linking of proteins on the carrier surface with bifunctional agents.

The most sophisticated technique for protein immobilization is their binding by covalent bonding to an activated carrier containing reactive groups that form covalent bonds with suitable immobilized protein moieties. The chemical linkage of enzymes or other proteins to insoluble carriers can be exploited by a variety of functional groups contained within the proteinaceous portion of the enzyme molecule, i.e., α-α-amino, εC, X 3 -carboxyl, cysteine, cysteine, aromatic tyrosine and tryptophan rings, imidazole groups of histidine and hydroxyl groups of serine, threonine and especially tyrosine. The basic types of reactions utilizing these functional groups for immobilization of proteins include amide bond formation, alkylation method, diazotation method, thiol-disulfide substitution reaction, Schiff base formation, utilization of reactive isonitriles primarily in conjunction with Ugi reactions, etc. relates to the properties of the carrier used and the purpose for which the immobilized enzyme or other bound protein is to be used. In general, protonellation can designate a particular mode of immobilization as the most suitable and the other unsuitable. The choice of carrier type and method of immobilization is largely determined by the method of application for which the enzymes or other proteins are immobilized.

Another proposed method of immobilizing proteins, e.g., enzymes and antibodies, is to activate the carrier containing hydroxyl, amino or sulfhydryl groups by treatment with perfluorinated olefins such as perfluoropropene, perfluoro-butadiene, perfluorocyclobutadiene, optionally in the presence of potassium fluoride. The activated carrier is then ligated with a protein by covalent bonding through its groups capable of nucleophilic addition to the perfluorinated olefin bound to the support. The advantage of the method of immobilizing the enzymes, antibodies and other proteins of the invention is mainly due to the strength of the covalent bond formed and the possibility of immobilizing a protein carrier which does not exhibit significant sorption properties, for example to the inner wall of glass capillaries or tubes and the like.

The invention is illustrated by the examples without limiting them.

Claims (1)

EXAMPLE 1 To 1 g of porous glass placed in a glass vial was added 3 ml of saturated solution of anhydrous potassium fluoride in acetone and after degassing on a vacuum line, 0.44 g of hexafluoro-1,3-butadiene was added. The vial was sealed and allowed to shake vigorously for 50 h. After the unreacted diene was opened and evaporated, the modified glass was decanted in acetone and the acetone residues were removed on a vacuum evaporator. To 200 mg of activated porous glass was added 10 mg of chymmotrypsin in 5 ml of 0.1 MTris-HCl buffer pH 7.8 containing 60% of dimethyl sulfoxide. After 16 h shaking at 4 ° C *, the conjugate with immobilized γmotripsin was aspirated, washed alternately with this buffer and 1 MNaCl in the same buffer. The conjugate obtained showed 1 µg activity per 1 g wet carrier (N-acetyl-L-phenylalanine methyl ester was used as substrate). EXAMPLE 2 A mixture of 3.8 g of Sephadex polysaccharide-200, 4 g of perfluorocyclobutane and 0.3 g of fluoride potassium in 8 ml of absolute acetonitrile was treated in the same manner as in Example 1. To 200 mg of activated carrier in 5 ml of 0.1 M phosphate buffer pH 7.2, 10 mg of glucose oxidase and 5 µl of catalase were added and the mixture was shaken for 16 h at 4 ° C. The mixture was then filtered. The heterogeneous biocatalyst obtained after thorough washing alternately with 0.1 M phosphate buffer pH 7.2 and 1 M NaCl in this buffer exhibited a glucose oxidase activity of 0.341 yucat / l g wet conjugate. Example 3 A mixture of 1 g of 1,6-diaminohexane modified glycidyl methacrylate polymer 0.6 g of perfluorohexatrilene in 3 ml of absolute acetonitrile was treated in the same manner as in Example 1. To 200 mg of activated carrier in 5 ml of 0.1 M pH 7 phosphate buffer , 2 yul catalase was added and the mixture was shaken for 16 h at 4 ° C. By the same procedure as in Example 2, a conjugate having a catalase activity of 5 µg / g wet carrier was obtained. Example 4 The inner wall of glass tubes or capillaries was activated by flowing solution of 3-chloro-nonafluoro-1,5-hexadiene in acetone saturated with anhydrous potassium fluoride. After washing with acetone with 0.1 M phosphate buffer pH 7.2, the inner wall was immobilized with glucose oxidase flowing through a tube or capillary at a concentration of 400 mgenzyme in 10 ml of 0.1 M phosphate buffer pH 7.2. After washing with the same buffer, the glucosoxidase-bound tubes or capillaries were used to determine glucose in the form of a flow reaction electrode in conjunction with a Clark type oxygen cell. OBJECT OF THE INVENTION A method of immobilizing proteins, for example, enzymes and antibodies for the preparation of heterogeneous biocatalysts, immunochemical assays and affinity chromatography on organic inorganic carriers containing hydroxyl amino or silihydryl groups, characterized in that the carrier is activated by the action of a perfluorinated olefin, e.g. fluoro-propene or perfluorocyclobutane or a fluorinated diene, something polyene, for example, perfluoro-1,3-butadiene or perfluorohexatriene or 3-chlorononluoro-1,5-hexadiene, optionally in the presence of potassium fluoride, and the activated enzyme carrier, CS 269 750 B1 3 antibodies or other proteins immobilize covalent bond-formed nucleophilic "addition groups, for example, sulfhydryl or hydroxyl or amino groups, to perfluorinated double bond present on the surface of the activated carrier.