CS267820B1 - A method for immobilizing glycoproteins, saccharides, polysaccharides, nucleotides, and other compounds containing a reactive carbonyl group - Google Patents

Abstract

The solution relates to a method for immobilizing glycoproteins, carbohydrates, polysaccharides and nucleotides and other compounds containing a reactive carbonyl group. The essence of the method for immobilizing glycoproteins, carbohydrates, polysaccharides, nucleotides and other compounds containing a reactive carbonyl group according to the solution lies in the fact that the carrier for immobilization is macroporous pearl cellulose with a bound carboxylic acid dihydrazide of the general formula I, where n is 0 or 1, and R is -(CH„).-, -(CH ),-, -C,H.- and X -OH, -NH-NH-CO-R-CO-NH-NH,. In this method, the sugar component is bound via a reactive carbonyl group either originally contained therein or introduced by selective oxidation with periodate or by another known reaction.

Description

The invention relates to a process for immobilizing glycoprotein, carbohydrates, polysaccharides, nucleotides and other compounds containing a reactive carbonyl group on macroporous bead cellulose with attached hydrazide groups.

Reactive groups of their amino acids have so far been used in the vast majority of cases when immobilizing enzymes or other protein proteins on a carrier by chemical bonding. However, such a procedure always carries the risk that the region essential for the specific binding or catalytic function of the protein may be damaged to varying degrees or at least its stability may be reduced. A living cell, on the other hand, often uses a sugar component to bind. For example, the proteolytic enzyme carboxypeptidase Y from yeast contains a sugar component by which it is bound in Saccharomyses cerevisiae cells to vacuole membranes. The stability of this enzyme is highly dependent on the content of the sugar component. 3iz Margolis et al. (3. Biol. Chem. 253 (1978) 7891) have shown that carboxypeptidase Y with a molecular weight of 49,000 to 65,500 can be isolated from different yeast species. The differences in molecular weights are only due to differences in sugar content, on which it then directly depends enzyme stability. Marshall and Rabinowitz (Biotechnol. Bioeng. 18 (1976) 1325 to 1329) have shown an increase in the stability of catalase, trypsin and 2-amylase by modification with dextran. The great importance of sugar components in localization and its dependent activity has already been demonstrated for a large number of different biologically active compounds (Gensburg V., Robins RW, eds. Biology of Carbohydrates, Vol. 2, Wiley, New York 1984; Brandley K.. K. , Schanaar 3. Cell-surface Carbohydrates in Cell Recognition and Response, 3. Leucocyte Biol. 40 (1986) 97-111; Liener IE, Sharon N., Goldstein I. 3., eds., The Lestins. in Biology and Medicine, Academic Press, New York 1986). Immobilization of enzymes, antibodies, nucleotides and other biologically active substances by means of aldehyde groups present or obtained by periodic oxidation on a solid support containing, for example, adipic acid hydrazide has a number of advantages over the most commonly used methods. By proceeding at a low pH (e.g. 4.5), at which the aldehyde groups do not react with the NH 4 groups on the surface of the proteins, it prevents polymerization and non-specific bonds. The presence of a sugar component between the bound biologically active compound and the solid support protects the activity of the immobilized compound from the possible adverse effect of the support and limits the possibility of loss of the sugar component by cleavage.

The present invention relates to a process for the immobilization of a glycoprotein, carbohydrates, polysaccharides, nucleotides and other compounds containing a reactive carbonyl group, the essence of which consists in that the carrier for immobilization is macroporous bead cellulose with bound dicarboxylic acid dihydrazide of general formula I.

Cel- (O-CH ₂ CH ₂ ) _n -OZ ^N 1 -NNO-NH-CO-R-CO-NN-NH ₂ (I)

wherein π is 0 or 1, and R is - (CH ₂ ) 4 -, - (CH 2) 8 -, and X -OH, -NH-NH-CO-R-CO-NH-NH ₂ .

The process according to the invention is based on the ability of a hydrazide group (-CO-NH-NH 2) to react under mild conditions with compounds containing reactive carbonyl groups (aldehyde, keto, free or suitable derivatives or certain carboxylic or other acid derivatives) to form a sufficiently hydrolytic bond. still. In this case, the attached compound either already contains the required carbonyl group, for example reducing mono- or oligosaccharides, and substances which contain them or are additionally introduced into them in a suitable manner, for example the selective oxidation of saccharides with periodate. Bonding by an alkylation reaction is also useful. Said reactions can also be used for coupling

CS 267 820 B1 hydrazide groups on the support skeleton if the dicarboxylic acid dihydrazide is taken for activation. This route is the most advantageous for the preparation of sorbents, since the corresponding introduction of reactive groups is known in principle and dihydrazides are also relatively readily available. The chain length between the carboxyls can influence the steric availability of the kydrazide group on the sorbents.

Agarose-based sorbents with adipic acid dihydrazide bound are described in the literature and are available on the market. But the mechanical and flow properties of agarose sorbents are far from optimal when working in a column. Also, the binding of the dihydrazide after the generally used cyanogen bromide activation is not sufficiently hydrolytically stable. These shortcomings are overcome by the sorbents according to the invention. Macroporous bead cellulose has very good mechanical and flow properties and adjustable pore size. Dihydrazide binding after activation with 2,4,6-trichlorotriazine has a hydrolytic stability comparable to that of the hydrazide group.

Example 1

To a stirred solution of 505 mg of adipic acid dihydrazide in 50 ml of 0.05M borate buffer pH 9 is added 50 g of water aspirated bead cellulose freshly activated with 2,4,6-trichlorotriazine (89.2 [mu] mol triazine / g dry matter). The reaction mixture was stirred at room temperature for 4.5 hours. The pH is maintained by the addition of sodium hydroxide (NaOH). The product is washed with water. It contains B, 5 [mu] mol of dihydrazide / ml of swollen material. Example 2

In an otherwise the same embodiment as in Example 1, only the activated cellulose contained 116 [mu] mol triazine / g and 657 mg of dihydrazide was used for the reaction. A sorbent containing 12.8 [mu] mol dihydrazide / ml of swollen material was prepared.

Example 3

To 100 mg of invertase dissolved in 100 ml of 50 mM acetate buffer pH 5 was added 10 ml of 50 mM sodium periodate (Na 2 SO 4). The mixture was stirred on a rotary shaker in the dark at 4 ° C for 5 hours. Unreacted NaOO 2 was removed with 0.2 mL of ethylene glycol. The oxidized invertase after 30 minutes of subsequent stirring was dialyzed against 0.1 M acetate buffer pH 4.5. The oxidized and dialyzed invertase was mixed with 10 ml of aspirated cellulose with bound adipic acid dihydrazide. The reaction mixture was stirred at 4 ° C for 16 hours. The immobilized invertase was transferred to a column where it was washed alternately with 0.1 M acetate buffer pH 4.5 and the same buffer containing 1 M NaCl until the invertase activity was detectable in the wash buffer. Immobilized invertase activity was determined by sucrose by the method described by Mark et al. (Biotechnol. Bioeng. 26 (19B4) 1223-1226) was 5.5 units (= .mu.mol of hydrolyzed sucrose per second) based on 1 g of dry conjugate. The amount of bound invertase determined by amino acid analysis of the acid hydrolyzate of the immobilized enzyme (6N HCl, 110 ° C, 20 hours) was 87 mg invertase / g dry conjugate.

Example 4

To 10 mg of carboxypeptidase Y dissolved in 6 ml of 0.1 M acetate buffer pH 5.0 was added 0.6 ml of 0.1 M sodium periodate (Na 2 SO 4), and the further procedure was the same as in Example 1. The amount of bound carboxypeptidase Y was 1. 0 mg carboxypeptidase per 1 g dry conjugate. The activity determined by N-carboxy-benzoxy-L-phenyl-alanyl-L-alanine was 210 units / g of dry conjugate (one unit is the hydrolysis of 1.0 μmol N-CB2-Phe-Ala to N-CBZ-L-phenyl- alanine and L-alanine per minute at 6.75 and 25 ° C according to Johansen et al.

CS 267 820 B1 (Carlsberg. Res. Kommun. 41 (1976) 1-14).

Example 5

One tenth of the volume of 0.1M sodium periodate (Na 2 SO 4) was added to a solution of polyclonal anti-ovalbumin antibodies (1-2 mg / ml) in 0.1M acetate buffer pH 5.5. The mixture was stirred on a rotary shaker at 4 ° C for 20 minutes. The reaction was stopped by the addition of glycerin to a concentration of 0.015M. The mixture was stirred under the same conditions for another 5 minutes. The reaction mixture was applied to a Sephadex G-25 column (30 x 1.6 cm) equilibrated with 0.1 M acetate buffer containing 0.5 M NaCl, pH 4.8. Oxidized immunoglobulin was eluted with the same buffer. 20 ml of the obtained desalted antibody fraction was mixed with 10 ml of cellulose with bound adipic acid dihydrazide equilibrated with the same buffer. Binding was performed again on a rotary shaker at 4 ° C for 48 hours. The reaction mixture was transferred to a column and washed with the same buffer until the absorbance of the eluate at 280 nm was less than 0.01. The amount of bound antibody determined by amino acid analysis as in Case 1 was 1.2 mg protein / g dry conjugate.

Example 6

To 60 mg of ovalbumin dissolved in 60 ml of 0.1 M acetate buffer pH 5.5 was added 6 ml of 0.1 M sodium periodate (Na 2 SO 4) and stirred on a rotary shaker in the dark for 20 minutes at 4 ° C. After oxidation, glycerin was added to a total concentration of 0.015M and stirred for another 5 minutes at 4 ° C. The ovalbumin solution after oxidation was desalted on a Sephadex G-25 column (3 x 3.5 cm) in 0.1 M acetate buffer containing 0.5 M NaCl pH 4.8. To the obtained 25 ml of ovalbumin solution (230230 = 0.540) was added 5 ml of bead cellulose with bound adipic acid dihydrazide equilibrated with the same buffer. Binding was performed by stirring on a rotary shaker for 48 hours at 4 ° C. After binding, the suspension was transferred to a column where it was washed with the same buffer until the absorbance at 280 nm of the eluted buffer was less than 0.01. The amount of bound ovalbumin determined by amino acid analysis as in case 1 was 18 mg / g of dry conjugate. The obtained immobilized ovalbumin was used to prepare a highly effective biospecific sorbent for concanavalin A lectin by isolating polyclonal antibodies against ovalbumin. These were coupled as described in Case 3. The resulting biospecific immunosorbent was used to isolate ovalbumin by adsorption outside the sugar region, complementary to the concanavalin A binding site. release of ovalbumin.

Example 7

To the ribonucleic acid transport solution (20 mg tRNA / 3 ml water) was added 20 mg sodium periodate (Na 2 SO 4) and stirred on a rotary shaker in the dark for 30 minutes at 0 ° C. A suspension of 5 ml of beaded cellulose with bound adipic acid dihydrazide and 5 ml of 0.1 M acetate buffer pH 4.5 was stirred at 0 ° C for 6 hours after the addition. After binding, the suspension was transferred to a column and washed with 2M NaCl - 0.1M TRIS-HCl buffer pH 8 until the eluate absorbance was less than 0.01. The amount of bound tRNA determined by Kjeldahl N analysis was 5.1 mg / g of dry conjugate.

Claims (1)

OBJECT OF THE INVENTION Method for immobilizing glycoproteins, carbohydrates, polysaccharides, nucleotides and other compounds containing a reactive carbonyl group, characterized in that the immobilization CS 267 Θ20 B1 is performed in an aqueous medium of a buffer at pH 4 to 7 on macroporous bead cellulose with bound dicarboxylic acid dihydrazide of general formula I Cel- (0-CH ₂ CH ₂ ) _n -O-Z X-NH-NH-CO-R-CO-NH-NH ₂ N X (I) wherein n is 0 or 1, R is - (CH 2) -, C.H.- and X is -OH or -NH-NH-CO-R-CO-NH-NH ", Wherein the sugar component either initially contains the reactive carbonyl group or is introduced into it by selective oxidation with periodate or another reaction known per se.