CS218702B1 - of high capacity protein immobilization - Google Patents

Abstract

The invention relates to a method for high-capacity immobilization of proteins. The essence of the invention, in which in the first stage pectin or a water-soluble salt of pectic acid is cross-linked in an alkaline medium with 2-chloromethyloxirane, consists in that in the second stage the carboxyl groups are activated with carbodiimides, hydrazine hydrate or hydrazine hydrate and then with sodium nitrite in the presence of hydrochloric acid and in the third stage the protein binding is carried out using known methods.

Description

The invention relates to a method of high throughput immunization of proteins on a modified pectin.

The covalent immobilization of proteins into polysaccharides requires a suitable activation of the polysaccharide in the first row. One of the frequently used compounds is the introduction of carboxyl groups. Thus, the jetoma, for example, in the case of commercial carboxymethyl rderivatives. cellulose (CM-cellulose Lovosa, What-man), agarose (CM-Sepharose®) ,. or stranded dextran (CM-Sephadex® C). Such carboxyl derivatives can be used directly for the immobilization of proteins upon activation of the carbodiimide. However, it is preferable to convert them into hydrazides, e.g. azides or isocyanates.

The introduction of carboxyl groups as a means of activating polysaccharides is not necessary when the polysaccharide already contains these groups. This is the case with cross-linked ipectic acid, which is prepared by pectinepichlorohydrin in alkaline medium (V. Ti-Jbensky, E. Kuniak, U.S. Patent 140,713 [1971]), and the cross-linked pectic acid is a macroporous, gelled material. swelling volume (water) of 4 to 14 ml / g. The degree of cross-linking can be adjusted by adjusting the crosslinking conditions. Cross-linked pectic acid has a high degree of substitution by carboxyl groups, DS-1. Crosslinked pectic acid and a carrier for covalent immobilization of proteins can already be used in such a form, using carbodiimides. Another possibility is to convert the cross-linked acid to hydrazides, respectively. on the azides. Whilst hydrazide derivatives permit the advantageous binding of glycoproteins through their carbohydrates, the azides bind proteins through their polypeptide chain.

The essence of the method of high-capacity immobilization of proteins, in which the pectin or soluble salt of pectic acid crosslinks in the first stage with 2-chloromethyl-oxirane in the first step, is that in the second stage the carboxyl group is activated with carbodiimides, hydrazine hydrate or hydrazine hydrate and then with sodium nitrite in the presence of sophoric acid and, in the third step, by using known methods of protein binding. {

For said protein immobilization procedures, the weakly cross-linked pectic acid (with epichlorohydrin), with a swelling volume (n. 3.7 ml of methanol / g. Such as; there is a high exclusion limit for the protein of 80,000 molecular weight). Also, the preparation of its derivatives, e.g. hydrazides, can be as high as DS 0.42, with the remainder of the carboxyl groups being in the ester form. The advantage of the proposed method of high-throughput immobilization of proteins to water-soluble pectic acid derivatives is that - the basic material (pectin) is readily available and natural biopolymer - pectin is simultaneously de-esterified while cross-linking - cross-linked pectic acid has DS Ni, what r 218702

It also makes it possible to prepare its other derivatives with the following DS - high protein-containing preparations (up to 100 mg protein / g carrier) can be prepared in conjunction with the above - cross-linked pectic acid, its derivatives as well as preparations with mobilized proteins are gel-like materials with good hydrodynamic properties; tami - makes it possible to produce preparations from cheap raw materials | EXAMPLES Example 1

To 20 g of pectin (citrus), 13 ml of 25% NaOH was added with stirring and allowed to react for 1 h at 20 ° C. 1.8 Mlepichlorohydrin was added and after 2 h stirring at 20 ° C, flowing over 1 h, the temperature was raised to 40 ° C. During this heat it was allowed to react for another 1 h. The cross-linked sodium salt of pectic acid was successively washed with water, ethanol and dried at 60 ° C. Sodium pectate was washed slowly with 0.25 N HCl, water was added to the reaction mixture with negative chlorides, ethanol and finally air-dried. 200 mg of cross-linked pectic acid (no 14 ml of H2O / g) were resuspended in 15 ml of a 1.5% solution of N-cyclohexyl-N '- (N-methylmorpholine) ethyl / carbodiimide-p-toluenesulfonate in acidified to pH 4.8 with distilled water, and the suspension was stirred for 1 h at room temperature. The activated gel was washed extensively with water and a bag of 10 mg acetylcholinesterase (acetylcholine * hydrolase, EC 3.1.1.7; 25.4 U-mg) solution was added in 8 ml of dehydrated water. The suspension was stirred for 2 h pp0 - 2 ° C. After filtration and washing of the insoluble portion with 100 ml of 2 M NaCl ·, the mobilized acetylcholinesterase showed a specific activity of 5.8 U / mg and the preparation had a total activity of 71.3 U / g. A 15% decrease in catalytic activity was observed during storage in a humidified condition at 4 ° C over 6 months. Example 2

Cross-linked pectic acid (1 g) prepared as in Example 1 was suspended in 100 mL of methanol. 0.6 ml of concentrated HCl and reflux were added. After washing with methanol and drying the saproduct (0.95 g) was resuspended in a mixture of 16.6 ml methanol and 16.6 ml 99% hydrazine hydrate. The suspension was maintained at 38 ° C for 72 hrs and mixed with 0.15 M after capture. NaCl to a negative reaction to tdrazine, 250 mL of water, 250 mL of ethanol, and the product dried. Cross-linked pectic acid hydrazide (0.28 g) contained 4.13% nitrogen. To a solution of 0.5 g / l of ktosylated bovine serum albuminΛ01ΟΟ ml of 0.1 M acetate buffer pH 5 was added 40 mg of sodium periodate and left to stand for 1.5 hours at room temperature. 0> 5 hrs for,

Claims (1)

4 'certain conditions. The mixture was dialyzed for 2 hours in 2 x 51 50 mM acetate buffer pH 5. 0% of pectic acid cross-linked hydrazide was suspended in the distalysed mixture, pH was 6.5 and allowed to stir for 24 hours. hours at room temperature. The preparation was washed with 250 µl of water, 500 ml of 0.1 M borate buffer pH 8.5 containing 1.075 M NaCl and Tween 20 (0.1% v / v), aspirated, resuspended in a known volume and stored at 4 ° C. ° C. For longer storage, a drop of chloroform or sodium azide was added to a concentration of 0.1% (w / v). The preparation contained 85.5 mg of Albumin / g of cross-linked pectic acid. i - Example 3 ·. Cross-linked pectic acid (4 ml H 2 O / g) was prepared as in Example 1 except that 4 ml of epichlorohydrin was used and the reaction time at room temperature was increased by 2 h. Hydrazidated pectic acid (0.59 g; 7.21% a) was then prepared as outlined in Example 2. 10 mg of pseudocholinesterase (10 * units) (acylcholine acyl hydrolase, EC 3.1.1.8) * were dissolved in 2 ml of 20 mM pH8 phosphate buffer and 1 ml of 4.35 mM periodate solution was added; potassium in the same buffer. The reaction mixture was kept for 5 h at 4 ° C, then the enzyme was precipitated twice with a 10-fold excess of ethanol. The precipitated enzyme was dissolved in 100 ml of 20 mM phosphate buffer pH 8 and 0.5 gf of pectic acid cross-linked hydrazide was added to the solution. The suspension was stirred for 18 h at 4 ° C. After washing with 100 ml of 20 mM NaCl, 200 ml of distilled water and 100 ml of 20 mM phosphate buffer pH 8, the immobilized pseudocholinesterase showed a specific activity of 1, 6U / mg and the preparation had an angular activity of 23 U / g? 218702 The preparation was stored as described in Example 1. {Example 4 The procedure was as in Example 3, except that the cross-linked pectic acid (no 7.4 µlH 2 O / g) was prepared as described in Example 1. 1 with 3 ml of epichlorohydrin used. The pre-wash contained 72.6 mg of albumin / g of cross-linked pectic acid. í - I f '' '5 Example 5; Cross-pectic acid hydrazide (0.25 'prepared as described in Example 2 is resuspended in 10 ml of 0.6 NHC1 cooled to 0 ° C. 1.25 ml of 5% aqueous solution is added slowly over 20 min with stirring) The sodium azide is stirred for a further 20 minutes at 0 DEG C. The azide is washed with 100 ml of ice-water, 50 ml of ice-cold 1 M NaCl, again with 100 ml of ice-water and suctioned. 0.1 g / ml of serum albumin in 10 ml of water. The pH is immediately adjusted to 8.7 with saturated sodium tetrabromide solution and the suspension is stirred for 20 minutes at this bath to check for pH. The suspension is stirred for a further 40 minutes, washed with 10 ml of water, 200 ml of 1 M NaCl, 100 ml of 50 mM acetic acid, 100 ml of 1 M NaCl, 100 ml of 0.5N NaHCO3, 100 ml of 1 M NaCl, 200 ml of water. The immobilized enzyme is suspended in water and glass The preparation contained 104.8 mg of albumin / g of cross-linking pectic acid. OBJECT OF THE INVENTION I Method of High Capacity Immobilization of Proteins, in which the pectic acid salt of cross-linking with 2-chloromethyloxirane in the first step is soluble or soluble, characterized in that carboxyl groups with carbodiimide hydrazine hydrate or hydrazine hydrate and nitrite sodium are activated in the second stage in the presence of phosphoric acid and, in the third step, protein preyedienation is utilized using known methods. Price: 2.40 K i s Printed Moravian Printing Works, Operation 12, Leninova 21, Olomouc