DE3910966A1 - Support material coated with enzymes - Google Patents

Abstract

The support material consists of acetylcholine esterase and choline oxidase and a copolymer based on vinyl acylate and/or vinyl alcohol units and units of a crosslinker. A material of this type is stable for at least 6 months. It is used for determining choline and acetylcholine, which are detected via the formation of hydrogen peroxide.

Description

The invention relates to a coated with enzymes Carrier material made from acetylcholine esterase and choline oxidase and a pearl-shaped macroporous support made from a Copolymer consists essentially of vinyl acetate and / or vinyl alcohol units of a crosslinking agent is constructed.

Acetylcholine is one of the most studied Neurotransmitters in the nervous system. The evidence was previously however quite labor intensive and included bioassay, Gas chromatography / mass spectrometry and radioenzymatic test.

Acetylcholine has been determined considerably more easily by using acetylcholine esterase and choline oxidase. In this process, the acetylcholine is converted into free choline by the esterase, which in turn is catalytically converted to betaine and hydrogen peroxide by the choline oxidase. The resulting hydrogen superoxide (H ₂ O ₂ ) was determined either by luminescence analysis or electrochemically (Potter et al., J. Neurochem. 41, 188-194 (1983)). The latter method offers the advantage that it is extremely specific and reliable using high pressure liquid chromatography (HPLC), which separates the two substances acetylcholine and choline before their determination. However, apparatus deficiencies were disadvantageous in this method, and the enzymes could not be recovered after the reaction.

Remedial action and considerable progress brought the Fixation to an ion exchange column using a so-called one-buffer process. A special one  The process describes the fixation of the two enzymes with bromine-activated Sepharose 4B (manufacturer Pharmacia, Uppsala, Sweden), where Sepharose is a large-pored, spherical agarose gel for coupling ligands, such as Represents enzymes (H. Stadler et al., Neurochem. Int., Vol. 9, pages 127-129 (1986)). The one in this work However, the immobilization method described has disadvantages. It is not stable to hydrolysis, which is the case when used with rapidly decreasing sensitivity. It it is stated that such a column with the fixed pair of enzymes only over a period of three Weeks can be used.

It is also a much more stable pairing method already (K.H. Burg et al., Angew. Makromol. Chemie 157, 105 (1987)). With this method, the Enzymes via oxirane groups on a pearl-shaped, macroporous support based on vinyl acetate and N, N'-divinyl coupled ethylene urea, one being exclusive stable secondary amines from the lysines of the enzyme or Obtains ether bonds from the tyrosines of the enzyme. The in However, the carrier described in this publication is only for the coupling of hydrolases such as trypsin and urease have been described. A note on the use of Immobilization of oxidoreductases and corresponding However, no attempts are made.

Oxydoreductases are generally considered to be unstable. So So far, for example, the glucose oxidase that leads to the Group of oxydoreductases counts and an important, technically usable enzyme is never satisfactory be fixed, i.e. that their use over a long period of time done reliably. For this reason, it's more technical Use in immobilized form has remained relatively small.

It was therefore the task of the activity of immobilized enzymes on the carrier material via a to sustain for a longer period of time, so a longer one Service life of the equipment used in the detection of to achieve called neurotransmitters.

The invention relates to a coated with enzymes Carrier material consisting of acetylcholine esterase and Choline oxidase and a porous, pear-shaped Support material made of a crosslinked copolymer, which in essentially from vinyl acetate and / or vinyl alcohol Units and units of a crosslinking agent, the units of the crosslinking agent being polymerized Compounds of the general formulas

and or

where R ₁ , R ₂ in formula (I) may be the same or different and are vinyl, 1-acyloxy-vinyl, allyl or 2-acyloxy-allyl, A is a divalent hydrocarbon radical with 2 to 8 C- Represents atoms, B in formula (II) represents a divalent hydrocarbon radical having 1 to 8 carbon atoms and X is acyloxy, the acyloxy group being a radical having 2 to 18 carbon atoms, the amount of crosslinking agent units being 1 to 60% by weight .-%, based on the polymer, and the acyloxy groups of the vinyl acylate units are present as such or partially or completely saponified to hydroxyl groups, and the average particle size of the beads 20 to 800 microns and the average pore diameter 2 to 10,000 nm.

It was surprising that the enzymes were immobilized in one Form after coupling to the carrier mentioned Show stability of at least 6 months. The production of the porous, pearl-shaped carrier materials used is known from DE-OS 33 44 912, with which reference is taken.

The anchoring reaction between the invention used enzymes and the carrier material is in performed in a known manner, such as in DE-OS 24 07 340 or in DE patents 22 15 687, 24 21 789 and 25 52 510.

The invention also relates to the use of the carrier material according to the invention for the detection of Acetylcholine and choline.

Contain the vinyl acylate units of the carrier polymer preferably 2 to 18 carbon atoms, in particular 2 to 6 carbon atoms in the acylate residue. This is preferably the acetate or Propionate residue. Various acylate residues can also be found in the Polymer is present, i.e. for its manufacture can also mixtures of the corresponding vinyl acylates be used.

In the crosslinking agent according to formula (I), A preferably a branched or unbranched aliphatic hydrocarbon radical with 2 to 5 carbon atoms, in particular 2 or 3 carbon atoms. Is particularly preferred this is the ethylene or propylene radical. If R₁ / R₂ this Formula (I) for 1-acyloxy-vinyl or 2-acyloxy-allyl , the acyloxy group preferably contains 2 therein up to 18 carbon atoms, in particular 2 to 6 carbon atoms. Prefers means acyloxy- the acetate or propionate residue. Preferably, the radicals R₁ / R₂ have the meaning of Vinyl. A preferred crosslinker unit in the  Polymer used according to the invention conducts accordingly from N, N'-divinyl ethylene urea. This Crosslinker is particularly resistant to hydrolysis Shortcut. Another preferred representative is N, N'-divinyl propylene urea.

In the crosslinking agent according to formula (II), B prefers the meaning of a divalent Hydrocarbon residue, especially a branched one or unbranched alkylene radical with 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms. The acyloxy group has here preferably the same meaning as above for the radical R described in formula (I). A preferred crosslinker This type is, for example, 3,3-dimethylpentadiene 2,4-diacetate, which is particularly light with the vinyl acylate copolymerized.

The amount of units of the crosslinking agent (II) is generally 0 to 100%, in particular 0 to 60%, based on the total amount of crosslinking units in Polymer.

The total amount of crosslinker units in the Carrier polymer is in the claimed ranges and depends on the particular application desired crosslink density. For example, at the application as a carrier material for enzyme reactions in Stirring kettle or a relatively small one for diagnostics Crosslinker density advantageous, which has a low content crosslinking monomer units. Crosslinker contents below 0.1% by weight lead to the in most cases to unusable products. As lower limit can therefore generally be about 1% by weight can be specified. Crosslinker contents above 60% by weight are basically possible; usually perform however no other advantages.  

The amount depends on the application Crosslinker units preferably at 1 to 50 wt .-%, and in particular from 1 to 40% by weight, based on the polymer. When used as a carrier material for biologically active substances such as acetylcholine esterase and The lower limit of choline oxidase is preferably 2.5 % By weight and particularly preferably 10% by weight. If only Crosslinker units of the formula (II) are present the lower limit of which is preferably 2.5% by weight.

It can be advantageous if the carrier polymer additionally monomer units one with vinyl acetate contains copolymerizable monomers, the amount of which in general 10% by weight, based on the total polymer, does not exceed, and preferably between 0.1 and 5 % By weight. Examples of such monomers that can optionally be used in a mixture are: N-vinyl pyrrolidone, vinylene carbonate, (meth) acrylic acid, (Meth) acrylonitrile, (meth) acrylamide, (meth) acrylic acid alkyl esters each having 2 to 12 carbon atoms, preferably 2 up to 4 carbon atoms in the alkyl radical, hydroxyalkyl esters (Meth) acrylic acid with 2 to 6 carbon atoms in the alkyl group, N-vinyl-N-alkylacetamide, styrene, a-methylstyrene and the like.

The crosslinked carrier polymer is preferably in the form of pearls in front, the predominantly spherical shape have, whose average particle size in dry, unswollen state 20 to 800 microns, preferably 50 to 300 microns and which is preferably a narrow Have particle size distribution. The respective optimum the particle size depends above all on the special one Area of application. With one carried out without pressure One of the column methods is particle size accordingly within the above limits  choose larger than with a printing process. The pearls of the Polymers used according to the invention are predominant macroporous. The average pore diameter is generally in the range of 2 to 10,000 nm, preferably 5 to 200 nm and in particular 20 to 200 nm.

The acylate groups of the vinyl acylate units in Polymer used according to the invention are as such before or are preferably partially or completely closed OH groups saponified. At least 10% by weight of the Acyloxy groups replaced by hydroxyl groups. The However, the degree of saponification is generally more than 50%, preferably more than 70% and in particular 90 to 100%. In the crosslinked polymer obtained by saponification (Polyvinyl alcohol) is preferably at least part of the OH groups occupied by so-called "spacer" groups (see "Spacer" below).

The carrier polymers are particularly notable for a high hydrolysis resistance with high Networking density. This high resistance to hydrolysis is used as a carrier material for biologically active Substances like enzymes of great importance. On carrier fixed enzymes are often in alkaline or for years acidic environment used. This is especially true for those "unspecific hydrolases" to the ester or Cleave carboxamide bonds, e.g. Proteases that cleave proteins non-specifically. Otherwise, the stable Crosslinking also during the saponification of the acylate groups OH groups in the polymers according to the invention advantageous.

The copolymer in the form of the polyvinyl acetate gel is not hydrophilic; for use in water the ester group be hydrolyzed. This can be alkaline in a known manner by swelling the product in an alcohol, e.g. Methanol  and addition of aqueous alkali such as sodium hydroxide or by transesterification of the alcohol-swollen product with catalytic amounts of acid or base while running e.g. removal of the ester formed by distillation (cf. DE-PS 15 17 935). The saponification can be at any level be canceled so that, depending on the intended use, the degree the hydrophilicity of the gel can be adjusted.

When using the pearl-shaped cross-linked Polyvinyl alcohol gel as a carrier for biologically active Substances such as acetylcholine esterase and choline oxidase, which be fixed to the support by a covalent bond in many cases, it is advisable to remove the gel beforehand to modify with so-called "spacers". Under "Spacer" one understands connections that both with the Carrier polymers as well as with the biologically active React substance and in a sense one between the two Build a bridge. The reaction of the bead polymer with the Spacer can either be directly or preferably after prior saponification of the acylate groups. The The degree of turnover depends on the bulkiness of the spacer and the accessibility of or from the acylate group secondary hydroxyl groups formed. As a spacer come according to the invention the known homo- and hetero-bifunctional compounds in question, the second functional group coupling with the one to be fixed biologically active substance (cf. the DE patents 24 21 789 and 25 52 510, and Ullmanns Encyclopedia of Technical Chemistry, 4th Edition, Vol. 10, Page 540 and "Characterization of Immobilized Biocatalysts ", Verlag Chemie, Weinheim, 1979, p. 53).

The spacers used are, for example for compounds that introduce the following groups:  

Preferred "spacers" are those which bring about hydrolysis-resistant chemical bonds, such as epichlorohydrin or its homologues ( α , β- epoxy- ω -haloalkanes). The reaction of the polyvinyl alcohols (polyvinyl acylates) takes place without a solvent or in the presence of a solvent, preferably in the presence of a catalyst. Depending on the temperature, which can be between room temperature and the reflux temperature of the epichlorohydrin (113-115 ° C.), the reaction time is generally between 30 minutes and 24 hours. Examples of suitable catalysts are NaOH (in powder form) or aqueous alkalis, dimethylformamide, triethylamine and other acid acceptors.

The implementation between those used according to the invention Enzymes and the carrier material are usually made with Room temperature or at + 40 ° C or below Temperatures. The latter especially if the to anchoring enzyme preparations are unstable; in this case then the temperatures are below + 10 ° C, preferably at 0 to + 5 ° C.  

The anchoring reaction is preferably done nearby a neutral pH value, for example at pH values of 5-9, since most of the biologically active substances have greatest stability, preferably in the presence of usual buffer mixtures. As a rule, it is not required, more acidic or alkaline conditions to be observed, since the porous used according to the invention Pearl polymers also in the neutral range with the most of the substances in question react quickly. The resulting bond offers enough stability for long storage and high operational stability.

The detection of acetylcholine and choline side by side is generally done according to the following procedure:
The mixture of substances in aqueous solution, in which acetylcholine and choline is present, is injected into an HPLC system and the two substances are separated chromatographically on modified silica gel. The outlet of the separation column is connected to a cartridge which contains acetylcholine esterase and choline oxidase, fixed on the pearl-shaped carrier material used according to the invention. Acetylcholine is converted into choline by the esterase and the oxidase then generates the actual measurement signal, namely hydrogen superoxide, which is formed in addition to betaine. The choline present in the mixture of substances in aqueous solution, which first emerges from the chromatography column, is of course converted directly into hydrogen superoxide, so that two peaks occur in offset times in the recorded chromatogram. At the outlet of the cartridge, an electrochemical detector is connected, which reacts to the hydrogen peroxide content and with which the hydrogen peroxide peak can be determined in each case.

Examples 1. Synthesis of the carriers

a) Suspension polymerization
In a 1.4 liter glass flask equipped with a stirrer, reflux condenser and thermometer, an organic phase consisting of a solution of 60.0 g of vinyl acetate, 40.0 g of N, N'-divinylethylene urea and 80.0 g of 2-ethylhexanol was added under a nitrogen atmosphere , 20.0 g polyglycol B 11/50 (Hoechst AG) and 2.0 g Azoisobutyronitril® Porofor N, Bayer AG) with stirring in an aqueous phase consisting of 3.2 g Na₂HPO₄, 8.0 g polyvinylpyrrolidone (MW ca . 360,000) and 800 ml of water suspended. The polymerization was started by heating to 75 ° C. in the heating bath. After two hours the temperature was raised to 85 ° C. and the polymerization was ended after two more hours. The suspension obtained was cooled to 25 ° C., suction filtered and stirred four times with 1 liter of water, three times with 1 liter of methanol and twice with 1 liter of acetone for 30 minutes, and suctioned off at 50 ° C. and 200 torr overnight in a vacuum drying cabinet dried under nitrogen. The yield was 75 g. There were cloudy pearls with a shiny surface. The bulk weight was 300 g / l. This results in a bulk volume of 3.3 ml / g.

b) partial saponification
50 g of dry product together with 150 ml of methanol and a solution of 17.5 g of NaOH in 150 ml of water were stirred for 3 hours at 30 ° C., filtered off with suction, neutralized in 500 ml of methanol with acetic acid, once with 500 ml of methanol and twice stirred with 500 ml of acetone, suction filtered, sieved and dried. The yield was 34.4 g (= 68.8% by weight) based on the polymer. The pearls were cloudy and had a shiny surface. The bulk density was 353 g / l (calculated bulk volume 2.8 ml / g).

Sieve distribution: <300 µm 19.0 g (55.2% by weight), 200-300 µm 8.9 g (25.9% by weight) 100-200 µm 6.1 g (17.9% by weight) and 50- 100 µm 0.4 g (1.2% by weight). Degree of saponification (IR, related on moles): 73%.

c) attachment of the spacer There were 10.0 g of the dry sieve fraction 50-200 microns Swollen in 100 ml epichlorohydrin at 25 ° C for 4 hours, then Heated to 115 ° C. for 4 hours with gentle stirring and after sucked off after cooling to 25 ° C. Then was twice with 200 ml of acetone stirred for 30 minutes, suction filtered and overnight in a drying cabinet under reduced pressure stored at 50 ° C with nitrogen blanket.

The weight resulted in 9.8 g of dry product with a Bulk density of 315 g and an epoxy equivalent of 350 µmol / g.

2. Binding the enzymes

0.64 mg acetylcholine esterase (161 units) and 5.36 mg Choline oxidase (80 units), each from Sigma Chemical Co. (Saint Louis, MO., USA) (No. C 5896 and C 3389), were taken together in 1 ml of 1 M potassium phosphate buffer, pH = 8.0, solved. The solution was made up to 1 ml of a moist Sieve fraction (50-100 µm) of that according to Example 1a) -1c) given carrier and slowly in centrifuge tubes rolls. After 3 days at 20 ° C, the carrier was first with 50 mM and then with 1 M potassium phosphate buffer, pH = 7.0, washed.

This was used to saturate remaining residual oxirane groups Product treated for 4 hours with 1 M glycine buffer, pH = 8.5. The storage was carried out by storage in 50 mM Potassium phosphate buffer, pH 7.0, the 0.02% sodium azide were added at 4 ° C.  

3. Detection of acetylcholine and choline

The carrier obtained according to Example 2 was miniaturized Stainless steel cartridges (MPLC system Brownlee, 4 × 0.2 cm, Bed volume 125 µl) filled and together with a appropriate cartridge holder with the outflow of a Separation column connected.

HPLC

It was a modular HPLC system consisting of a Double piston pump (type 300B, manufacturer: Gynkotek, Germering, Federal Republic of Germany) with Pulsation damper with an electrochemical detector Platinum electrode (Gynkotek M20) and one with compressed air operated sample application valve with 50 µl sample loop used.

A ®Nucleosil 5SA (manufacturer: Macherey and Nagel, Düren, Federal Republic of Germany) filled steel prefabricated column (60 × 4.6 mm) used. The mobile phase consisted of a 0.1 molar sodium Phosphate buffer of pH 7.6, the 7 mM tetramethylammonium perchlorate contained. The buffer was made with ultrapure water prepared through a cellulose acetate filter with 0.2 µm Pore size filtered, degassed and under a helium atmosphere held. The flow in the separation column was 0.7 ml / min set (pressure 25-30 bar). At this Flow rates were the retention times for Choline at 3-5 minutes and for acetylcholine at 7 to 10 Minutes.

There was an oxidation potential at the electrochemical detector set from +0.5 volts for the platinum electrode that Gain was for the detection of acetylcholine and Choline in the pico-mol range 0.02-0.04 nA / 1V.

Under these conditions, a standard mixture of choline and acetylcholine with a concentration of 0.4 pmol per 50 μl buffer solution was examined and the following chromatogram was obtained ( FIG. 1).

The figure shows the retention times for choline (CH) and Acetylcholine (ACH) clearly recognizable, they prove the sharp Separation of the two substances. The first peak in the figure refers to the solvent. The Detection sensitivity for both substances lies with the described arrangement usually at <0.5 Pico-mol / injection (see chromatogram), under optimal Conditions are still detectable by 0.1 pmol.

The separation column showed no after three months of use Waning of the wearer's activity after six months almost 20% loss of activity was found.

Claims (9)

1. Enzyme-coated carrier material consisting of acetylcholine esterase and choline oxidase and a porous, pearl-shaped carrier material made of a crosslinked copolymer which consists essentially of vinyl acetate and / or vinyl alcohol units and units of a crosslinking agent, the units of the crosslinking agent being polymerized Compounds of the general formula and or where R ₁ , R ₂ in formula (I) may be the same or different and are vinyl, 1-acyloxy-vinyl, allyl or 2-acyloxy-allyl, A is a divalent hydrocarbon radical with 2 to 8 C- Represents atoms, B in formula (II) represents a divalent hydrocarbon radical having 1 to 8 carbon atoms and X is acyloxy, the acyloxy group being a radical having 2 to 18 carbon atoms, the amount of crosslinking agent units being 1 to 60% by weight .-%, based on the polymer, and the acyloxy groups of the vinyl acetate units are present as such or partially or completely saponified to hydroxyl groups, and the average particle size of the beads 20 to 800 microns and the average pore diameter 2 to 10,000 nm. 2. Support material according to claim 1, characterized in that the vinyl acylate units of the copolymer Are vinyl acetate units.   3. Support material according to claim 1 or 2, characterized in that in the copolymer the acyloxy group in the radicals R ₁ , R _{2 of} the formula (I) or the acyloxy radical in formula (II) has 1 to 6 carbon atoms. 4. Support material according to one or more of claims 1 to 3, characterized in that in the copolymer the radicals R ₁ / R _{2 of} the formula (I) are each vinyl. 5. Support material according to one or more of claims 1 to 4, characterized in that in the copolymer the amount of crosslinking agent units 1 to 50% by weight is. 6. Support material according to one or more of claims 1 to 5, characterized in that in the copolymer at least 10% by weight of the acyloxy groups Vinyl acylate units replaced by hydroxyl groups are. 7. carrier material according to claim 6, characterized in that in the copolymer at least part of OH groups are occupied by spacer groups. 8. Support material according to one or more of claims 1 to 7, characterized in that in the copolymer still up to 10% by weight, based on the polymer, Units of another with the vinyl acylate copolymerizable hydrolysis-resistant usual Monomers are present. 9. Use of the carrier material according to claim 1 for Detection of acetylcholine and choline.