FI57616B - FOERFARANDE FOER AKTIVERING AV CHOLESTEROLOXIDE - Google Patents

Description

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Patent- och ragiitarrtyraltan AnsOksn utisgd och utUkrtften pubiicmd 30.05.80 (32) (33) (31) Privilege claimed —Begird prlorltet 07.12.73 16.08.7¾ Federal Republic of Germany-Förbunds-republiken Tyskland (DE) P 2363969.0 P 2 ) Boehringer Mannheim GmbH., Sandhofer Strasse 112-132, 6800 Mannheim-Waldhof, Federal Republic of Germany-Förbundsrepubliken Tyskland (DE) (72) Peter Roeschlau, Tutzing / Obb., Gunter Lang, Tutzing / Obb.,

Klaus Beaucamp, Tutzing / Obb., Erich Bernt, Munich,

Wolfgang Gruber, Tutzihg-Unterzeismering, Federal Republic of Germany Förbundsrepubliken Tyskland (DE) (7U) Berggren Oy Ab (5U) Method for activating cholesterol oxidase - Förfarande för aktivering av cholesteroloxidas

The invention relates to a method by which cholesterol oxidase can be activated.

German Offenlegungsschrift No. 2,222,132 discloses a method for determining cholesterol by incubating cholesterol in an aqueous medium with cholesterol oxidase and then determining either oxygen consumption or formed or cholestenone. Also known from this method is a reagent for the determination of cholesterol, which consists of cholesterol oxidase and a system for the determination of HjOj or a system for the determination of cholestenone. It has now been found that cholesterol oxidase is not sufficiently stable on storage but is inactivated relatively rapidly. When this instability was investigated, it was found that the inactivation is caused by negligible amounts of detergent from the enzyme manufacturing step and accompanying it as impurities. This preparation is carried out, for example, according to the method described in German Application 2 22-131 by soaking a cholesterol-modifying microorganism by disrupting the cell walls with a buffer solution containing 2 57616 nonionic surfactants and extracting, centrifuging the extract and discarding the precipitate thus obtained. The residue is poured into an anion exchanger, the enzyme is eluted with a buffer solution containing a non-ionic surfactant and isolated from the eluate. After removing these detergent residues, it was achieved that the enzyme lasted completely sufficiently for storage. But at that time it was found that the enzyme released from the detergent residues had clearly lost its activity. If, for example, the enzyme is used for the determination of cholesterol, it follows that either a freshly prepared preparation must be used or significantly higher amounts of enzyme must be used for the test or the measurement time must be significantly extended.

It is therefore an object of the invention to provide a method for activating cholesterol oxidase. The object of the invention is in particular to provide a method in which it is possible, firstly, to use cholesterol oxidase which is completely free of detergents and has sufficient storage stability and, secondly, to obtain the activity of the enzyme used corresponding to freshly prepared enzymes.

According to the invention, this is achieved by a method of activating cholesterol oxidase, characterized in that about 0.005-20% by weight of at least one surfactant having lipophilic and hydrophilic properties is added to the cholesterol oxidase before it is used in the assay.

As the surfactant having lipophilic and hydrophilic properties, nonionic detergents having at least one OH group in the molecule are mainly used. Particularly preferred are polyoxyethylene derivatives of alkyl, aryl and aralkyl alcohols. Examples of such particularly popular surface-active compounds are polyoxyethylene-alkyylieette-esters, polyoksietyleenialkyylikarbonihappoesterit, polyoxyethylene-sorbitan-alkyylikarbonihappoesterit, polyoxyethylene-glycerin-alkyylikarbonihappoesterit, polyoxyethylene alkyl amines, polyoxyethylene-polyoxypropylene lohkarepolymerisaatit, polyoxyethylene alkyl thioether, polyoxyethylene alkyl aryl ether. Specific examples are hydroxypolyethoxy-dodecane, ethylene oxide coupling products of alkylphenols, polyethoxyethylene derivatives of sorbitan anhydrides, etc. 5761 6, etc. Mercaptan-modified polyoxyethylene derivatives have also proved to be somewhat suitable.

When such particularly preferred surfactants are used, it is usually sufficient to add them in an amount of approximately 0.005 to 0.1% by weight.

Compounds belonging to these categories are usually marketed in complete series, which differ in the number of oxyethylene groups. Most useful are water-soluble agents with a moderate number of oxyethylene groups (about 5-20). Types with a lower number of oxyethylene groups are most often only dispersible and therefore less useful. Even more ethoxylated compounds (greater than 25 in number) are water-soluble, but so hydrophilic that it reduces their effect.

Equally good results are obtained with physiological surfactants which meet the above definition, for example deoxycholates.

In addition, other surfactants which have both lipophilic and hydrophilic properties and which contain at least one OH group have also proved useful. Examples are ethanol, butyl diglycol and hexylene glycol. However, lower mono- and dial-alcohols of this kind must be added in relatively large amounts, 10-20% by volume, in order to achieve the desired activation, and their suitability must be determined experimentally in each case. But, for example, methanol, ethylene glycol, polyethylene glycol, cyclohexanol, glycerin, lecithin and saponin do not cause any activation. Also suitable are surfactants which have lipophilic and hydrophilic properties but no OH groups.

Of the anionic wetting agents, salts of bile acids such as cholic acid, tauric acid and deoxycholic acid are particularly suitable, further salts of fatty acids and fatty acid sarcosides. Of particular importance are sulfuric acid derivatives, which are also known to have the property of stabilizing the colored radical oxide products of several indicators (e.g. o-tolidine, heterocyclic azines). These include, for example, the following classes: sulfomeric resin acid esters, alkylarylsulfonates, alkyl sulfates, alkyl polyoxyethylene sulfates.

From the cationic wetting agents, for example, alkylpyridine or trimethylammonium salts can be used, but also compounds with a more complex structure, such as, for example, benzethonium chloride.

Of the amphoteric wetting agents, for example, imidazole lumetaines can be used.

Specific examples are sodium di (2-ethylhexyl) sulfosuccinate, sodium dodecyl sulfate, sodium oleate, benzalkonium chloride and cetylpyridine chloride.

Such ionic or amphoteric detergents can be advantageously used in combination with the above-mentioned nonionic detergents. Suitable amounts correspond to the amounts of nonionic detergents.

Nonionic surfactants containing at least one OH group are considered to be in the most preferred position because they provide about 5 ~ 10 times more potent activation and thus a corresponding increase in reaction rate, i.e. a reduction in reaction time, compared to other useful surfactants. They also have an effect in smaller amounts.

Alkyl groups as used herein mean those having up to about 20 C atoms, primarily 12-18 C atoms.

The inactivation of cholesterol oxidase in the presence of detergent residues is particularly evident when the enzyme is in ammonium sulphate solution. Table 1 below shows the effect of different amounts of detergent on the stability of the enzyme during storage in 1 M ammonium sulfate at 33 ° C. The experiments were performed using an octylphenol-ethylene oxide coupling product.

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Cholesterol oxidase activity calculated as a percentage of the freshly prepared enzyme

Detergent Days without 0.02> ί 0.05 ί 0.15? 0.6% 10 d 89% 60% 10 5? 7 5? 3 5? 22 d 85 5? 48 5? 15? 15? 15?

The effective amount of surfactant compound added in the process of the invention depends on the molecular weight of the compound and the degree of hydrophobic and hydrophilic properties. Too little amount does not activate at all, too much amount causes denaturation of the enzyme, which is a well-known effect in the case of surfactants. Suitable concentration ranges as well as optimal concentrations for each suitable surfactant can be determined by experiments. For example, suitable limits for hydroxypolyethoxydodecane are 0.15-0.6% by weight, for octylphenol ethylene oxide coupling products 0.03-0.1% by weight, for polyoxyethylene derivatives of sorbitan anhydrides 0.15-0.6% by weight, for sodium deoxycholate 0.03-0.05% by weight for connection products (e.g. Sterox SE) 0.005-0.02% by weight.

Of the nonionic polyoxyethylene surfactants, those in which the ratio of the hydrophobic residue to the polyoxyethylene chain is balanced and which are water-soluble are particularly useful.

With respect to the hydrophobic residue and the polyoxyethylene chain, the measure is the so-called HLB value (cf. W.C. Griffin, J. Soc. Cosmetic. Chem. 1, 311 (1950) and 5, 249 (1954)).

Surfactants with HLB values between 10 and 17 · are particularly suitable. However, these values must only be understood as guide values, since the effect also depends on the nature of the hydrophobic residue.

The following examples illustrate the invention.

Example 1 0.05 ml of serum is poured into 10 ml of 0.5 M potassium phosphate buffer, pH 7.5, containing 0.4 5? hydroksipolyetoksidodekaania.

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Read the extinction (E 2) at 240 nm on a suitable spectral photometer and start the reaction by adding 0,02 ml (= 0,1 U) of storage-stable, completely detergent-free cholesterol oxidase in 1 M ammonium sulphate solution.

After 3 minutes, the extinction (Eg) is read again. The concentration of cholestenone formed and the cholesterol formed by it is obtained from the difference between the first and second readings, taking into account the molar extinction constant of cholestenone at 240 nm.

When measured on a typical sample, 62 mg% free cholesterol and 167 mg% total cholesterol (after saponification) were obtained.

A comparative measurement without the addition of surfactant required a reaction time of 15 minutes.

Removal of detergent residues to better preserve the enzyme is conveniently performed using hydrophobic adsorbent resins. For example, the company Biorad under the trade name Bio-beads as well as Röhm u. Products I sold under the name XAD resins.

Example 2 0.05 ml of serum is added to 10 ml of 0.5 M potassium phosphate buffer, pH 7.5, containing 0.02% hydroxypolyethoxydodecane and 0.03% sodium deoxycholate. Read the extinction (E 2) at 240 nm on a suitable spectral photometer and start the reaction by adding 0,02 ml (= 0,1 U) of storage-free, completely detergent-free cholesterol oxidase in 1 M ammonium sulphate solution.

After 3 minutes, the extinction (Eg) is read again. The concentration of cholestenone formed and the cholesterol formed by it is obtained from the difference between the first and second readings, taking into account the molar extinction constant of 2 cholestenones at 240 nm (ε = 15.5 cm / μmol).

When measured on a typical sample, 65 mg% free cholesterol and 170 mg% total cholesterol were obtained (after saponification).

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Example 3 0.05 ml of serum is poured into any 0.5 M potassium phosphate buffer, pH 7.5, containing 0.02% hydroxypolyethoxydodecane and 0.1% secondary alkyl sulfate. Read the extinction (E 2) at 2 * 10 nm with a suitable spectral photometer and start the reaction by adding 0,02 ml (= 0,1 U) of storage-stable, completely detergent-free cholesterol oxidase in 1 M ammonium sulphate solution.

After 3 minutes, read the extinction (E2) again. The concentration of cholestenone formed and the cholesterol formed by it is obtained from the difference between the first and second readings, in which case the molar extinction constant of cholestenone at 2 * 10 nm must be taken into account.

When measured on a typical sample, 62 mg% free cholesterol and 167 mg% total cholesterol (after saponification) were obtained.

Example * 1 0.5 ml of serum is poured into 10 ml of 0.5 M potassium phosphate buffer, pH 7.5, containing 0.0 * 1% N-lauryl diethanolamide. Read the extinction (E1) at 2 * 10 nmr on a suitable spectral photometer and start the reaction by adding 0,02 ml (= 0,1 U) of storage-stable, detergent-free cholesterol oxidase in 1 M ammonium sulphate solution.

The extinction (E2) is read again after 3 minutes. The concentration of the cholestenone formed and thus the cholesterol is obtained from the difference between the first and second readings, taking into account the molar extinction constant of o cholestenone at 2 * 10 nm (= 15.5 cm / μmol).

Measurement with a typical sample gave 62 mg% free cholesterol and I67 mg% total cholesterol (after saponification). A reference measurement without surfactant required a reaction time of 15 minutes.

Example 5 0.5 ml of serum is poured into 10 ml of 0.5 M potassium phosphate buffer, pH 7.5, containing 0.2% dodecyltrimethylammonium chloride.

Read the extinction (E1) at 2 * t0 nm on a suitable spectral photometer and start the reaction by adding 0,02 ml (= 0,1 U) of storage-stable, detergent-free cholesterol oxidase in 1 M ammonium sulphate solution.

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The extinction (E2) is read again after 3 minutes. The concentration of cholestenone formed and thus the cholesterol is obtained from the difference between the first and second readings, taking into account the molar extinction constant of cholestenone at 240 nm (= 15.5 cm -1).

Measurement with a typical sample gave 62 mg% free cholesterol and 167 mg% total cholesterol (after saponification). A control measurement without surfactant required a reaction time of 15 minutes.

Example 6 0.5 ml of serum is poured into 10 ml of 0.5 M potassium phosphate buffer, pH 7.5, containing 0.08% dioctyl sodium sulfosuccinate. Read the extinction (E 2) at 240 nm on a suitable spectral photometer and start the reaction by adding 0.02 ml (= 0.1 U) of storage-stable, detergent-free cholesterol oxidase in 1 M ammonium sulphate solution.

The extinction (E2) is read again after 3 minutes. The concentration of cholestenone formed and thus the cholesterol is obtained from the difference between the first and second readings, taking into account the molar extinction constant of cholestenone at 240 o nmr (= 15,5 cm / μmol).

Measurement with a typical sample gave 62 mg% free cholesterol and 167 mg% total cholesterol (after saponification). A control measurement without surfactant required a reaction time of 15 minutes.