DD237326A1 - STABILIZATION OF ENZYMES AND SO STABILIZED MEDIUM - Google Patents

Abstract

A method is described for stabilizing various enzymes used for analytical purposes, as control or comparative solutions or as reagents. Stabilization takes place by addition of polyalcohols to the medium containing the enzyme and proteins and / or amino acids or derivatives of these substances and optionally substrates and / or coenzymes.

Description

Application of the invention

Various analytical laboratory tests, especially in clinical laboratory diagnostics, require enzymes. They are commercially available in the form of control and standard solutions to control or calibrate the routine determinations of enzymes in body fluids. On the other hand, they are also used in many forms as tools to determine other parameters. In any case, the reagents must be as stable as possible, because to a large extent depends on the safety of the results.

Since many enzymes are very sensitive to various influences, it must always be ensured in the production of enzyme-containing reagents that their durability is made as safe as possible.

Characteristic of the known technical solutions

The hitherto published, very numerous proposals for the stabilization of enzymes for use in analytics use the most diverse additives, which generally concern only one enzyme or groups of similar acting enzymes. For various reasons, however, it would be desirable to have a uniform type of stabilizing medium for various enzymes.

Object of the invention

The aim of the invention was therefore to find a type of stabilizing medium that was unspecific but applicable to different enzymes.

Essence of the invention

In the trial of various variants, it has surprisingly been found that the stability of numerous enzymes in solutions of proteins, protein derivatives, amino acids or amino acid derivatives in the presence or absence of their substrates or coenzymes considerably increases when polyalcohols are added to the medium. This applies in principle to all enzymes used in the analysis, eg. B. those from the class of hydrolases, eg. B. phosphatases, the transferases, e.g. 8. aminotransferases, the oxidoreductases, eg. As peroxidases, lyases, z. As aldolases, isomerases and ligases. The optional addition of substrates or coenzymes contributes in various cases to improve the stability, but is by no means always effective and not always fully satisfactory with an existing effectiveness. The proteins are of animal, vegetable or microbial origin, as well as their derivatives. Of the soluble proteins, especially serum proteins of humans and animals are suitable. The serum can be added natively or in an edited state. The partial or complete removal of the fat components in a conventional manner, for. B. on the adsorption of Aerosil ^R , is often a favorable pre-treatment. On the other hand, serum fractions can also be used successfully, for example those in which the proteins of lower molecular weight, especially albumin, have been removed. Surprisingly, pure albumin of different origin was suitable in the process described here, e.g. For peroxidase stabilization, in some cases not good; on the other hand, it was well suited for other enzymes. In order to suppress any possible adverse effects of the serum enzymes, the transport proteins or other substances, it proves to be advantageous, if necessary, to partially or completely destroy these activities by heating. Similarly, you can remove if necessary, low molecular weight substances in a conventional manner, for. B. by adsorption on activated carbon, by dialysis or by other methods.

It is possible to use additives of serum or serum fractions in liquid or solid form which give a protein content of the medium of more than 0.1%. Very good effects are often achieved with more than 2%.

Another source of soluble protein is z. B. animal milk. For their use, in principle, the said in serum applies.

Protein derivatives are understood here to mean those compounds which, by breaking down the proteins, extend through fragments down to the amino acids as well as aggregates which arise from proteins or their degradation products.

Protein degradation can be done in several ways, e.g. already purely thermal, with some oxidizing agent (hydrogen peroxide) are added, but especially by hydrolysis with acids, bases or enzymes. Such degradation products are to be prepared in a conventional manner of all proteins. For technical use, it is above all those products which are commercially available at low cost, e.g. Gelatin, hydrolyzed gelatin and peptones.

Gelatine is obtained from scaffold proteins of animals by acid hydrolysis, the peptones are degraded by enzymatic degradation, e.g. B. made of gas, meat, gluthene or soybean protein. The molecular weights of such protein degradation products are very different, depending on the method of preparation, and they are also generally very inhomogeneous. The number of peptide bonds per molecule can range from a few thousand to zero. The molecular sizes of both products are somewhat better defined.

While in gelatin the number of peptide bonds is of the order of a thousand, in the so-called hydrolyzed (or partially hydrolyzed) gelatin used for infusion purposes, molecular weights of about 20,000 to 40,000 occur; the smaller molecules are previously obtained by dialysis away.

In the case of the various commercially available peptones, molecular weights of less than 5,000, sometimes even less than 2,000, usually occur, and free amino acids are always present.

Although such degradation products are generally relatively inhomogeneous, they also have the advantage over serum that they do not possess the properties of antibodies, specific transport proteins, hormones or enzymes and often also do not contain lipids. They are therefore better defined in this respect than serum, which may be important for some purposes.

Aggregates of proteins are gradually formed in the storage of liquid, frozen or lyophilized solutions, but especially quickly on heating. Such aggregates can be composed of two to twenty or more molecules, their bonding form is not well known but relatively strong. It can therefore be partially separated by column chromatography. The properties of the aggregates may differ significantly from those of the individual molecules. Protein aggregates bound to arise, inter alia, whenever used serum for stabilization and a large portion of the potential interfering substances by the so-called. Inactivating (. Heating, e.g., at 50 to 6O ⁰ C for 60 minutes) can be made ineffective

As end products of protein hydrolysis amino acids occur, for. Glycine, alanine, valine, leucine, phenylalanine, tyrosine, serine, cysteine, lysine, aspartic acid, glutamic acid, proline and others. In addition to the naturally occurring amino acids, one can also use synthetic, not occurring in nature, and in addition to α-amino acids also those in which the carboxyl and the amino group are separated by a gap, e.g. ε-aminocaproic acid, or in which two amino groups next to each other. 8. diamino succinic acid. The main derivatives of the amino acids here are the peptides in question, which are obtained in addition to the degradation of proteins by synthesis, but also N-acyl amino acids.

Polyalcohols are understood here in particular to be simple and compound saccharides up to polysaccharides, e.g.

Arabinose, glucose, fructose, sucrose, maltose and others as far as dextran, but also glycosides, sugar alcohols, amines and acids, eg. As methyl glucoside, sorbitol, glucosamine and gluconic acid, and condensation and polymerization of alcohols, eg. For example, polyethylene glycol and polyvinyl alcohol.

Very low is z. As cane sugar, because it is cheap and in great purity everywhere available. An effect occurs at a concentration in the medium of more than 1%, for the stabilization and lyophilization 5 to 15% are particularly favorable.

For the adjustment and maintenance of the desired pH-value an addition of buffer is important. The type of buffer ions and their concentration depends on the enzyme to be stabilized. Phosphate buffer and the so-called Good buffer in concentrations of 0.005 to 0.25 mol / l have proved favorable in various cases. But you can also use citrate, tartrate, succinate and other anions. As cations in addition to sodium, potassium and ammonium and Tris buffer and amines in question. pH values between 4.0 and 9.0 are usable in principle, but in most cases a value of 6.0 to 7.8 am

best. ·

The lyophilization of the enzyme solutions to be stabilized takes place in a conventional manner by z. B. bottlings of 0.1 to 5.0 ml in bottles, this cooled to a temperature below -30 ⁰ C and the ice sublimated under reduced pressure. Thereafter, the bottles are provided with a protective gas or sealed in a vacuum air and moisture-tight.

The stabilization occurs z.T. even in the liquid solution,

Embodiment 1

A. alanine aminotransferase (from pig heart) was (deiipidiert 1 hour at 56 ⁰ C heated) in bovine serum buffered with morpholineethanesulfonic acid to pH 7.2 with addition of 10% sucrose lyophilized. Activity 1,24, umol / l · s

B. Like Approach A, but without cane sugar.

After 21 days of storage at 37 ^° C., 98% of the activity obtained after lyophilization was recovered in A, and in B 86%.

Embodiment 2 '

A. Aspartataminotransferase (from porcine heart) was lyophilized in human serum (deiipidated, heated to 56 ° C for 1 hour) buffered with morpholineethanesulfonic acid to pH 7.2 with the addition of 10% cane sugar. Activity 1.28, umoi / · s

B. Like Approach A, but without. Cane sugar.

After 21 days of storage at 37 ° C, 99% of A and B of 83% of the activity obtained after lyophilization were recovered.

Embodiment 3

A. Lactate dehydrogenase (from yeast) was lyophilized in human serum buffered with morpholine propanesulfonic acid to pH 7.0 with the addition of 10% glucose. Activity 4.66, u.mol / l.s.

B. Like Approach A but without glucose.

After 21 days of storage at 37 ° C., 99% of A and B 95% of the activity obtained after lyophilization were recovered.

Embodiment 4

A. As in Example 3 A, but instead of lactate dehydrogenase, alkaline phosphatase (from calf intestine) was used. Activity 3,80 / imol / l · s

B. Like Approach A but without glucose.

After 21 days of storage at 37 ° C., 93% of A and B 86% of the activity obtained after lyophilization were recovered.

Embodiment 5

A. peroxidase (from horseradish) was lyophilized in the following solution: 2% casein peptone, 4% dextran, 0.01 mol / l phosphate buffer, pH 6.5. The peroxidase content was 800ng / ml.

B. Like Approach A but without Dextran.

After 6 days of storage at 37 ^° C., 59% were recovered for A and 6% for B after the lyophilization.

Exemplary embodiment 6

A. As in Example 5, Approach A, but 10% cane sugar was used instead of dextran.

3. Like Approach A, but without cane sugar.

After 14 days of storage at 37 ° C, A recovered 92% and B 6% of the activity obtained after lyophilization.

Exemplary embodiment 7

A. Progesterone peroxidase conjugate (prepared by the mixed anhydride method) was lyophilized in the following solution: 0.5% partially hydrolyzed gelatin (Geiafusal ^R ), 10% sorbitol, phosphate buffer 0.01 mol / l, pH 6.5 , The peroxidase content was about 80 ng / ml.

B. Like Approach A, but without sorbitol.

After 8 days storage at 37 ^° C., A showed 80%, B 12% of the activity obtained after lyophilization.

Embodiment 8

A. 17-hydroxyprogesterone peroxidase conjugate was lyophilized in the following solution: 2% glycine, 10% cane sugar, 0.01 mol / l phosphate buffer, pH 6.5. The peroxidase content was about 30 ng / ml.

B. Like Approach A, but without cane sugar.

After 6 days of storage at 37 ^° C., 64% of the activity obtained in the case of B and 12% of the activity obtained in the course of the lyophilization were recovered.

Embodiment 9

A. Human serum was in a conventional manner pre-treated (one hour at 56 ⁰ C heated delipidated and treated with activated charcoal) and then with ammonium sulfate solution precipitated (final 2,0mol / l of ammonium sulphate). After centrifugation, the supernatant was discarded and the precipitate was washed twice with 2.0 mol / L ammonium sulfate solution. The thus pre-cleaned precipitate was taken up in 0.05 mol / l phosphate buffer, pH 6.5, membrane-filtered and washed in an ultrafiltration plant with 0.05 mol / l phosphate buffer. Finally, the protein content was adjusted to 50 g / l. This ultrafiltrate was used in the following experiments. Progesterone peroxidase conjugate prepared by the mixed anhydride method was lyophilized in a solution of the following composition: 80% of the above ultrafiltrate, 10% cane sugar, 0.05 mol / l phosphate buffer, pH 6.5. The peroxidase content was about 60 ng / ml.

B. Like Approach A, but without cane sugar.

After 10 days of storage at 37 ^° C., 92% of the activity obtained after lyophilization was found in A, and in B 63%.

Claims (8)

claims: 1. A process for the stabilization of enzymes from the classes of hydrolases, transferases, oxidoreductases, lyases, isomerases or lyases in a protein and / or amino acids or derivatives of these substances and optionally substrates and / or coenzymes containing medium, characterized in that the medium Polyalcohols admits. 2. The method according to claim 1, characterized in that the proteins of animal, vegetable or microbial origin. 3. The method according to claim 1, characterized in that the protein derivatives are degradation products or aggregates of proteins. 4. The method according to claim 1, characterized in that the derivatives of amino acids are peptides or N-acyl amino acids. 5. The method according to claim 1 to 4, characterized in that the content of proteins and / or amino acids or the derivatives of these substances in the medium more than 1 g / l, preferably 20 to 60 g / l. 6. The method according to claim 1, characterized in that the polyalcohols are simple, composite or polysaccharides, sugar alcohols, amines or acids, glycosides, condensation or polymerization of alcohols. 7. The method according to claim 1 and 6, characterized in that the addition of polyalcohols between 10und400g / l, preferably between 50 and 150g / l. 8. Stabilized enzymes in a protein and / or amino acids or derivatives of these substances and optionally substrates and / or coenzymes containing medium, characterized in that the medium contains polyalcohols.