CS210467B1 - Stabil enzyme preparation for fixing the urea - Google Patents

Description

The present invention provides a stable urea enzyme preparation.

The determination of urea is of considerable analytical but especially clinical importance since urea is the most important end product of protein metabolism.

In clinical biochemistry, the determination of urea in biological material is most often carried out photometrically, for example by reaction with alpha-diketones and their oximes. Recently, often by the enzyme method, urea is specifically hydrolysed by urease to carbon dioxide and ammonia, which is then determined by a suitable analytical reaction, for example as colored indofenol. The urea enzyme assay is specific and does not require high temperature heating, unlike the reaction of urea with alpha-diketones or their oximes. The enzymatic reaction of urea with urease proceeds with optimum efficiency in aqueous solutions of pH 6.0 to 8.5. These solutions must be suitably buffered to ensure optimum pH of the enzyme reaction even after addition of the sample to be analyzed, and to avoid pH changes due to ammonia resulting from the hydrolysis of urea.

A major disadvantage of the known urease enzyme-containing agents is their low stability, since urease is rapidly inhibited, especially in aqueous dew210467 streams, already by traces of heavy metals. Similarly, agents containing solid urease enzyme are poorly stable when exposed to atmospheric moisture.

Undesirable heavy metals may be introduced into the reaction mixture as trace impurities together with substances used as buffers or in the analyzed material.

In order to overcome the aforementioned drawbacks, a stable enzyme preparation according to the invention is characterized in that it comprises 0.05 to 40 parts by weight of urease with a specific activity of 1000 to 1500 000 U / g, 1 to 10 parts by weight of a buffering component, e.g. 5'-diethylbarbiturate of alkali metal, alkali metal phosphates, for example sodium or potassium, 1 to 20 parts by weight of complexate, for example in the form of free nitrilotriacetic acid, ethylenediaminetetraacetic acid or its disodium salt, and optionally 90 to 99.9 parts by weight of water. The composition further comprises 3 to 120 parts by weight of an alkali metal azide, for example sodium azide.

The stable enzyme preparation of the present invention contains both urease enzyme and buffer, which not only adjusts the pH optimal for the enzyme reaction, but also stabilizes the en210487 zym, both in the solid mixture and in solution.

By combining the buffering component with the complexan free acid form with sodium azide, the formulation is perfectly stable not only as a solid mixture but also as an aqueous solution for direct use. Unlike previously known and industrially produced forms of urease in diagnostic kits, i.e., suspensions with limited stability of up to 6 months at reduced temperature, the enzyme preparation of the invention contains the enzyme together with buffer and stabilizers and can be used directly for the enzyme reaction without it is necessary to modify its reaction conditions.

The enzyme preparation can be taken either in solid form and dosed into the reaction, for example, in the form of a tablet, or by measuring a powder dose, or as a solution. In both cases, the reaction mixture is metered in such an amount that the reaction mixture contains a suitable amount of urease enzyme, preferably 0.5 to 5 U. Under these conditions, the reaction mixture simultaneously has a pH optimal for the enzyme reaction.

As has been experimentally verified, the solid composition of the composition of the invention is stable practically indefinitely, the aqueous solution for several months. Buffer solution based on buffering component and complexan or even azide eliminates the effect of heavy metals combined by the effect and at the same time eliminates the inhibition of the enzyme by possible bacterial contamination in the case of azide. At the same time, the buffering capacity and the resulting optimal pH setting are also advantageous. The agent of the invention can be prepared in a simple manner from cheap and available raw materials.

He did

A solid mixture is prepared containing 50 U urease, 0.04 g of sodium 5,5'-diethylbarbiturate, 0.03 g of ethylenediaminotetraacetic acid disodium salt and 0.03 g of sodium azide. For the determination of urea, 0.01 g of the preparation is added to the reaction mixture.

Example 2

A solid mixture of 500 U urease, 2.8 g of potassium 5,5'-diethylbarbiturate and 1 g of ethylenediaminetetraacetic acid is prepared. For the determination, 0.006 g is added or the dry mixture is dissolved in 0.25 kg of water and 0.5 ml for the determination.

Example 3

A solid mixture is prepared containing 247500 U urease with a specific activity of at least 15,000 U / g, 50 g of sodium 5,5'-diethylbarbiturate and 100 g of ethylenediaminetetraacetic acid disodium salt. For the determination, 0.0044 g of the mixture is dosed.

Example 4

A solid mixture was prepared containing 1 g of disodium ethylenediaminetetraacetic acid, 0.1 g of disodium hydrogen phosphate and 45,000 U of urease. For the determination, dissolve the mixture in 450 ml of distilled water and measure 1.0 ml of this solution for analysis.

Example 1 a d 5

Prepare a solid mixture of urease (500 UJ, 2.8 g of sodium 5,5'-diethylbarbiturate and 0.7 g of nitrilotriacetic acid) and add 0.006 g of this mixture.

Claims (2)

SUBJECT 1 to 10 parts by weight of a buffering component, for example alkali metal 5,5'-diethylbarbiturate, alkali metal phosphates, for example sodium or potassium, 1 to 20 parts by weight of complexate, for example in the form of free nitrilotriacetic acid up to 99.9% by weight of water. A stable enzyme preparation for the determination of urea, characterized in that it contains 0.05 to 40 parts by weight of urease with a specific activity of 1 000 to 1 500 000 U / g, 2. A stable enzyme preparation for the determination of urea according to claim 1, characterized in that it contains from 3 to 120 parts by weight of an alkali metal azide, for example sodium azide.