JPS6255081A - Stable xanthine oxidase composition - Google Patents

Abstract

PURPOSE:To improve the stability of xanthine oxidase, by adding phosphoric acid or its salt and one or more compounds selected from protein, acidic amino acid or its salt, aliphatic carboxylic acid, aromatic carboxylic acid, etc. CONSTITUTION:There is no particular restriction in the method for compounding the stabilizer. For example, xanthine oxidase is dissolved in a buffer solution containing phosphoric acid or a phosphate such as sodium phosphate, potassium phosphate, etc., and the solution is compounded with one or more compounds selected from a protein, an acidic amino acid or its salt, an aliphatic carboxylic acid, an aromatic carboxylic acid, an alicyclic carboxylic acid and their salt. The buffer solution is preferably a phosphoric acid buffer solution or tris buffer solution having a pH of 6-9. The protein is e.g. animal or vegetable albumin such as serum, albumen, etc., and the acidic amino acid or its salt is e.g. glutamic acid, aspartic acid, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a stable xanthine oxidase composition.

Recently, in the field of clinical diagnostic reagents, there have been high hopes for the development of reagents that use this enzyme to measure guanase activity, inorganic phosphorus, adenosine deaminase activity, etc. in serum.

Xanthine oxidase (hereinafter sometimes abbreviated as XOD) has an enzyme number (EC1, 2, 3, 2) and is a metal (
It is a flavoenzyme containing iron, molybdenum) that oxidizes purine bases such as xanthine and hypoxanthine to produce uric acid. Also, aldehydes, NADH, butyryl/
, pyrimidine can also be a substrate. This enzyme is most commonly found in milk, but it is also present in various animal organs and bacteria.

The reaction of this enzyme is as follows.

Quinanthin 10HtO+O-XOD9Uric acid+HtO*(
Prior Art) Conventionally, the above-mentioned arithmetic element is considered to be in the most stable state as an ammonium sulfate suspension, and most enzyme products are also in this form. Since this suspension is in liquid form, it is relatively stable when stored at a low temperature below 10°C, but if left at room temperature or high temperature, the enzyme activity is significantly deactivated, making it difficult to preserve the enzyme product. Reagents containing oxidizing enzymes also have poor stability, which poses a practical problem.

(Problems to be Solved by the Invention) Even with the ammonium sulfate suspension product, which is an enzyme product with a small unit volume and high specific activity, the enzyme activity is significantly inactivated above room temperature, and the reagent structure is The stability deteriorates as the specific activity per unit volume decreases, so with conventional ammonium sulfate suspensions, enzyme activity is significantly deactivated even when clinical test drugs are added.
Since the reagents are severely degraded, it is difficult to store them and at the same time has a large effect on a single measurement value, posing a practical problem.

(Another Means to Solve the Problem) The present inventors conducted various intensive studies on stabilizers for xanthine oxidase and found that the intended purpose could be achieved by using phosphoric acid or its salt in combination with a specific compound. , arrived at the present invention. That is, the present invention provides xanthine oxidase with (1) phosphoric acid or its salt and <1) protein, acidic amino acid or its salt, aliphatic carboxylic acid, aromatic carboxylic acid,
A stable xanthine oxidase composition containing at least one compound selected from alicyclic carboxylic acids or salts thereof.

Xanthine oxidase of the present invention enzyme number (EC1, 2
, 3, 2) is a flavoenzyme containing iron and molybdenum, and oxidizes purine bases such as xanthine and hypoxanthine to produce uric acid. Aldehydes, N
ADH, pterin, and pyrimidine' can also be substrates. This enzyme is commonly found in milk, but it also exists in various animal organs and in microorganisms.

Examples of the phosphoric acid and its salts used in the present invention include phosphoric acid and salts of phosphoric acid such as potassium and sodium. The concentration of addition is not particularly limited, but is 10 mM or more in the enzyme composition, and there is no upper limit, but it is limited by solubility and PHK.

Proteins used in the present invention include simple proteins such as animal and plant albumins such as serum and egg white, and globulins, and complex proteins such as glycoproteins. The concentration is 0.1 in the enzyme composition
It is 8% or more by weight, preferably 5% or more and 10% or less by weight. If it exceeds 10% by weight, solubility will deteriorate.

The acidic amino acids or their salts used in Zero-Hatsu 8AK include glutamic acid, aspartic acid, and their salts. The concentration in the enzyme composition is 1 (f'-"M or more, preferably 10-"
"M or more. There is no upper limit, but it is limited by solubility.

The aliphatic carboxylic acids used in the present invention include acetic acid, citric acid, and ? There are bottles of α-ketoglutaric acid and uronic acid.
Examples of aromatic carboxylic acids include salicylic acid and benzoic acid, and examples of alicyclic carboxylic acids include cyclobutanedicarboxylic acid, cyclopropanedicarboxylic acid, and cyclohexanedicarboxylic acid. The concentration is 1 in the enzyme composition.
0-"M or more, preferably l O-"M or more,
The upper limit is the solubility x'a*ga''. There is no particular restriction on the method of blending the stabilizer of the present invention.

For example, a method in which xanthine oxidase is dissolved in a buffer solution containing phosphoric acid or a phosphate salt, and then other stabilizing agents are added; There are several methods, such as a method in which an enzyme and a stabilizer are combined in a buffer solution at the same time.

The buffer may be phosphate buffer, Tris buffer, or any other buffer used in biochemistry, but the pH is 6 to 9, preferably 7 to 9.

Further, the enzyme composition may contain other enzymes in addition to the stabilizing agent, the agent-relaxing solution, and the xanthine oxidase, and the other enzymes may be mixed in multiple forms instead of just one type. The enzyme composition may be in liquid form, but it is preferably freeze-dried and crystallized to improve stability.

(Effect of the invention) The present invention provides (+) for xanthine oxidase! By adding a compound of acid or a salt thereof and (ii) a protein, an acidic amino acid or a salt thereof, or a fat, the stability could be significantly improved compared to the conventional sulfur suspension. Therefore, it has become possible to preserve xanthine oxidase for a long time and to create a clinical test drug using xanthine oxidase.

(Example) The present invention will be explained below using Example 'A'.

In the examples, enzyme activity was determined according to the following method.

Enzyme activity measurement method 98 mM phosphate buffer (pH 7,6) 9.8 m
After heating 2.95 mm of substrate solution containing M k hoxanthine 9.8 mM EDTA at 25°C for 5 minutes, add 0.05 d of sample containing xanthine oxidase and measure the change in absorbance at 25°C for 1 minute. Measure.

The activity is determined by the formula.

Example 1 Xanthine oxidase was dissolved in an aqueous solution containing the additives shown in Table 1 below, and the resulting lyophilized product was stored at 30°C for 4 weeks, and then redissolved to determine the enzyme activity. was measured. Table 1 shows the enzyme activity after freeze-drying relative to the enzyme activity before freeze-drying as residual activity (Q).

For reference, bovine santhin oxidase was dissolved in ammonium sulfate suspension, trisamitumethane-hydrochloric acid buffer, borate buffer, and Linling buffer instead of the aqueous solution in which the additive of the present invention was dissolved. Table 1 also shows the cases in which phosphoric acid and neutral amino acids, alkaline acids, or class m are added.

As is clear from Table 1, xanthine oxidase can be stabilized when phosphoric acid is used in combination with acidic amino acids, proteins, and aliphatic carboxylic acids.

Claims (1)

[Claims] xanthine oxidase (i) phosphoric acid or its salt and (
ii) Stable xanthine oxidation comprising at least one compound selected from the group consisting of proteins, acidic amino acids or salts thereof, aliphatic carboxylic acids, aromatic carboxylic acids, alicyclic carboxylic acids, or salts thereof. Enzyme composition.