JPH0574359B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a reagent for enzymatically measuring N-acetylhexosamine in a sample, particularly an aqueous liquid. Enzymatic measurement of N-acetylhexosamine in body fluids can be performed using a number of sugar-related compounds, such as N-acetylhexosamide, sialic acid, the mucopolysaccharide hyaluronic acid, chondrotin sulfate, dermatan sulfate,
Heparin, heparan sulfate, ketalan sulfate and enzyme activities such as N-acetyl hexasominidase,
Used for quantifying muramidase, sialidase, etc. The clinical significance of these activities is that measurement of N-acetylhexosaminidase activity is useful for early detection of renal diseases, especially proximal tubular disorders, renal transplant rejection, etc., and measurement of muramidase activity is useful for the early detection of renal diseases, especially proximal tubular disorders, renal transplant rejection, etc. Quantification of sialic acid is used to diagnose atypical tuberculosis, etc. Sialidase activity is used to diagnose inflammatory diseases, etc.
Measurement of mucopolysaccharide provides information effective in diagnosing bacterial infections and the like, as well as in diagnosing mucopolysaccharidoses. (Prior Art) Conventionally, there has been no suitable method for quantifying N-acetylhexosamine, and enzyme activities such as N-acetylhexosaminidase have been determined by measuring other reaction products. For example, N-acetylhexosaminidase activity, β-glocuronidase activity, etc. can be determined by colorimetrically quantifying the chromophore produced by the enzymatic reaction using a chromodiic substrate bound to a chromophore such as p-nitrophenyl. The enzyme activity was determined by This method is complicated because the optimum pH of the enzyme and the pH of the chromophore are different. In addition, muramidase activity is not quantitative because it is determined by the rate of lysis using live bacterial cells as a substrate, and there is considerable variation. Sialic acid is
Chemical methods and thiobarbituric acid methods have low specificity and are complicated to operate. Furthermore, the enzymatic method for determining the amount of sialic acid by measuring the reaction product pyruvate has a problem in accuracy due to endogenous pyruvate. Regarding sialidase, the aminoff method is known, which measures sialic acid released using sialyllactose as a substrate, but it has problems in terms of sensitivity and accuracy. Mucopolysaccharides have drawbacks such as the lack of suitable quantitative methods. Taking these circumstances into consideration, the inventors have
We discovered an accurate, simple, and quick method for quantifying sugar-related compounds and enzyme activities and applied for a patent (patent application filed in 1983).
No. 42652). In other words, N-acetylhexosamine oxidase, which reacts with N-acetylhexamine in the presence of water and oxygen to produce hydrogen peroxide, is applied to the sample, and the hydrogen peroxide produced or the oxygen consumed is measured. This is a method for quantifying N-acetylhexosamine using an enzymatic method. This method is more accurate, simple, and
Although the problem of speed was solved, it was found that the problem of reactivity was still insufficient. (Problems to be Solved by the Invention) The purpose of the present invention is to improve the reactivity of an enzymatic determination method for N-acetylhexasomine,
This also increases practicality. (Means for Solving the Problems) In order to achieve the above-mentioned object, the present inventors conducted various studies and found that the effect of promoting the conversion of the anomer of N-acetylhexosamine (from α type to β type) By using mutarotase as a conjugate enzyme in the presence of N-acetylhexosamine oxidase, the reactivity of N-acetylhexosamine oxidase is promoted and N-acetylhexosamine is rapidly quantified with more accuracy and simple operation than ever before. This discovery led to the present invention. That is, the present invention is characterized in that it contains N-acetylhexosamine oxidase and mutarotase that has the effect of promoting the conversion of the anomer of N-acetylhexosamine (from α type to β type).
-A reagent for measuring acetylhexosamine. N-acetylhexosamine to be measured in the present invention includes N-acetylglucosamine, N-
Examples include acetylgalactosamine and N-acetylmuramic acid. N-acetylhexosamine may be derived from sugar-related compounds by chemical or enzymatic methods. For example, N-acetylhexosaminide, N-
There is N-acetylhexosamine produced by the action of acetylhexosaminidanse. In addition, N-acetylglucosamine and N-acetylglucosamine and N-
There is acetylhexosaminic acid. Furthermore, neuraminidase is allowed to act on the bound sialic acid to generate N-acetylneuraminic acid, thereby obtaining N-acetylmannosamine and pyruvic acid. Next, there is N-acetylglucosamine, which is obtained by treating N-acetylmannosamine with acylglucosamine-2-epimeranase. For example, in the case of N-acetylglucosamine, it is known that N-acetylhexosamine exists in β-type and α-type as shown in the figure below.

[Formula] N-acetyl-β-D-glue Cosamine

[Formula] N-acetyl-α-D-glucosamine The mutarotase used in the present invention is the conversion of the anomer of N-acetylhexosamine (α type → β type).
It is an enzyme that has the effect of promoting D-
This effect cannot be confirmed in the known mutarotase (Methods Enzymol 41 , 471 (1975)) whose substrates are pentaose and hexaose, which have the configuration of glucose. On the other hand, known N-acetylhexosamine oxidase is known to have substrate specificity in the form of an anomer, and has particularly high specificity for the β type. In the present invention, due to the presence of mutarotase which has the effect of promoting the conversion of the anomer of N-acetylhexosamine (from α type to β type), the α type and β type N-acetylhexosamine coexisting in the aqueous solution are converted to the β type. Since N-acetylhexosamine oxidase, which is converted into the β form and has high substrate specificity, acts more easily, the reactivity is improved. The mutarotase used in the present invention may be of any origin as long as it has the effect of promoting the anomeric conversion of N-acetylhexosamine (from α type to β type), but for example, mutarotase obtained from pig kidney can be used. There is. The concentration of mutarotase is preferably 17 μ/ml or more. The N-acetylhexosamine oxidase used in the present invention may be of any origin, but for example, N-acetylhexosamine oxidase produced by a microorganism belonging to the genus Pseudomonas can be used.
-There is acetylhexosamine oxidase. N
-The purification and properties of acetylhexosamine oxidase were presented at the 1988 annual meeting of the Japanese Society of Agricultural Chemistry. The N-acetylhexosamine oxidase concentration is preferably 0.1 unit/ml or more. In addition to N-acetylhexosamine oxidase and mutarotase, the reagent of the present invention may optionally contain peroxidase or catalase, a coloring agent such as a phenol derivative or an aniline derivative, and further, ascorbic acid oxidase. Furthermore, the measurement reagent of the present invention has a pH of 4.0.
~10.0 is preferred. The conditions under which the reagent is allowed to act on the sample are not particularly limited, but it is appropriate to use an appropriate buffer that maintains the pH between 4.0 and 10.0, and at a reaction temperature of 20 to 50°C. The type of buffer solution is not particularly limited. Just select the buffer solution that is most suitable for the pH used. For example, in a neutral range, phosphate buffer or Gutt's buffer is used, in an acidic range, an acetate buffer is used, and in an alkaline range, a Tris-HCl buffer is used. The concentration of the buffer solution is not particularly limited, but is usually 0.01m to 1
It is about m. Further, a surfactant or the like may be added as necessary. As a method for quantifying N-acetylhexasomine using the reagent of the present invention, hydrogen peroxide produced by reacting a sample with the reagent is conjugated with, for example, peroxidase or catalase and a coloring agent, and the absorbance is measured; Alternatively, it can be measured directly electrochemically using a hydrogen peroxide electrode. Ascorbic acid in the sample, which interferes with the determination of hydrogen peroxide, can be removed by the action of ascorbate oxidase. In this case, ascorbate oxidase may be removed by acting prior to other enzyme reactions or by acting simultaneously with other enzymes. Ascorbic acid oxidase may be derived from microorganisms or plants, but those derived from plants such as pumpkin and cucumber are particularly preferred. (Action) In a reagent for measuring N-acetylhexosamine using N-acetylhexosamine oxidase,
By coexisting mutarotase, which has the effect of promoting the conversion of the anomer of N-acetylhexosamine (from α type to β type) as a conjugate enzyme, reactivity is improved and N-acetylhexosamine in the reagent and chemical and enzymatic Accurately, easily,
Can be quantified quickly. (Example) Hereinafter, the present invention will be explained in detail with reference to Examples. Reference example 1 285g of pig kidney cortex was homogenized with 570ml of 0.3M potassium phosphate buffer (PH7.6) and centrifuged (8000γpm
30 minutes), add the same amount of distilled water and 19.2 ml of 2% protamine sulfate to the resulting extract (640 ml), centrifuge (same as above), and add the resulting precipitate to 0.01 M potassium phosphate buffer (PH7. 6) The first wash was performed with 427 ml, and the second and third washes were performed with 192 ml of 0.025M potassium phosphate buffer (PH7.6). As a final step, 150 ml of the resulting protamine extract contained 2.565 g of bentonite.
28.5 ml of 0.001 MEDTA solution was added, centrifuged (8000 γ pm x 20 minutes), and 170 ml of the obtained bentonite extract was concentrated by ultrafiltration, yielding 4.4 ml of mutarotase. Example 1 The amount of N-acetylhexosamine in a specimen was determined using the following reagent and the following method. 1 Reagent N-acetylhexosamine oxidase 1 unit 4-aminoantipyrine 0.5mg N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium 2mg Mutarotase (manufactured in Reference Example 1) 170μ 0.1M phosphorus Acid buffer solution (PH7.0) total volume: 10 ml 2. Measurement method A solution or 20 µ of N-acetylhexosamine with a known concentration was taken, and the absorbance was measured using the above reagent. For comparison, absorbance measurements were performed in the same manner using a reagent to which mutarotase was not added. The results are shown in FIG. (Effects of the Invention) The present invention, which contains mutarotase which has the effect of promoting the conversion of the anomer of N-acetylhexosamine (from α type to β type), has higher reactivity than when no additive is used.

[Brief explanation of the drawing]

FIG. 1 shows a reaction curve between a reagent of the present invention containing mutarotase and a reagent without mutarotase.

Claims (1)

[Claims] 1. A reagent for measuring N-acetylhexosamine, which contains N-acetylhexosamine oxidase and mutarotase, which has the effect of promoting the conversion of the anomer of N-acetylhexosamine (from α type to β type).