Method for stabilizing ascorbic acid oxidase

Abstract

Provided are: a method for stabilizing an ascorbic acid oxidase; a method for preserving an ascorbic acid oxidase; and a stabilized composition of an ascorbic acid oxidase. A method for stabilizing an ascorbic acid oxidase and a method for preserving an ascorbic acid oxidase, each of the methods comprising allowing an ascorbic acid oxidase to coexist with nitrous acid or a salt thereof, or a nitrous acid ester in an aqueous medium; and a stabilized composition of an ascorbic acid oxidase, which comprises an ascorbic acid oxidase being allowed to coexist with nitrous acid or a salt thereof, or a nitrous acid ester in an aqueous medium. The method for stabilizing an ascorbic acid oxidase, the method for preserving an ascorbic acid oxidase, and the stabilized composition of an ascorbic acid oxidase according to the present invention are useful for clinical diagnosis and the like.

Claims

1. A method for stabilizing an ascorbic acid oxidase, which comprises allowing the ascorbic acid oxidase to coexist at 5 to 45 C. for at least 10 days with nitrous acid or a salt thereof, or a nitrous acid ester in an aqueous medium, wherein the salt is selected from the group consisting of a lithium salt, a sodium salt, a potassium salt, an ammonium salt, a calcium salt and a magnesium salt, and the nitrous acid ester is selected from the group consisting of methyl nitrite, ethyl nitrite, propyl nitrite, butyl nitrite, pentyl nitrite and isopentyl nitrite.

2. A method for preserving an ascorbic acid oxidase, which comprises allowing the ascorbic acid oxidase to coexist at 5 to 45 C. for at least 10 days with nitrous acid or a salt thereof, or a nitrous acid ester in an aqueous medium, wherein the salt is selected from the group consisting of a lithium salt, a sodium salt, a potassium salt, an ammonium salt, a calcium salt and a magnesium salt, and the nitrous acid ester is selected from the group consisting of methyl nitrite, ethyl nitrite, propyl nitrite, butyl nitrite, pentyl nitrite and isopentyl nitrite.

Description

EXAMPLES

Example 1

(1) The effects of a salt of nitrous acid and a nitrous acid ester for stabilizing an ascorbic acid oxidase were evaluated by the following method.

(1) Specimen

(2) Specimen A (specimens A0 to A4) and specimen B (specimens B0 to B4) consisting of the following compositions were prepared.

(3) TABLE-US-00001 <Specimen A (specimens A0 to A4)> MOPS (pH 7.0) 20 mmol/L BSA 4 g/L Chemically modified ascorbic acid oxidase 4 kU/L

(4) A salt of nitrous acid or a nitrous acid ester (see Table 1, specimen A0 containing neither salt of nitrous acid nor a nitrous acid ester)

(5) TABLE-US-00002 <Specimen B (specimens B0 to B4)> MOPS (pH 7.0) 20 mmol/L BSA 4 g/L Ascorbic acid oxidase 4 kU/L

(6) A salt of nitrous acid or a nitrous acid ester (see Table 1, specimen B0 containing neither salt of nitrous acid nor a nitrous acid ester)

(2) Buffer Solution for Measuring of Ascorbic Acid Oxidase Activity

(7) A buffer solution for measuring ascorbic acid oxidase activity, consisting of the following composition was prepared.

(8) TABLE-US-00003 Potassium dihydrogen phosphate (pH 6.0) 67 mmol/L Sodium acetate 67 mmol/L BSA 1 g/L

(3) Substrate Solution of Ascorbic Acid Oxidase

(9) An ascorbic acid aqueous solution (3 g/L) was used as a substrate solution of ascorbic acid oxidase.

(4) Ascorbic Acid Oxidase Activity in Specimen Immediately After Preparation

(10) The reagent (3 mL) for measuring ascorbic acid oxidase activity in above (2) was added to a reaction cuvette and incubated at 37 C. for 5 minutes. The specimen A1 (0.04 mL) was added thereto, and then the substrate solution for ascorbic acid oxidase (0.1 mL) in above (3) was added to start reaction. The absorbance (E1) at 292 nm of the reaction solution at 2 minutes after reaction and the absorbance (E2) at 292 nm of the reaction solution at 3 minutes after reaction were measured, and activity V.sub.A1(immediately after preparation) of an ascorbic acid oxidase in the specimen A1 was determined by the aforementioned equation (I).

(5) Ascorbic Acid Oxidase Activity in Specimen After Preservation at 30 C. for 10 Days

(11) Activity V.sub.A1(after preservation) of an ascorbic acid oxidase in the specimen A1 after preservation at 30 C. for 10 days was determined in the same way as in above (4) except for using the specimen A1 after preservation at 30 C. for 10 days in place of the specimen A1 immediately after preparation.

(6) Residual Ratio of Ascorbic Acid Oxidase Activity in Specimen after Preservation at 30 C. for 10 Days

(12) The residual ratio of ascorbic acid oxidase activity in the specimen A1 after preservation at 30 C. for 10 days relative to activity of an ascorbic acid oxidase in the specimen A1 immediately after preparation was determined by the aforementioned equation (II) from V.sub.A1(immediately after preparation) determined in above (4) and V.sub.A1(after preservation) determined in above (5). The results are shown in Table 1.

(13) In the same way as in above (1) to (6) except for using each of the specimens A2 to A4 and B1 to B4 in place of the specimen A1 as a specimen, the residual ratio of ascorbic acid oxidase activity in each of the specimens after preservation at 30 C. for 10 days relative to ascorbic acid oxidase in each of the specimens immediately after preparation was determined. The results are shown in Table 1.

(14) In the same way as in above (1) to (6) except for using each of the specimens A0 and B0 in place of the specimen A1 and using the aforementioned equation (III) in place of the aforementioned equation (II), the residual ratio of ascorbic acid oxidase activity in each specimen after preservation at 30 C. for 10 days relative to ascorbic acid oxidase activity in each specimen immediately after preparation was further determined. The results are shown in Table 1.

(15) TABLE-US-00004 TABLE 1 Salt of Nitrous Acid or Ester of Residual Specimen Nitrous Acid (Concentration) Ratio (%) A0 52 A1 Sodium Nitrite (7 mmol/L) 72 A2 Sodium Nitrite (14 mmol/L) 72 A3 Potassium Nitrite (7 mmol/L) 68 A4 Ethyl Nitrite (7 mmol/L) 70 B0 48 B1 Sodium Nitrite (7 mmol/L) 87 B2 Sodium Nitrite (14 mmol/L) 89 B3 Potassium Nitrite (7 mmol/L) 85 B4 Ethyl Nitrite (7 mmol/L) 83

(16) It is apparent from Table 1 that the residual ratio of ascorbic acid oxidase activity was 60% or more under the coexistence of a salt of nitrous acid or a nitrous acid ester compared to that under the absence of a salt of nitrous acid or a nitrous acid ester even in the case where a chemically modified ascorbic acid oxidase was used as an ascorbic acid oxidase (A0 to A4) or even in the case where a non-modified ascorbic acid oxidase was used (B0 to B4). In contrast, the residual ratio thereof was less than 60% under the absence of a salt of nitrous acid or a nitrous acid ester. Thus, it proved that the coexistence of a salt of nitrous acid or a nitrous acid ester with an ascorbic acid oxidase in an aqueous medium stabilizes the ascorbic acid oxidase.

INDUSTRIAL APPLICABILITY

(17) According to the present invention, a method for stabilizing an ascorbic acid oxidase, a method for preserving an ascorbic acid oxidase, and a stabilized composition of an ascorbic acid oxidase are provided. The method for stabilizing an ascorbic acid oxidase, the method for preserving an ascorbic acid oxidase, and the stabilized composition of an ascorbic acid oxidase according to the present invention are useful for clinical diagnosis and the like.