Method for stabilizing cholesterol oxidase

Abstract

The present invention provides a method for stabilizing a cholesterol oxidase, a method for preserving a cholesterol oxidase, and a stabilized composition of cholesterol oxidase. A method for stabilizing a cholesterol oxidase and a method for preserving a cholesterol oxidase which comprises allowing the cholesterol oxidase to coexist with an -keto acid in an aqueous medium, and, a stabilized composition of cholesterol oxidase which comprises the cholesterol oxidase being allowed to coexist with an -keto acid in an aqueous medium. The method for stabilizing a cholesterol oxidase, the method for preserving a cholesterol oxidase, and a stabilized composition of cholesterol oxidase according to the present invention are useful for clinical diagnosis such as metabolic syndrome.

Claims

1. A method for stabilizing a cholesterol oxidase which comprises the steps of: preparing an aqueous solution of cholesterol oxidase; adding an -keto acid or a salt thereof to the aqueous medium to prepare an aqueous solution comprising the -keto acid or a salt thereof and the cholesterol oxidase; and preserving the aqueous solution comprising the -keto acid or a salt thereof and the cholesterol oxidase at 5 to 45 C. for at least two weeks, wherein a concentration of the -keto acid or a salt thereof in said aqueous solution is from 0.05 to 40 mmol/L, and the -keto acid is selected from the group consisting of pyruvic acid, -ketoglutaric acid and oxaloacetic acid.

2. The method for stabilizing a cholesterol oxidase according to claim 1, wherein said cholesterol oxidase is isolated.

Description

EXAMPLES

Example 1

(1) The effect of an -keto acid for stabilizing a cholesterol oxidase was evaluated by the following method. (1) Specimen

(2) Specimens A (specimens A0 to A3) and specimens B (specimens B0 to B3) comprising the following compositions were prepared.

(3) <Specimens A (specimens A0 to A3)>

(4) TABLE-US-00001 MOPS (pH 7.0) 20 mmol/L CHO-CE 2 kU/L

(5) -Keto acid (see Table 1, no -keto acid in specimen A0)

(6) <Specimens B (specimens B0 to B3)>

(7) TABLE-US-00002 MOPS (pH 7.0) 20 mmol/L Triton X-100 1% CHO-CE 2 kU/L

(8) -Keto acid (see Table 1, no -keto acid in specimen B0) (2) Reagent For Measuring Cholesterol Oxidase Activity

(9) Reagent A and Reagent B comprising the following compositions were prepared.

(10) <Reagent A>

(11) TABLE-US-00003 MOPS (pH 7.0) 50 mmol/L EMSE 0.3 g/L 4-AA 0.1 g/L BioAce 0.3 g/L PGM-50 10 g/L Sodium cholate 5 g/L Peroxidase 15 kU/L
<Reagent B>

(12) Solution of cholesterol in 2-propanol (5 mg/mL)

(13) Reagent B (2 mL) was added to Reagent A (50 mL) and stirred well to thereby provide a reagent for measuring cholesterol oxidase activity. (3) Difference in the Absorbances for a Specimen Immediately After Preparation

(14) The specimen A1 immediately after preparation (2.4 L) and the reagent for measuring cholesterol oxidase activity (225 L) of (2) described above were added to a reaction cell. After warming at 37 C. for 5 minutes, the absorbance of the reaction solution, E1.sub.A1(immediately after preparation), was measured at a main wavelength of 546 nm and a sub-wavelength of 800 nm. The reaction solution was further warmed at 37 C. for 5 minutes, and the absorbance of the reaction solution 10 minutes after reaction, E2.sub.A1(immediately after preparation), was measured at a main wavelength of 546 nm and a sub-wavelength of 800 nm. E1.sub.A1(immediately after preparation) was subtracted from E2.sub.A1(immediately after preparation) to provide E.sub.A1(immediately after preparation).

(15) Similarly, the reaction was carried out using distilled water as a specimen. The absorbance of the reaction solution 5 minutes after reaction, E1.sub.0, was subtracted from the absorbance of the reaction solution 10 minutes after reaction, E2.sub.0, to provide E.sub.0. E.sub.0 was subtracted from E.sub.A1(immediately after preparation) to provide the difference in the absorbances, E.sub.A1(immediately after preparation) for the specimen A1 immediately after preparation. (4) Difference in the Absorbances for a Specimen After Preservation at 5 C. for 2 Weeks

(16) Except that the specimen A1 after preservation at 5 C. for 2 weeks was used instead of the specimen A1 immediately after preparation, the difference in the absorbances, E.sub.A1(after preservation) for the specimen A1 after preservation at 5 C. for 2 weeks was determined by the same method as in (3) described above. (5) Residual Ratio of a Cholesterol Oxidase in the Specimen After Preservation at 5 C. for 2 Weeks

(17) The residual ratio of the cholesterol oxidase in the specimen after preservation at 5 C. for 2 weeks relative to the cholesterol oxidase in the specimen immediately after preparation was determined from E.sub.A1(immediately after preparation) determined in (3) described above and E.sub.A1(after preservation) determined in (4) described above, using the expression (II) described above. The results are shown in Table 1.

(18) The residual ratio of the cholesterol oxidase in each of the specimens after preservation at 5 C. for 2 weeks relative to the cholesterol oxidase in each of the specimens immediately after preparation was determined by the same method as in (1) to (5) described above except that the specimens A2 and A3 and specimens B1 to B3 were each used as the specimen instead of the specimen A1. The results are shown in Table 1.

(19) Furthermore, the residual ratio of the cholesterol oxidase in each of the specimens after preservation at 5 C. for 2 weeks relative to the cholesterol oxidase in each of the specimens immediately after preparation was determined by the same method as in (1) to (5) described above except that the specimen A0 or the specimen B0 was used instead of the specimen A1 and the expression (III) described above was used instead of the expression (II) described above. The results are shown in Table 1.

(20) TABLE-US-00004 TABLE 1 Residual Specimen -Keto acid (Concentration) ratio (%) A0 61 A1 Sodium pyruvate (0.5 g/L) 88 A2 -Ketoglutaric acid disodium 91 (0.5 g/L) A3 Oxaloacetic acid (0.5 g/L) 80 B0 71 B1 Sodium pyruvate (0.5 g/L) 95 B2 -Ketoglutaric acid disodium 85 (0.5 g/L) B3 Oxaloacetic acid (0.5 g/L) 79

(21) As seen from Table 1, it was found that in case the specimens containing no surfactant (A0 to A3) were used or in case the specimens containing a surfactant (B0 to B3) were used, the residual ratio of the cholesterol oxidase in the coexistence of an -keto acid was higher than that in the absence of an -keto acid and was 75% or more. In contrast, in the absence of an -keto acid or a salt thereof, the residual ratio was as low as less than 75%. Thus, it was found that allowing a cholesterol oxidase to coexist with an -keto acid or a salt thereof in an aqueous medium stabilizes the cholesterol oxidase.

INDUSTRIAL APPLICABILITY

(22) The present invention provides a method for stabilizing a cholesterol oxidase, a method for preserving a cholesterol oxidase, and a stabilized composition of cholesterol oxidase. The method for stabilizing a cholesterol oxidase, the method for preserving a cholesterol oxidase, and a stabilized composition of cholesterol oxidase according to the present invention are useful for clinical diagnosis such as metabolic syndrome.