Glycated proteins are contained in biological samples such as body fluid and hair, and body fluid includes blood in a living body, and such. The concentration of glycated proteins present in the blood depends on the concentration of sugars such as glucose dissolved in the serum, and in the field of clinical diagnosis, measurement of the concentration of hemoglobin A1c (herein after, HbA1c; Non-Patent Document 1), which is a glycated protein in the blood, is being used to diagnose and monitor diabetes mellitus. Hemoglobin is a heme protein consisting of two of each of the two types of subunits, the α-chain and the β-chain, and has a molecular weight of 64,000. HbA1c is defined as hemoglobin in which particularly the N-terminal valine residue of the β-chain is glycated. As a method for measuring this HbA1c, instrumental analytical methods using high performance liquid chromatography (HPLC) (Non-Patent Document 2), immunoassays using antigen-antibody reactions (for example, Non-Patent Document 3), and such had been known, but in recent years, enzymatic assays have been developed, and for example, a method using a protease and a glycated peptide oxidase (Patent Document 1) has been developed. Enzymatic assays can be applied to versatile automated analyzers, and since the operations are also simple, they are being developed actively.
The glycated peptide oxidase used in enzymatic assays is an enzyme that catalyzes the reaction which produces a sugar osone (an α-keto aldehyde), a peptide, and hydrogen peroxide by oxidatively cleaving, in the presence of oxygen molecules, the C—N bond in the ketose derivative produced by Amadori rearrangement of glucosylamine produced by the reaction between the hemiacetal of glucose and the N-terminal amino group of a peptide.
In the case of enzymatic assays, a method is known in which HbA1c is first degraded with a protease, and α-glycated valyl histidine (hereinafter, denoted as α-FVH) is produced from the N terminus of the β-chain of hemoglobin; next, glycated peptide oxidase is made to act on the produced α-FVH to produce hydrogen peroxide, a quinone dye is produced in the presence of peroxidase by the produced hydrogen peroxide, and the produced amount is determined by colorimetry using a spectrophotometer (Patent Document 1).
However, ϵ-glycated lysine (herein after denoted as ϵ-FK), in which a sugar is bound to the ϵ-amino group of lysine, and glycated peptides containing the ϵ-FK are produced as byproducts by the protease treatment, and it has been pointed out that there is a risk that, in case glycated peptide oxidase acts on them, the measured values of HbA1c may be higher than the true values (Patent Document 2).
Glycated peptide oxidase has been found from bacteria, fungi, and plants. For example, glycated peptide oxidase derived from the genus Achaetomiella, the genus Chaetomium (Patent Document 3), the genus Curvularia (Patent Document 2), the Rosaceae family, the Vitaceae family, the Apiaceae family (Patent Document 5), and such are known.
However, glycated peptide oxidases reported so far had drawbacks, such as:
(1) activity towards α-glycated valyl histidine (hereinafter denoted as α-FVH) which is an HbA1c-derived α-glycated dipeptide, in comparison to an α-glycated amino acid (for example, α-glycated valine (hereinafter denoted as α-FV)), is not necessarily high;
(2) as described above, in addition to the N-terminal α-glycated dipeptide, it also acts on ϵ-FK, and increases the measured values in HbA1c measurements; and
(3) in the case of measurement methods using enzymes, the enzymes become unstable during measurement or storage.
To overcome these drawbacks, enzymes with decreased reactivity towards ϵ-FK (Patent Document 4), enzymes with increased thermostability (Non-Patent Document 4), and such, as a result of artificial introduction of mutations into glycated peptide oxidase have been reported. Furthermore, enzymes that have simultaneously overcome the above-mentioned drawbacks of (1) to (3) have been reported (Patent Document 6).
Conventionally, as an HbA1c measurement method set forth by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), a known method for determining HbA1c concentration is the method, in which HbA1c β-chain is digested using Glu-C protease to dissociate a peptide fragment consisting of six amino acids including the glycated N-terminal amino acid [α-glycated hexapeptide: Fru-Val-His-Leu-Thr-Pro-Glu (SEQ ID NO: 134) (hereinafter denoted as α-F6P)], and this is measured using HPLC-capillary electrophoresis (HPLC-CE) or HPLC-mass spectrometry (HPLC-MS) (Non-Patent Document 5). This method is widely used to date as a practical standard method with excellent specificity, but it requires a special apparatus for detection, and has a problem that it requires complicated operations.
In comparison of the above-described practical standard method in the HbA1c measurement (which uses HPLC-CE or HPLC-MS) with an enzymatic assay, the subject of measurement of the former is a glycated hexapeptide, whereas the subject of measurement of the latter is mainly a glycated dipeptide. This is because most known glycated peptide oxidases are highly reactive towards relatively short glycated peptides. Development of an enzymatic assay based on the same principles as the practical standard method, in which α-F6P, a glycated hexapeptide derived from HbA1c, is measured and thereby HbA1c is measured, is believed to be very meaningful in industry also from the viewpoint of increasing the correlation between the two methods.
Known glycated hexapeptide oxidases that act on the glycated hexapeptide α-F6P corresponding to the N terminus of the β-chain of HbA1c are glycated peptide oxidases derived from Zingiberaceae plants (Patent Document 7), glycated peptide oxidases derived from Rosaceae, Vitaceae, and Apiaceae plants (Patent Document 5), glycated peptide oxidases derived from microorganisms (Patent Document 8), and chimeric enzymes consisting of two types of microorganism-derived glycated peptide oxidase sequences (Non-Patent Document 6); however, they have problems such as requiring a long time for reaction with glycated hexapeptides or insufficient reactivity with glycated hexapeptides.
Furthermore, as described above, conventional glycated peptide oxidases can only act on peptides with up to six amino acids, and enzymes having oxidase activity on longer peptide chains and glycated hemoglobin are not known. Therefore, in case measuring glycated hemoglobin by an enzymatic assay, peptide fragments have to be dissociated by a protease as described above, and then the glycated peptide oxidase is made to act on the fragments; however, in case other measurements besides the glycated hemoglobin measurements are taken simultaneously using an automatic analyzer or such, the protease in the reagent for measuring glycated hemoglobin may act on other reagents and may affect the measured values.
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