A glycated protein is a protein that has been glycosylated in a nonenzymatic manner and produced as a result of nonenzymatic covalent binding between an aldehyde group on a saccharide, i.e., an aldose (a monosaccharide potentially having an aldehyde group or a derivative thereof), and an amino group on the protein. These glycated proteins are also referred to as so-called “Amadori compounds” since they are formed through Amadori rearrangement of a Schiff's base produced as a reaction intermediate.
Glycated protein is contained in biological samples including hair and body fluid such as blood in an organism. The concentration of glycated protein that is present in blood strongly depends on the concentration of saccharides such as glucose dissolved in blood serum. With a diabetic condition, production of glycated protein is accelerated and the concentration of glycated hemoglobin contained in erythrocyte or the concentration of glycated albumin in blood serum reflect the average blood glucose level of the specific past period. Thus, the assay of those glycated proteins is important in diagnosis or control of diabetic symptoms.
Conventional methods known as methods for quantitatively analyzing a glycated protein include a method utilizing high performance liquid chromatography (Chromatogr. sci., 10, 659 (1979)), a method utilizing a column in which a solid prepared by binding a boric acid is packed (Clin. Chem., 28, 2088-2094 (1982)), a method utilizing electrophoresis (Clin. Chem., 26, 1598-1602 (1980)), a method utilizing an antigen-antibody reaction (JJCLA, 18,620 (1993)), a method for performing colorimetric measurement of reducibility using tetrazolium salt (Clin. Chim. Acta, 127, 87-95 (1982)), and a method for performing colorimetric measurement after oxidation with thiobarbituric acid (Clin. Chem. Acta, 112, 197-204 (1981)). An enzymatic method is currently proposed as a method for assaying a glycated protein in which the procedure is carried out in a simpler and more cost-effective manner within a shorter period of time with higher accuracy compared to the above-described methods (Japanese Patent Publication (kokoku) No. 33997/1993 (Hei5-33997), Japanese Patent Laid-Open No. 127895/1999 (Hei11-127895), and WO 97/13872).
In these enzymatic methods, a glycated protein is decomposed with protease and a fructosyl amino acid oxidase is allowed to act on a liberated glycated amino acid to assay the produced hydrogen peroxide. Examples of oxidases disclosed as usable in the enzymatic assay methods include an oxidase produced from a bacteria belonging to the genus Corynebacterium (Japanese Patent Publication (kokoku) Nos. 33997/1993 (Hei5-33997) and 65300/1994 (Hei6-65300)), oxidase produced from a fungus belonging to the genus Aspergillus (Japanese Patent Laid-Open No. 155780/1991 (Hei3-155780)), an oxidation produced from a fungus belonging to the genus Gibberella (Japanese Patent Laid-Open No. 289253/1995 (Hei7-289253)), an oxidase produced from a fungus belonging to the genus Fusarium (Japanese Patent Laid-Open Nos. 289253/1995 (Hei7-289253) and 154672/1996 (Hei8-154672)), an oxidase produced from a fungus belonging to the genus Penicillium (Japanese Patent Laid-Open No. 336386/1996 (Hei8-336386)), and a ketoamine oxidase (Japanese Patent Laid-Open No. 192193/1993 (Hei5-192193)). These enzymes effectively react with a glycated amino acid. However, the above enzymes do not react with a glycated peptide in which an amino group of a peptide has been glycated.
At present, various glycated proteins are used as an index for diagnosing diabetes. Examples of such glycated proteins include those in which an ε-amino group of an internal lysine residue in a protein is glycated (for example, glycated albumin) and those in which an α-amino group of an amino acid at the amino terminus of the protein is glycated (for example, glycated hemoglobin (HbAlc)). Currently, however, some glycated proteins cannot be decomposed to quantitatively liberate a glycated amino acid even with the use of conventional protease. In addition, the above-described fructosyl amino acid oxidase, which is currently employed, has a high reactivity to a liberated glycated amino acid although it does not substantially react with a glycated peptide. Thus, the above enzymatic method is not always accurate.
For example, glycated hemoglobin (HbAlc) is produced by glycating an α-amino group of an amino acid at the amino terminus of hemoglobin β-subunit. However, even though various proteases react with the glycated protein, the α-glycated amino acid (an α-amino group of the amino acid is glycated) cannot be liberated. Thus, even with the use of the above-described fructosyl amino acid oxidase, glycated hemoglobin (HbAlc) cannot be assayed.
At present, methods proposed for assaying glycated hemoglobin (HbAlc) include a method in which glycated hemoglobin is directly assayed in that state by electrosprayionization mass spectrometry (Clinical Test, 42, 304-343 (1997)), a method in which endoproteinase Glu-C is allowed to act on glycated hemoglobin, a liberated α-glycated hexapeptide derived from β-subunit (α-amino group of an amino acid at the amino terminus of hexapeptide is glycated) is fractionated by reversed phase high performance liquid chromatography, and the content thereof is determined through mass spectrometry analysis to assay (Clin. Chem., 43, 1944-1951 (1997)), and the like. However, a highly sensitive and expensive assay device is necessary for these methods, and the operation of the assay is complicated, costly, and time-consuming.
The object of the present invention is to overcome the drawbacks of the conventional methods for assaying a glycated protein and to provide a novel method for assaying a glycated protein by a simple procedure based on a principle different from that of the conventional enzymatic methods in a cost-effective manner within a short period of time with high accuracy.
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