USOO6867O12B2 (12) United States Patent (10) Patent No.: US 6,867,012 B2 Kishimoto et al. (45) Date of Patent: Mar. 15, 2005
(54) DETERMINATION METHOD OF FOREIGN PATENT DOCUMENTS BIOLOGICAL COMPONENT AND REAGENT EP O 437 373 A 7/1991 KIT USED THEREFOR EP O 477 OO1 A 3/1992 JP P2001-17198 A 1/2001 (75) Inventors: Takahide Kishimoto, Tsuruga (JP); WO WO 99/47559 A 9/1999 Atsushi Sogabe, Tsuruga (JP); Shizuo WO WO OO/28071 A 5/2000 Hattori, Tsuruga (JP); Masanori Oka, Tsuruga (JP); Yoshihisa Kawamura, OTHER PUBLICATIONS Tsuruga (JP) van Dijken et al (Archives of microbiol, V.111(1-2), pp (73) Assignee: Toyo Boseki Kabushiki Kaisha, Osaka 77-83,(Dec. 1976)(Abstract Only).* (JP) (List continued on next page.) (*) Notice: Subject to any disclaimer, the term of this Primary Examiner Louise N. Leary patent is extended or adjusted under 35 (74) Attorney, Agent, or Firm-Kenyon & Kenyon U.S.C. 154(b) by 330 days. (57) ABSTRACT The present invention provides novel glutathione-dependent (21) Appl. No.: 09/998,130 formaldehyde dehydrogenase that makes possible quantita (22) Filed: Dec. 3, 2001 tive measurement of formaldehyde by cycling reaction, and a determination method of formaldehyde and biological (65) Prior Publication Data components, Such as creatinine, creatine, and homocysteine, US 2002/0119507 A1 Aug. 29, 2002 which produces formaldehyde as a reaction intermediate. In addition, the present invention provides a reagent kit for the (30) Foreign Application Priority Data above-mentioned determination method. The present inven tion provides a novel determination method of a homocys Dec. 5, 2000 (JP) ...... 2OOO-370445 teine using transferase utilizing homocysteine and other Mar. 29, 2001 (JP) ...... 2001-096724 compound as a pair of Substrates. Particularly, the present (51) Int. Cl...... C12Q 1/48 invention provides a determination method of homocysteine (52) U.S. Cl...... 435/15; 435/26; 435/25; which includes bringing betaine-homocysteine methyltrans 435/28; 435/975 ferase and dimethylglycine oxidase into contact with a (58) Field of Search ...... 435/15, 26, 25, Sample and measuring produced hydrogen peroxide or form 435/28,975 aldehyde. Moreover, the present invention provides novel dimethylglycine oxidase stable to thiol compound, which is (56) References Cited Suitably used for the measurement. The present invention provides a reagent kit used for any of the above-mentioned U.S. PATENT DOCUMENTS determination methods of homocysteine. 5,668,173 A 9/1997 Garrow ...... 514/557 5.998,191 A 12/1999 Tan et al...... 435/32 73 Claims, 2 Drawing Sheets
0.25 g 0.20 o St cy - 0.15
30, 10 O 0.05
0.00 O 100 200 300 400 500 formaldehyde concentration (M) US 6,867,012 B2 Page 2
OTHER PUBLICATIONS Lasse Uotila, et al., “A Steady-State-Kinetic Model for Mori et al., “Purification and Properties of Dimethylglycine Formaldehyde Dehydrogenase from Human Liver”, Bio Oxidase from Cylindrocarpon didymum M-1”, Agricultural chem. J., 177, 869-878, (1979). and Biological Chemistry, 44(6): 1383-1390 (1980). Bernard Vinet," An Enzymic Assay for the Specific Deter Meskys, R. et al., “Peculiarities of creatinline catabolism in mination of Methanol in Serum", Clin. Chem., 33(12), Some Arthrobacter Strains”, Biologija, 1:61-64 (1997). Kerr R. G. et al., “An Enzyme-Based Formaldehyde ASSay 2204–2208 (1987). and Its Utility in a Sponge Sterol Biosynthetic Pathway”, J. Yuzo Kayamori, et al., “A Sensitive Determination of Uric Nat. Prod., 62(1):201–202 (1999). Acid in Serum Using Uricase/Catalase/Formaldehyde Dehy Kiba N. et al., “Determination of nano-molar levels of drogenase Coupled with Formate Dehydrogenase', Clin. formaldehyde in drinking water using flow-injection System Biochem., 27(2), 93–97 (1994). with immobilized formaldehyde dehydrogenase after off-line Solid phase extraction', Analytica Chimica ACTA, Yutaka Teranishi, et al., “Catalase Activities of Hydrocar 378(1-3):169-175 (1999). bon-utilizing Candida Yeasts' Agric. Biol. Chem. 38(6), Van Ophem P. W. et al., “NAD-and co-substrate (GSH or 1213–1220 (1974). factor)-dependent formaldehyde dehydrogenases from methylotrophic microorganisms act as a class III alcohol Jacob W. Dubnoff, et al., “Dimethylthetin and dehyrogenase”, FEMS Microbiology Letters, 116(1):87–93 Dimethyl-3-Propiothetin in Methionine Synthesis”, J. Biol. (1994). Chem., 176, 789–796 (1948). Kato N. et al., “Formaldehyde Dehyrdogenase from Methy Raymond F. White, et al., “Betaine-Homocysteine Transm lotrophic Yeasts', Methods in Enzymology, 188:455-459 ethylase in Pseudomonas denitrificans, a Vitamin B Over (1990). M.S. Quesenberry, et al., “A Rapid Formaldehyde Assay producer”, J. Bacteriol., 113(1), 218–223, (1973). Using Purpald Reagent: Application under Periodation Con Timothy A. Garrow, “Purification, Kinetic Properties, and ditions”, Anal. Biochem., 234(1), 50–55 (1996). cDNA Cloning of Mammaliam Betaine-Homocysteine Kensa to Gijutsu, 27(8): 973-980 (1999). Methyltransferase” J. Biol. Chem., 271 (37), 22831–22838 Enzyme Handbook 9, 1.1.1.1, “Alcohol Dehydrogenase” (1996). Springer-Verlag, pp. 1-23, (1995). Masayoshi Yasuhara, et al., “A New Enzymatic Method to Ingo Neben, et al., “Studies on an Enzyme, S-Formylglu Determine Creatine', Clin. Clim. Acta, 122, 181-188 tathione Hydrolase, of the Dissimilatory Pathway of Metha (1982). nol in Candida Boidinii', Biochlm. Biophys. Acta, 614, O. Sugita, et al., “Reference values of Serum and urine 81-91 (1980). creatine, and of creatinine clearance by a new enzymatic method”, Ann. Clin. Biochem, 29, 523-528 (1992). Horst Schütte, et al., “Purification and Properties of Form Anders Andersson, et al., “Homocysteine and Other Thiols aldehyde Dehydrogenase and Formate Dehydrogenase from Determined in Plasma by HPLC and Thiol-Specific Post Candida Boidinii”, Eur: J. Biochem. 62(1), 151-150 (1976). column Derivatization', Clin. Chem., 39, 1590–1597, (1993). * cited by examiner U.S. Patent Mar. 15, 2005 Sheet 1 of 2 US 6,867,012 B2 F.G. 1
0.25
0, 2 O
0.15
0, 10
0. O 5
O O. O O 100 200 300 400 500 formaldehyde concentration (puM)
FIG 2
0.O 6
0. O4
0, O2 0.00 O O 20 30 40 50 formaldehyde concentration (lM) U.S. Patent Mar. 15, 2005 Sheet 2 of 2 US 6,867,012 B2 EIG 3
0.12 : 0. O
0.02
0.00 O 20 40 60 8O 00 Creatinine concentration (uM)
FIG 4
0.8 0.6 0.14 0.12 0.10 0.08 0.06 0.04 0.02
0.00 O O 20 30 40 50 L-homocysteine concentration (pM) US 6,867,012 B2 1 2 DETERMINATION METHOD OF pound and thio-NADH compound. The representative BIOLOGICAL COMPONENT AND REAGENT enzyme of the alcohol dehydrogenase used here, which KIT USED THEREFOR catalyzes the following reaction, is the enzyme of EC1.1.1.1: alcohol-i-NAD(P)'' (Paldehyde--NAD(P)H TECHNICAL FIELD OF THE INVENTION D. Schomburg, D. Stephan Eds., Enzyme Handbook 9 The present invention relates to a convenient and highly (Springer-Verlag) States that this enzyme oxidizes not only Sensitive determination method, using glutathione methanol as a Substrate but also its product (formaldehyde). dependent formaldehyde dehydrogenase, of formaldehyde Since formaldehyde exists as a hydrate in an aqueous or a compound forming formaldehyde as a reaction Solution and as alcohol, it is a Substrate oxidized with intermediate, and a reagent kit therefor. More particularly, alcohol dehydrogenase allowing oxidation to acetic acid. the present invention relates to a determination method of a Thus, a highly sensitive determination of formaldehyde biological component, Such as creatinine, creatine, and using this enzyme is unattainable. In addition, Since an homocysteine and via formaldehyde as an intermediate, and aldehyde dehydrogenase, Such as glutathione-independent a reagent kit therefor. The present invention also relates to a 15 formaldehyde dehydrogenase, irreversibly oxidizes determination method of homocysteine using a transferase formaldehyde, it cannot be applied to a highly Sensitive utilizing homocysteine and other Substance as a pair of determination by a cycling reaction. Substrates and a reagent kit therefor. In the field of analytical Science, Some compounds are known to be measured via formaldehyde as an intermediate. BACKGROUND OF THE INVENTION Of these, useful is the determination of creatinine, creatine and homocysteine in clinical tests. Formaldehyde is known to be a cytotoxin rich in reactiv Creatinine is a major diagnostic marker of kidney func ity with protein, membrane, and DNA, which causes various tion in clinical tests, and determination of creatine is used for disorders by being inhaled or oral administration. Formal the analysis of disease State of muscular dystrophy, hyper dehyde contained in atmosphere, waste water, and food has 25 thyroidism. As the determination method therefor, the Jaffe been considered problematic in recent years, and a conve method is dominantly used, but this method is pointed out to nient method for accurately measuring formaldehyde has be insufficient in the Specificity. Recently, enzymatic meth been demanded. ods using creatinine amidohydrolase, creatine As a determination method of formaldehyde, a method amidinohydrolase, Sarcosine oxidase and peroxidase having comprising calorimetric analysis using Hanz reagent, CTA high Specificity are increasingly used, but it has been pointed reagent (J. Biol. Chem, 231, 813 (1958)), Purpald reagent out that these methods may be influenced by reducing (Anal. Biochem, 234(1), 50 (1996)) and an analysis method substances present in the body. There are reported some using a reagent containing phenylhydrazine, potassium enzymatic methods wherein glutathione-independent form ferricyanide, chloroform and methanol in combination are aldehyde dehydrogenase is used instead of peroxidase to known. These methods are associated with problems in that 35 analyze formaldehyde derived from Sarcosine oxidase reac they require complicated manipulation and a long time for tion by the above-mentioned method (Clin. Clim. Acta, 122, the determination, or use of harmful reagents. As a means for 181 (1982), Ann. Clin. Biochem., 29,523 (1992)). However, Solving Such problems, enzymatic methods using these methods are not necessarily Sufficient for the measure glutathione-independent formaldehyde dehydrogenase (EC ment of a trace amount, as mentioned above. 2.1.1.46) have been disclosed (JP 5-42000 A, JP 2000 40 Homocysteine is an amino acid having an SH group 225000A). These enzymatic methods analyze reduced nico produced by metabolism of methionine which is an essential tinamide adenine dinucleotide Simultaneously produced amino acid in the body and is generally present at a low when forming formic acid from formaldehyde, based on the concentration in the body. It is known that homocystinuria, enzymatic reaction or a pigment produced by the reaction of which is a genetic disease of these metabolizing enzymes the reduced nicotinamide adenine dinucleotide. The deter 45 causing increased blood homocysteine concentrations, mination Sensitivity is, nevertheless, not necessarily Suffi relates to arteriosclerosis. In recent years, the connection of cient for the measurement of a trace amount of homocysteine concentration of the level Somewhat higher formaldehyde, because it depends on the molar absorption than the normal value, to cerebral infarction, myocardial coefficient of the reduced nicotinamide adenine dinucleotide infarction and depths vein thrombosis (so-called economy or pigment. 50 class Syndrome) has been clarified, and the present under As a highly Sensitive assay of a trace amount of a Standing is that blood homocysteine concentration is an Substance by the use of an enzyme, a method is known independent risk factor of these diseases. The Standard wherein the determination object Substrate and a coenzyme method for determination of homocysteine has been here of an enzyme acting on a Substrate are amplified by cycling tofore a method using HPLC (Clin. Chem, 39, 1590 (1993)), reaction and quantitatively measured (Kensa to Gijutsu, 55 and various modifications have been also reported. The 27(8), July, 1999). As one of the cycling methods, a deter method using HPLC requires Sophisticated analyzing mination method by an enzyme cycling method utilizing a devices and is inconvenient for processing a number of test reversible reaction using dehydrogenase and two kinds of samples. As a method without separation by HPLC, a coenzymes (thio-NAD compound and NADH compound, or method for determining homocysteine has been proposed to NAD compound and thio-NADH compound) has been 60 react homocysteine with S-adenosylhomocysteinase in the reported (JP 6-61278 B, JP 6-73477 B, JP 6-73478 B, JP presence of a de no Sine and fluoresce in-labeled 6-73479 B, JP 3023700, JP 3034969, JP 3034979, JP S-ade no Sylhomoc y Steine, and CSU C 3034984, JP 3034986, JP 3034987, JP 3034988, JP S-adenosylhomocysteine present in the reaction System by 8-103298 A). fluorescence polarization immunoassay using an anti-S- JP 4-341198. A discloses a highly sensitive assay of 65 adenosylhomocysteine antibody (JP 8-506478 A). Recently alcohols or aldehydes using alcohol dehydrogenase and proposed enzymatic methods include a method comprising thio-NAD compound and NADH compound, or NAD com reacting homocysteine with homocysteine desulfrase and US 6,867,012 B2 3 4 measuring ammonia, C.-keto acid or hydrogen Sulfide pro nation System, formaldehyde is produced as a reaction duced thereby (JP 2000-502262 A), a method comprising intermediate, which enables use of the above-mentioned measuring hydrogen Sulfide or thiol compound-Substituted formaldehyde dehydrogenase for the determination of homocysteine produced in the presence of thiol compound, homocysteine. using L-methionine Y-lyase and O-acetylhomoserine-lyase The present inventors have further Succeeded, after capable of decomposing homocysteine (JP 2000-166597 A), Screening dimethylglycine oxidase having properties Suit a method comprising quantitatively measuring able for use in the determination System, in isolation and Y-Substituted-C-aminobutylic acid or hydrogen Sulfide purification of novel dimethylglycine oxidase resistant to derived from homocysteine by a Y-substituted-C.- thiol compound and having Smaller Kim Value to dimeth aminobutyrate Synthase in the presence of a nucleophilic ylglycine as compared to conventional enzyme. Application of this enzyme to the above-mentioned determination Sys reagent Substitutable with Y-mercapto group of homocys tem led to the highly Sensitive and highly accurate determi teine (JP 2000-228998 A) However, it is a general under nation of homocysteine. Standing that immunoassay requires a longer time and higher Accordingly, the present invention provides the follow cost than do typical biochemical tests, and analysis methods Ing. using enzymatic reactions are not entirely Satisfactory for 15 (1) A method for determining formaldehyde, which com the accurate measurement of homocysteine amount, Since prises bringing glutathione-dependent formaldehyde the blood homocysteine is in a trace amount and its con dehydrogenase having a ratio of reactivity with thio-NAD centration is about 10 uM or lower by normal value, leading to reactivity with NAD of not less than 30%, glutathione to insufficient determination Sensitivity, or Substrate Speci and an oxidized coenzyme into contact with a Sample, and ficity of the enzyme used causes croSS-reaction with Sub analyzing a compound resulting from the enzymatic reac stances other than homocysteine in the test Sample. tion. As a homocysteine determination method utilizing an (2) The method of the above-mentioned (1), wherein the enzyme cycling method, a method for measuring pyruvic ratio of reactivity with thio-NAD to reactivity with NAD acid and ammonia produced by cyStathionine B-Synthase of glutathione-dependent formaldehyde dehydrogenase is and cystathionine B-lyase (U.S. Pat. No. 6,174,696), a 25 not less than 60%. method for analyzing NAD compound or thio-NAD com (3) The method of the above-mentioned (1) or (2), wherein pound produced or consumed using homocysteine des the glutathione-dependent formaldehyde dehydrogenase ulfrase and 2-keto-butylate dehydrogenase are known. In has the following physico-chemical properties: action: View of the homocysteine amount in the body, however, a production of S-formylglutathione and reduced coenzyme method capable of assaying with higher Sensitivity is by action on formaldehyde in the presence of one oxi desired. dized coenzyme Selected from the group consisting of It is therefore an object of the present invention to enable NADs, NADPs, thio-NADs and thio-NADPs and reduced highly Sensitive and convenient determination of formalde glutathione optimal pH: about 7.5-about 8.5 pH stability: hyde or a compound that produces formaldehyde as a about 6.0-about 9.0, and heat stability: about 40 C. or reaction intermediate. The present invention also aims at 35 less (pH 7.5, 30 min). providing a method for determining homocysteine highly (4) The method of any of the above-mentioned (1) to (3), sensitively and conveniently without using HPLC or immu wherein the glutathione-dependent formaldehyde dehy noassay. drogenase is derived from microorganism. SUMMARY OF THE INVENTION (5) The method of the above-mentioned (4), wherein the 40 glutathione-dependent formaldehyde dehydrogenase is The present inventors took note of glutathione-dependent derived from methylotrophic yeast. formaldehyde dehydrogenase (EC 1.2.1.1) as an enzyme (6) The method of the above-mentioned (5), wherein the that acts reversibly on formaldehyde and Succeeded in glutathione-dependent formaldehyde dehydrogenase is Screening novel glutathione-dependent formaldehyde derived from Hansenula yeast. dehydrogenase, wherein two kinds of coenzymes can be 45 (7) The method of the above-mentioned (6), wherein the used and the reactivity balance with the both permits pro glutathione-dependent formaldehyde dehydrogenase is gression of cycling reaction. They have then found out that derived from Hansenula nonfermenians IFO1473. formaldehyde or a compound that produces formaldehyde as (8) A method for determining formaldehyde, which com a reaction intermediate of multi-step measurement, Such as prises bringing glutathione-dependent formaldehyde creatine, and creatinine, can be measured highly Sensitively 50 dehydrogenase having a ratio of reactivity with thio-NAD and conveniently by reacting this enzyme, glutathione, oxi to reactivity with NAD of not less than 30%, glutathione, dized coenzyme and other reduced coenzyme with a Sample one compound Selected from the group consisting of and measuring changes in the coenzyme amount of any of thio-NADs and thio-NADPs, and one compound selected them due to the enzymatic reaction. from the group consisting of reduced NADS and reduced In addition, the present inventors have found that 55 NADPs into contact with a sample to allow cycling homocysteine can be measured highly Sensitively and highly reaction and analyzing changes in the amount of a com accurately by reacting transferase utilizing homocysteine pound due to the reaction. and other Substance as a pair of Substrates with a Sample (9) A method for determining formaldehyde, which com containing homocysteine together with the other Substance prises bringing glutathione-dependent formaldehyde and measuring the compound produced. Particularly, the 60 dehydrogenase having a ratio of reactivity with thio-NAD present inventors have found that homocysteine can be to reactivity with NAD of not less than 30%, glutathione, measured highly Sensitively and highly accurately by using one compound Selected from the group consisting of betaine-homocysteine methyltransferase as transferase and reduced thio-NADS and reduced thio-NADPs, and one betaine as other Substrate, further decomposing the produced compound Selected from the group consisting of NADS dimethylglycine into Sarcosine, formaldehyde and hydrogen 65 and NADPs into contact with a sample to allow cycling peroxide by the use of dimethylglycine oxidase, and mea reaction and analyzing changes in the amount of a com Suring any of these compounds. According to this determi pound due to the reaction. US 6,867,012 B2 S 6 (10) The method of the above-mentioned (8), wherein the group consisting of dithiothreitol, dithioerythritol, amount of the reduced thio-NADP or reduced thio-NAD 2-mercaptoe thanol, 2-mercaptoethane Sulfonate, compound is analyzed. 2-mercaptoethylamine, cysteine, homocy Steine, (11) The method of any of the above-mentioned (1)–(10), N-acetylcysteine, thioglycerol, thioglycolic acid, reduced wherein a minimum detection limit of the formaldehyde glutathione and Salts thereof. is not more than 1 limol/L. (22) The method of the above-mentioned (21), wherein the (12) A method for determining a biological component, thiol compound is dithiothreitol. which comprises, in the determination of a biological (23) The method of any of the above-mentioned (20)-(22), component that produces formaldehyde as a reaction wherein the dimethylglycine oxidase shows an enzyme intermediate, measuring produced formaldehyde by the activity retained at least by 50% in the presence of 0.05 method of any of the above-mentioned (1)–(11). mmol/L dithiothreitol relative to the enzyme activity in (13) A method for determining homocysteine, which com the absence of dithiothreitol. prises bringing betaine, be taine-homocysteine (24) The method of any of the above-mentioned (20)-(23), methyltransferase, dimethylglycine oxidase and, where wherein the dimethylglycine oxidase is an enzyme having necessary, Sarcosine oxidase into contact with a Sample, 15 the following physico-chemical properties: action: acting and measuring, according to the method of any of the on dimethylglycine in the presence of oxygen to produce above-mentioned (1)–(11), formaldehyde produced by the Sarcosine, formaldehyde and hydrogen peroxide, and Km enzymatic reactions. value for dimethylglycine: not more than 15 mM. (14) A method for determining creatine or creatinine, which (25) The method of any of the above-mentioned (20)-(24), comprises reacting creatine amidinohydrolase, Sarcosine wherein the dimethylglycine oxidase is derived from a Oxidase, and, where necessary, creatinine amidohydrolase microorganism. and measuring, according to the method of any of the (26) The method of the above-mentioned (25), wherein the above-mentioned (1)–(11), formaldehyde produced by the dimethylglycine oxidase is derived from a microorganism enzymatic reactions. belonging to the genus Arthrobacter or the genus Strep (15) A method for determining homocysteine, which com 25 tomyces. prises bringing transferase utilizing homocysteine and (27) The method of the above-mentioned (26), wherein the other compound as a pair of Substrates and Said other dimethylglycine oxidase is derived from Arthrobacter compound into contact with a Sample and measuring the nicotianae IFO14234 or Streptomyces mutabilis resulting compound. IFO128OO. (16) The method of the above-mentioned (15), wherein the (28) The method of any of the above-mentioned (20)-(27), transferase and Said other compound is a combination wherein a minimum detection limit of the homocysteine selected from the group consisting of betaine is not more than 1 limol/L. homocysteine methyltransferase and betaine, betaine (29) Glutathione-dependent formaldehyde dehydrogenase homocysteine methyltransferase and dimethylthetin, having the following physico-chemical properties: action: homoc y Steine methyltransferase and 35 acting on formaldehyde in the presence of one coenzyme S-adenosylmethionine, and N5-methyltetrahydrofolate selected from the group consisting of NADs, NADPs, homoc y Steine methyltransferase and thio-NADs and thio-NADPs, reduced glutathione to pro N5-methyltetrahydrofolate, and the resulting compound duce S-formylglutathione, a reduced coenzyme, a ratio of is methionine. reactivity with thio-NAD to reactivity with NAD: not less (17) The method of the above-mentioned (15), wherein the 40 than 30%, optimal pH: about 7.5-about 8.5, pH stability: betaine, betaine-homocysteine methyltransferase, dimeth about 6.0-about 9.0, and heat stability: about 40 C. or ylglycine oxidase and, where necessary, Sarcosine oxidase less (pH 7.5, 30 min) are brought into contact with a Sample, hydrogen peroxide (30) The glutathione-dependent formaldehyde dehydroge produced by the enzymatic reactions is reacted with nase of the above-mentioned (29), which is derived from hydrogen donor chromogenic reagent and, where 45 a microorganism. necessary, coupler, in the presence of peroxidase, and the (31) The glutathione-dependent formaldehyde dehydroge resulting pigment is measured. nase of the above-mentioned (30), which is derived from (18) The method of the above-mentioned (15), wherein the methylotrophic yeast. betaine, betaine-homocysteine methyltransferase, dimeth (32) The glutathione-dependent formaldehyde dehydroge ylglycine oxidase and, where necessary, Sarcosine oxidase 50 nase of the above-mentioned (31), which is derived from are brought into contact with a Sample, formaldehyde Hansenula yeast. produced by the enzymatic reactions is reacted with (33) The glutathione-dependent formaldehyde dehydroge formaldehyde dehydrogenase and oxidized coenzyme and nase of the above-mentioned (32), which is derived from the resulting reduced coenzyme is measured. Hansenula nonfermenians IFO1473. (19) The method of the above-mentioned (15), wherein the 55 (34) Dimethylglycine oxidase having the following physico betaine, betaine-homocysteine methyltransferase, dimeth chemical properties: ylglycine oxidase and, where necessary, Sarcosine oxidase action: acting on dimethylglycine in the presence of oxygen are brought into contact with a Sample, formaldehyde to produce Sarcosine, formaldehyde and hydrogen peroxide, produced by the enzymatic reactions is reacted with an enzyme activity is retained at least by 50% in the glutathione, glutathione-dependent formaldehyde dehy 60 presence of 0.05 mmol/L dithiothreitol relative to the drogenase and oxidized coenzyme and the resulting enzyme activity in the absence of dithiothreitol, and Km reduced coenzyme is measured. value for dimethylglycine: not more than 15 mM. (20) The method of any of the above-mentioned (17)-(19), (35) The dimethylglycine oxidase of the above-mentioned wherein the dimethylglycine oxidase is an enzyme stable (34), which is derived from a microorganism. to thiol compound. 65 (36) The dimethylglycine oxidase of the above-mentioned (21) The method of the above-mentioned (20), wherein the (35), which is derived from a microorganism belonging to thiol compound is at least one kind Selected from the the genus Arthrobacter or the genus Streptomyces. US 6,867,012 B2 7 8 (37) The dimethylglycine oxidase of the above-mentioned (49) The reagent kit of the above-mentioned (48), wherein (36), which is derived from Arthrobacter nicotianae the reagent for measuring hydrogen peroxide comprises IFO14234, or Streptomyces mutabilis IFO12800. peroxidase, hydrogen donor chromogenic reagent and, (38) A reagent kit for formaldehyde determination, which where necessary, coupler. comprises at least buffer, glutathione, glutathione-dependent 5 (50) A reagent kit for homocysteine determination, which formaldehyde dehydrogenase having a ratio of reactivity comprise S be taine, be taine - homoc y Steine with thio-NAD to reactivity with NAD of not less than 30% methyltransferase, dimethylglycine oxidase and, where and a reagent for analyzing a compound produced by the necessary, Sarcosine oxidase, and a reagent for determi enzymatic reaction. nation of formaldehyde produced by the enzymatic reac (39) The reagent kit for formaldehyde determination of the tions. above-mentioned (38), wherein glutathione-dependent (51) The reagent kit of the above-mentioned (50), which formaldehyde dehydrogenase is an enzyme of any of the comprises formaldehyde dehydrogenase and oxidized above-mentioned (29)–(33). coenzyme as reagents for determination of the formalde (40) A reagent kit for formaldehyde determination, which hyde. comprises at least buffer, glutathione, glutathione 15 (52) The reagent kit of the above-mentioned (50), which dependent formaldehyde dehydrogenase having a ratio of comprises glutathione, glutathione-dependent formalde reactivity with thio-NAD to reactivity with NAD of not hyde dehydrogenase and oxidized coenzyme as reagents less than 30%, one compound Selected from the group for determination of the formaldehyde. consisting of thio-NADs and thio-NADPs, and one com (53) The reagent kit of any of the above-mentioned (48)- pound Selected from the group consisting of reduced (52) for homocysteine determination, wherein the dim NADS and reduced NADPs. ethylglycine oxidase is for the enzyme of any of the (41) The reagent kit for formaldehyde determination of the above-mentioned (34)–(37). above-mentioned (40), where in the glutathione dependent formaldehyde dehydrogenase is an enzyme of BRIEF DESCRIPTION OF THE DRAWINGS any of the above-mentioned (29)–(33). 25 (42) A reagent kit for formaldehyde determination, which FIG. 1 shows the relationship between absorbance and comprises at least buffer, glutathione, glutathione formaldehyde concentration in Example 4. dependent formaldehyde dehydrogenase having a ratio of FIG. 2 shows the relationship between absorbance and reactivity with thio-NAD to reactivity with NAD of not formaldehyde concentration in Example 5. less than 30%, one compound Selected from the group FIG. 3 shows the relationship between absorbance and consisting of reduced thio-NADs and reduced thio creatinine concentration in Example 6. NADPs, and one compound selected from the group FIG. 4 shows the relationship between absorbance and consisting of NADs and NADPs. homocysteine concentration in Example 7. (43) The reagent kit for formaldehyde determination of the above-mentioned (42), where in the glutathione 35 DETAILED DESCRIPTION OF THE dependent formaldehyde dehydrogenase is an enzyme of INVENTION any of the above-mentioned (29)–(33). The determination method of formaldehyde of the present (44) A reagent kit for homocysteine determination, which invention is characterized in that novel glutathione comprises, in addition to the reagent of any of the dependent formaldehyde dehydrogenase capable of utilizing above-mentioned (38)-(43), betaine, betaine 40 homocysteine methyltransferase, dimethylglycine oxi two kinds of coenzymes of thio-NADs or thio-NADPs, and dase and, where necessary, Sarcosine oxidase. NADs or NADPs and having higher ratio of reactivity with (45) A reagent kit for creatinine or creatine determination, thio-NAD to reactivity with NAD when compared to those which comprises, in addition to the reagent of any of the known in the art is reacted with a Sample together with above-mentioned (38)-(43), creatine amidinohydrolase, 45 glutathione and any of the above-mentioned oxidized Sarcosine oxidase and, where necessary, creatinine ami coenzymes, and the amount of the compound produced or dohydrolase. consumed is measured. (46) A reagent kit for homocysteine determination, which To be exact, the glutathione-dependent formaldehyde comprises at least buffer, transferase utilizing homocys dehydrogenase (EC 1.2.1.1) used in the present invention is teine and other compound as a pair of Substrates, Said 50 an enzyme reversibly catalyzes the production of other compound, and a reagent for analyzing a compound S-formylglutathione and reduced coenzyme in the presence produced by the enzymatic reaction. of oxidized coenzyme, using, as a Substrate, (47) The reagent kit for homocysteine determination of the S-hydroxylmethylglutathione non-enzymatically produced above-mentioned (46), wherein the transferase and said from glutathione and formaldehyde. other compound are a combination Selected from the 55 According to the present invention, glutathione group consisting of betaine-homocysteine methyltrans dependent formaldehyde dehydrogenase can use, as a ferase and betaine, betaine-homocysteine methyltrans coenzyme, thio-NADs or thio-NADPs, and NADs or ferase and dimethylthetin, homocysteine methyltrans NADPs. Examples of NADs include nicotineamide adenine ferase and S-ade nosyl methio nine and dinucleotide, acetylpyridine adenine dinucleotide, acetylpy N5-methyltetrahydrofolate-homocysteine methyltrans 60 ridine adeninehypoxanthine dinucleotide, nicotineamide ferase and N5-methyltetrahydrofolate, and the produced hypoxanthine dinucleotide, and examples of NADPs include compound is methionine. nicotineamide adenine dinucleotide phosphate, acetylpyri (48) A reagent kit for homocysteine determination, which dine adenine dinucleotide phosphate, acetylpyridine comprises buffer, betaine, betaine-homocysteine adeninehypoxanthine dinucleotide phosphate, and nic methyltransferase, dimethylglycine oxidase and, where 65 otineamide hypoxanthine dinucleotide phosphate. Examples necessary, Sarcosine oxidase, and a reagent for measuring of thio-NADS include thionicotine amide adenine hydrogen peroxide produced by the enzymatic reactions. dinucleotide, thionicotineamide hypoxanthine dinucleotide, US 6,867,012 B2 9 10 and examples of thio-NADPs include thionicotineamide Satisfies any of the above-mentioned conditions and is adenine dinucleotide phosphate, and thionicotineamide capable of catalyzing the cycling reaction. Preferably, this hypoxanthine dinucleotide phosphate. enzyme further has the following physico-chemical proper In a preferable embodiment, the determination method of ties. formaldehyde of the present invention is characterized in 5 (a) action: acting on formaldehyde in the presence of one that glutathione, glutathione-dependent formaldehyde coenzyme Selected from the group consisting of NADS, dehydrogenase, one compound Selected from the group NADPs, thio-NADs and thio-NADPs, and reduced glu consisting of thio-NADs and thio-NADPs, and one com tathione to produce S-formylglutathione and reduced pound Selected from the group consisting of reduced NADS coenzyme. and reduced NADPs are reacted on a sample to allow (b) optimal pH: about 7.5-about 8.5 cycling reaction, and the amount of the compound after (c) pH stability: about 6.0-about 9.0 (pH range affording the change due to the reaction is analyzed. residual activity of not less than 80% after treatment at In another preferable embodiment, the determination 25 C. for 24 hr. method of formaldehyde of the present invention is charac (d) heat stability: about 40° C. or less (temperature range terized in that glutathione, glutathione-dependent formalde 15 affording the residual activity of not less than 90% after hyde dehydrogenase, one compound Selected from the group treatment at pH 7.5 for 30 min). consisting of reduced thio-NADs and reduced thio-NADPs, The glutathione-dependent formaldehyde dehydrogenase and one compound Selected from the group consisting of one of the present invention is free of any particular limitation on compound Selected from the group consisting of NADS and its derivation as long as it Satisfies the above-mentioned NADPS are reacted on a Sample to allow cycling reaction, conditions necessary for catalyzing the cycling reaction. and the amount of the compound after change due to the Preferably, it is derived from a microorganism, more pref reaction is analyzed. erably derived from methylotrophic yeast, most preferably In this cycling reaction, reduced coenzyme is produced derived from Hansenula yeast, particularly derived from from oxidized coenzyme, or oxidized coenzyme is produced Hansenula nonfermenians strain IFO1473. The strain from reduced coenzyme in proportion to the reaction time 25 IFO1473 is available from the Institute for Fermentation, and upon amplification relative to the amount of a Substrate. Osaka (IFO) (17-85, Juso-honmachi, 2-chome, Yodogawa When the coenzyme is NAD compound or NADP ku, Osaka 532-8686 Japan). compound, the absorbance around 340 nm and when it is The glutathione-dependent formaldehyde dehydrogenase thio-NAD compound or thio-NADP compound, the absor of the present invention may be genetically engineered to bance around 400 nm are measured for highly Sensitive improve enzymatic properties Such as Specific activity and quantitative measurement of formaldehyde. The Stability, when retaining a balance of the reactivity with two glutathione-dependent formaldehyde dehydrogenase to be kinds of coenzymes, which is necessary for catalyzing the used for the cycling reaction desirably contains no contami cycling reaction, by inducing at random or site-specific nant enzyme that decomposes formaldehyde to release same mutation. outside the cycling System, Such as S-formylglutathione 35 The glutathione-dependent formaldehyde dehydrogenase hydrolase, or contains Said enzyme in a trace amount unin of the present invention can be obtained by isolation and fluenceable on the measures. It is also desirable that NAD(P) purification of, as a material, a cell or tissue culture produc degrading enzyme is not contained or contained in a trace ing the enzyme, or by isolating a gene encoding the enzyme amount uninfluenceable on the measures. protein and allowing expression thereof in a Suitable host For efficient cycling reaction, glutathione-dependent 40 according to genetic recombination techniques. Preferable formaldehyde dehydrogenase to be used in the present embodiment of the former is the following method. invention should have sufficient reactivity with thio-NADs Any of the above-mentioned glutathione-dependent form or thio-NADPs. The ratio of reactivity with thio-NAD to aldehyde dehydrogenase-producing microorganisms, Such reactivity with NAD is not less than 30%, preferably not less as Hansenula nonfermenians strain IFO1473, is cultured in than 60%. When thio-NAD(P) compound is used as an 45 a nutrient medium. The nutrient medium to be used may be oxidized coenzyme, in a reverse reaction, glutathione a Synthetic medium or a natural medium that contains dependent formaldehyde dehydrogenase shows Sufficiently carbon Source, nitrogen Source, mineral and other necessary higher reactivity with reduced NAD(P) compound added nutrients in Suitable amounts that the Strain to be used can than the reactivity with reduced thio-NAD(P) compound utilize. AS the carbon Source, for example, malic acid, and produced by forward reaction. On the other hand, when 50 Succinic acid can be used. AS the nitrogen Source, for NAD(P) compound is used as an oxidized coenzyme, in a example, nitrogen-containing natural products Such as reverse reaction, glutathione-dependent formaldehyde dehy peptones, beef extract, and yeast extract, and inorganic drogenase shows Sufficiently higher reactivity with reduced nitrogen-containing compounds Such as ammonium thio-NAD(P) compound added than the reactivity with chloride, and ammonium citrate can be used. AS the mineral, reduced NAD(P) compound produced by forward reaction. 55 for example, potassium phosphate, Sodium phosphate, and Glutathione-dependent formaldehyde dehydrogenase is magnesium Sulfate can be used. known to exist in a vast range of from higher animals to The culture is generally Shaking culture or aeration microorganisms, and as an enzyme having relatively high agitation culture. The culture temperature is about 20-about reactivity with thio-NAD, one derived from human liver, has 40° C., preferably about 25-about 37 C., and the culture pH been reported (Biochem. J., 177, 869-878 (1979)). 60 is about 5-about 9, preferably controlled to about 6-about 8. However, glutathione-dependent formaldehyde dehydroge AS long as the Strain in use can grow, other conditions may nase of a higher animal generally shows lower Specific be employed. The culture period is generally about 1-about activity as compared to enzyme derived from 7 days, glutathione-dependent formaldehyde dehydrogenase microorganism, and there is no report on Successful cycling is generally produced and accumulated in the cell. reaction using this enzyme. 65 The glutathione-dependent formaldehyde dehydrogenase Therefore, the present invention provides a novel of the present invention can be purified by a purification glutathione-dependent formaldehyde dehydrogenase that method generally used. For example, after recovery of the US 6,867,012 B2 11 12 cells, disruption by ultraSonication, mechanical disruption 1.1.1.49) (e.g., derived from yeast or Leuconostoc bacteria) with glass beads, disruption with French Press, and lysis and glucose-6-phosphate, glucose dehydrogenase (EC with Surfactant are applied to extract intracellular fractions. 1.1.1.47) (e.g., derived from Bacillus bacteria, Pseudomonas The obtained extract is Subjected to Salting out with ammo bacteria) and B-D-glucose, glutamate dehydrogenase (EC nium Sulfate, Sodium Sulfate, metal aggregation with mag 1.4.1.3) (e.g., derived from bovine liver) and L-glutamic nesium chloride, calcium chloride, aggregation with acid. protamin, polyethyleneimine, ion-exchange chromatogra The determination of formaldehyde using the glutathione phy with DEAE (diethylaminoethyl)-sepharose, CM dependent formaldehyde dehydrogenase of the present invention can be also carried out without a cycling reaction (carboxymethyl)-Sepharose, to purify glutathione-dependent as follows. formaldehyde dehydrogenase. That is, glutathione, glutathione-dependent formaldehyde The determination method of formaldehyde of the present dehydrogenase and one coenzyme Selected from the group invention using a cycling reaction of the glutathione consisting of thio-NADs, thio-NADPs, NADs and NADPs dependent formaldehyde dehydrogenase is described in are brought into contact with a Sample, and produced detail by referring to an example wherein thio-NAD(P) S-formylglutathione is measured using absorbance of UV compound is used as an oxidized coenzyme. When reduced 15 region as an indeX for the determination of formaldehyde. glutathione, glutathione-dependent formaldehyde Alternatively, S-formylglutathione hydrolase (EC 3.1.1.12) dehydrogenase, oxidized thio-NAD(P) compound and is further reacted and the produced formic acid is decom reduced NAD(P) compound are brought into contact with a posed to carbon dioxide by formate dehydrogenase sample, S-formylglutathione and reduced thio-NAD(P) (EC1.2.1.2; EC 1.2.1.43), and the produced reduced coen compound are produced from formaldehyde, reduced glu Zyme is measured for the determination of formaldehyde. tathione and oxidized thio-NAD(P) compound contained in S-Formylglutathione hydrolase is known to be present in the Sample. Then, glutathione-dependent formaldehyde animal organs, methylotrophic yeast, and bacteria, and can dehydrogenase uses an excess reduced NAD(P) compound be obtained from these sources and used. Formate dehydro as a coenzyme to convert the produced S-formylglutathione genase is an enzyme present in plant Seed, methylotrophic to formaldehyde and glutathione. Thereafter, forward reac 25 yeast, and bacteria and can be obtained from these Sources tion using oxidized thio-NAD(P) compound as a coenzyme and used. Commercially available enzymes (e.g., FOR and reverse reaction using reduced NAD(P) compound as a MATE DEHYDROGENASE (Sigma) ) may be used. coenzyme are repeated, as a result of which a number of The reduced coenzyme (NADH or NADPH compound) molecules of reduced thio-NAD(P) compound is produced produced by the above-mentioned Series of enzymatic reac relative to 1 molecule of formaldehyde. On the other hand, tions can be analyzed by measuring absorbance in the UV reduced NAD(P) compound is consumed as the reaction region or fluorescence. The reduced coenzyme can be also cycle proceeds. Therefore, low concentration formaldehyde measured by measuring formazan pigment produced when can be detected with high Sensitivity by monitoring increase reducing tetrazolium Salt in the presence of an electron in reduced thio-NAD(P) compound or decrease in reduced carrier Such as diaphorase and methylphenazinium methyl NAD(P) compound, based on the above-mentioned absor 35 Sulfate. bance as an indeX. The use of glutathione-dependent formaldehyde dehydro In the above-mentioned determination System, when other genase of the present invention enables detection of a trace enzyme that can use NAD(P)S as a coenzyme, but Substan amount of formaldehyde contained in a Sample at a concen tially cannot use thio-NAD(P)S as a coenzyme, and a Sub tration of not more than 1 umol/L. strate of this enzyme are further added, oxidized NAD(P) 40 The determination method of formaldehyde of the present compound can be reproduced as a reduced form. In this case, invention is preferably used as a final Step of the determi formaldehyde is measured using increase in the reduced nation of various biological components, wherein formal thio-NAD(P) compound as an index. The combination of the dehyde is produced as a reaction intermediate for multi-step other enzyme and a Substrate thereof that can be used for measurement. such determination system when NADs can be used is, for 45 For example, the determination of creatinine or creatine example, malate dehydrogenase (EC 1.1.1.37) (e.g., derived comprises reacting creatine amidinohydrolase, Sarcosine from porcine or bovine cardiac muscle) and L-malic acid, oxidase and, where necessary, creatinine amidohydrolase glycerol-3-phosophate dehydrogenase (EC 1.1.1.8) (e.g., with a Sample to decompose creatinine or creatine into derived from rabbit muscle) and L-glycerol-3-phosphate, glycine, formaldehyde and hydrogen peroxide, and measur glyceraldehyde-phosphate dehydrogenase (EC 1.1.1.12) 50 ing hydrogen peroxide produced using color developing (e.g., derived from rabbit skeletal muscle or liver, yeast or E. Substrate. Thus, the glutathione-dependent formaldehyde coli) and D-gly Seraldehyde phosphate and phosphoric acid, dehydrogenase of the present invention can be used instead glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (e.g., of a reagent for quantitative measurement of hydrogen derived from Leuconostoc bacteria) and glucose-6- peroxide for the determination of formaldehyde, thereby to phosphate, glucose dehydrogenase (EC 1.1.1.47) (e.g., 55 measure creatinine or creatine. Therefore, the present inven derived from Bacillus bacteria, Pseudomonas bacteria) and tion also provides a determination method of creatinine or f-D-glucose, glutamate dehydrogenase (EC 1.4.1.3) (e.g., creatine, which method comprising bringing creatine derived from bovine liver) and L-glutamic acid, and Such amidinohydrolase, Sarcosine oxidase and, where necessary, combination when NADPs can be used is, for example, creatinine amidohydrolase into contact with a Sample to glyoxylate dehydrogenase (EC 1.2.1.17) (e.g., derived from 60 produce formaldehyde, bringing glutathione-dependent Pseudomonas Oxalaticus) and CoA and glyoxylic acid, formaldehyde dehydrogenase of the present invention, phosphogluconate dehydrogenase (EC 1.1.1.44) (e.g., glutathione, and one oxidized coenzyme Selected from the derived from rat liver, brewer's yeast or E. coli) and group consisting of thio-NADs, thio-NADPs, NADs and 6-phospho-D-gluconic acid, glyceraldehyde-phosphate NADPS, and, where necessary, a reduced coenzyme of a dehydrogenase (EC 1.1.1.13) (e.g., derived from plant 65 different kind from the oxidized coenzyme into contact with chloroplast) and D-glyceraldehyde-3-phosphate and phos the Sample, and measuring the produced or consumed com phoric acid, glucose-6-phosphate dehydrogenase (EC pound. US 6,867,012 B2 13 14 The creatine amidinohydrolase, Sarcosine oxidase and copper, Zinc, and manganese are exemplified. AS the protein, creatinine amidohydrolase to be used are not particularly those uninfluenceable on the enzymatic reaction are limited, and can be obtained from the microorganism that desirable, Such as bovine Serum albumin, Ovalbumin, and produce these enzymes by conventional methods or obtained gelatin. Examples of the amino acid include glycine, lysine, from commercial Sources. glutamic acid, glycylglycine, and polylysine. Examples of AS described in detail in the following, Since formalde the Saccharide include monosaccharide, disaccharide, hyde can be produced by reacting betaine, betaine oligosaccharide, polysaccharide and their derivatives. homocysteine methyltransferase and dimethylglycine oxi Examples thereof include glucose, fructose, galactose, dase with a Sample containing homocysteine, by the mannose, Xylose, lactose, Sucrose, maltose, trehalose, malto triose, maltotetraose, maltosylcyclode Xtrin, determination of formaldehyde using the glutathione C-cyclodextrin, B-cyclodextrin, Y-cyclodextrin, dextrin, dependent formaldehyde dehydrogenase of the present amylose, glycogen, Starch, inulin, glucosamine, inositol, invention, homocysteine can be measured. Wen Sarcosine mannitol, Sorbitol, ribitol, and deoxyglucose. Examples of oxidase is reacted with Sarcosine produced by dimethylgly the organic acid include C.-keto-glutaric acid, malic acid, cine oxidase, a 2-fold amount of formaldehyde can be fumaric acid, gluconic acid, cholic acid, and desoxycholic produced, thereby increasing the detection Sensitivity. 15 acid. In addition, boric acid, borax, Sodium chloride, potas As a different measurement object that produces formal sium chloride, ammonium Sulfate, glycerol, and Ficoll can dehyde as a reaction intermediate, for example, methanol, be used. and uric acid are exemplified (Clin. Chem., 33/12, The reagent constituting the reagent kit of the present 2204–2208 (1987), Clin. Biochem., 27/2, 93–97 (1994)). invention may contain antiseptic and Surfactant as long as That is, methanol produces formaldehyde and hydrogen they do not adversely affect the reagent properties. Examples peroxide due to alcohol oxidase (EC 1.1.3.13). Because uric of the antiseptic include Sodium azide, chelating agent, acid produces formaldehyde by reacting hydrogen peroxide various antibiotics, antibacterial agent, and an antifugal produced by the reaction of uricase (EC 1.7.3.3) with agent. Specifically, Sodium azide, EDTA and its Salt catalase (EC 1.11.1.6) in the presence of methanol, the (inclusive of metal chelate), chelating agents Such as above-mentioned determination method can be used to 25 Cy DTA, GHEG, DPTA-OH, DTPA, EDDA, EDDP, analyze formaldehyde. EDDPO, EDTA-OH, EDTPO, EGTA, HBED, HDTA, The present invention also provides a reagent kit to be HIDA, IDA, Methyl-EDTA, NTA, NTP, NTPO, TTHA used for the determination method of the above-mentioned (these are commercially available from Dojindo), BND, formaldehyde. The reagent kit for formaldehyde determina CAA, HPO, IZU, MIT (commercially available from tion of the present invention contains at least buffer, Roche), ProClin150, ProClin300 (commercially available glutathione, glutathione-dependent formaldehyde dehydro from Rohm & Haas), antibacterial (antifugal) agents Such as genase and a reagent for analyzing the compound produced ben Zalkonium chloride, Kath on CG, by the enzymatic reaction. Glutathione-dependent formal p-hydroxymethylbenzoate, antibiotics Such as amphotericin dehyde dehydrogenase is exemplified by those mentioned B, amplicillin, blasticidin S, chloramphe nicol, above, but is not particularly limited. 35 dihydroStreptomycin, clindamycin, cycloheximide, filipin, A different embodiment of the reagent kit for formalde G418, gentamycin, hygromycin, kanamycin, lincomycin, hyde determination of the present invention contains at least neomycin, polyoxin, penicillin, Sulfamethizol, and tetracy buffer, glutathione, glutathione-dependent formaldehyde cline can be used. Examples of the Surfactant include dehydrogenase, one compound Selected from the group non-ionic Surfactant, cationic Surfactant, anionic Surfactant consisting of thio-NADs and thio-NADPs, and one com 40 and amphoteric Surfactant. Specifically, non-ionic Surfac pound Selected from the group consisting of reduced NADS tants such as Adekanol 720N, B-795, B-797, LO-7, NP-690, and reduced NADPs. NP-695, NP-720, PC-8, SO-120, SO-145, Brij 35, 98,700, In another embodiment, it contains at least buffer, EMULGEN 109P, 430, 460, 707, 709,810, 911, 935, 950, glutathione, glutathione-dependent formaldehyde A-60, B66, n-dodecylmaltoside, Genapol X-080, MEGA-7, dehydrogenase, one compound Selected from the group 45 8, 9, 10, NIKKOL BL-9EX, BL-20TX, HCD-100, MGO, consisting of reduced thio-NADs and reduced thio-NADPs, MYO-6, MYL-10, NP-18TX, OP-10, TL-10, TMGO5, and one compound Selected from the group consisting of SL-10, octyl C. -glucoside, octyl B-glucoside, NADs and NADPs. As the NADs, NADPs, thio-NADs and octylthioglucoside, octylthiogalactoside, pentaethylenegly thio-NADPs, those mentioned above can be used. col dodecylether, polyethylene ether W-1, Pluronic F-68, The buffer to be used for the reagent kit of the present 50 L-71, P-103, Nonidet P40, RHEODOL 460, TWL-103, invention is exemplified by Tris-HCl buffer, phosphate TWL-106, saponin, sarcosinate PN, SPAN 20, 85, SM1080, buffer, borate buffer, and GOOD buffer. While Tris-HCl Sucrose monolaurate, Tetronic 704, Thesit, Triton X-100, buffer and phosphate buffer are easily Show changes in pH X-114, X-305, Tween 20, 40,80, cationic Surfactants Such as due to concentration and temperature, but advantageously bis(hydroxymethyl)-(stearoylaminomethylcarbonyloxy) economical. On the other hand, GOOD buffer is exemplified 55 ethylamine chloride, benzyllaurylmethylsulfonium by MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, methylsulfate, 2-(4-t- octylphe no Xy) ethoxy HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS, ethylmorpholine chloride, laurylpyridinium chloride, lauryl and are widely used as clinical diagnostic. The kinds, (tri-p-tolyl)phosphonium chloride, laurylphenylcyclotet concentration and pH of these buffers may be one kind or ramethylenephosphonium bromide, cetylpyridium chloride, plural kinds according to the object Such as preservation and 60 cetyltrimethylammonium chloride, (polyoxyethylene) enzymatic reaction of each reagent component. Any of these laurylamine, anionic Surfactants Such as Sodium cholate, bufferS may be preferably used at a pH during enzymatic desoxycholic acid, N-lauroyl Sarcosine, taurotaurocholic reaction within the range of 5.0-10.0. acid, and amphoteric Surfactants such as CHAPS, CHAPSO, The reagent constituting the reagent kit of the resent N,N'-bis(octylaminoethyl)glycine, N-carboxymethyl-N- invention may contain metal Salt, protein, amino acid, 65 (Stearyloxymethyl)pyridiumbetaine, N-palmitylsulfotaurin, Saccharide, and an organic acid as a Stabilizer. AS the metal lauryldimethylamine-oxide, and N-(laurylthioethoxy) Salt, Salts of Sodium, potassium, magnesium, calcium, iron, methyl-N,N-dimethylbetaine can be used. US 6,867,012 B2 15 16 The reagent kit of the present invention can preserve each When sarcosine oxidase (EC 1.5.3.1) is further added, of the above-mentioned reagent component or two or more moreover, Sarcosine is decomposed into glycine, formalde components in a single reagent. Therefore, the reagent kit of hyde and hydrogen peroxide, which affords Sensitively the present invention may be a single reagent composition higher measurement. Furthermore, measurement of produc containing all of the above-mentioned reagent components. tion amount of hydrogen peroxide by reaction with formal However, when interfering components are present or a dehyde oxidase affords improved Sensitivity. component that cannot be ensured Stability under Single Dimethylglycine oxidase is an enzyme that acts on dim preservation conditions is present, the constituent compo ethylglycine produced by betaine-homocysteine methyl nents are desirably divided and preserved. transferase to decompose into Sarcosine, formaldehyde and The present invention also provides a reagent kit for hydrogen peroxide. For example, it can be obtained from a creatinine or creatine determination, which contains, besides microorganism Such as the genus Cylindrocarpon, the genus the above-mentioned reagent, creatine amidinohydrolase, Achromobacter, and the genus Arthrobacter. Sarcosine oxidase and, where necessary, creatinine amidohy The Sarcosine oxidase (EC 1.5.3.1) that acts on Sarcosine drolase. The enzymes mentioned above can be used as produced by dimethylglycine oxidase to produce glycine, appropriate. 15 formaldehyde and hydrogen peroxide can be obtained from The present invention further provides a reagent kit for various animals, and a microorganism Such as the genus homocysteine determination containing, besides the above Arthrobacter, the genus Penicillium), the genus Bacillus. mentioned reagent, at least betaine, betaine-homocysteine Alternatively, a commercially available enzyme can be also methyltransferase and dimethylglycine oxidase. The used. enzymes mentioned above can be used as appropriate. The above-mentioned enzyme may be one obtained by The present invention Still yet provides a determination isolating a gene encoding each enzyme protein from a method of homocysteine, which method comprises bringing producing microorganism and allowing expression by transferase utilizing homocysteine and other compound as a genetic engineering. In addition, for example, one geneti pair of Substrates, and Said other compound into contact with cally modified to achieve improvement in enzymatic a Sample, and measuring a compound produced by the 25 properties, Such as Specific activity and Stability of the reaction of the transferase without chromatography or enzyme protein can be also used. antigen-antibody reaction. Hydrogen peroxide produced by dimethylglycine oxidase The transferase to be used for the above-mentioned deter and/or Sarcosine oxidase can be measured by direct mea mination method may be, for example, an enzyme that Surement of absorbance in the UV region, calorimetric assay transfers methyl to thiol of homocysteine. Preferably, it is using catalase and titanium oxide reagent (Agric. Biol. betaine-homocysteine methyltransferase (EC 2.1.1.5), Chem., 38, 1213 (1974)), titration assay using potassium homocysteine methyltransferase (EC 2.1.1.10), and permanganate. For highly sensitive measurement, it is N5-methyltetrahydrofolate-homocysteine methyltransferase reacted with a hydrogen donor chromogenic reagent in the (EC 2.1.1.13), more preferably betaine-homocysteine meth presence of peroxidase and, where necessary, reacted with a yltransferase. 35 coupler Such as 4-aminoantipyrine, 3-methyl-2- The betaine-homocysteine methyltransferase (EC 2.1.1.5) benzothiazolinone and the produced pigment is measured to to be used in the present invention produces dimethylglycine preferably afford a quantitative measurement. The chro and methionine by reacting with homocysteine when betaine mogenic reagent to be used is not particularly limited and is a different substrate. For example, it can be obtained from various commercially available ones can be used. Examples mammal and microorganisms. Such as the genus 40 thereof include N-ethyl-N-sulfopropyl-m-anisidine, Pseudomonas, and the genus Aspergillus. N-ethyl-N-sulfopropyl-aniline, N-methyl-N-sulfopropyl When betaine-homocysteine methyltransferase is used, aniline, N-butyl-N-sulfopropyl-aniline, N-ethyl-N- betaine is used as the other Substrate, which is commercially sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5- available at a low cost in the form of hydrochloride. dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5- The dimethylglycine, which is the product of the above 45 dimethylaniline, N-ethyl-N-sulfopropyl-m-toluidine, mentioned enzymatic reaction, can be measured by the N-ethyl-N-sulfopropyl-o-toluidine, N-ethyl-N-sulfopropyl following method. p-toluidine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m- Dimethylglycine is decomposed by dimethylglycine oxi anisidine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, dase (EC 1.5.3.10) into sarcosine, formaldehyde and hydro N-ethyl-N-(2-hydroxy-3-sulfo propyl)-3,5- gen peroxide according to the following formula: 50 dimethoxyaniline, N-(2-hydroxy-3-sulfopropyl)-3,5- dimethylglycine + H2O+O - Sarcosine+formaldehyde-- dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3, HO 5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)- Therefore, by measuring the amount of oxygen consumed m-toluidine, N-Sulfopropylaniline, p-chlorophenol, during the reaction or by measuring the production amount p-bromophenol, 2,4-dichlorophenol, p-hydroxybenzoate, of hydrogen peroxide using a hydrogen peroxide Sensor, or 55 3,3',5,5'-tetramethylbenzidine, N-(3-sulfopropyl)3,3',5,5'- using a redox indicator directly and in the presence of tetramethylbenzidine, and N,N,N',N',N',N"-hexa(3- peroxidase, dimethylglycine can be measured. Alternatively, Sulfopropyl)-4,4',4'-triaminotriphenylmethane. In addition, produced formaldehyde can be indirectly measured by mea hydrogen peroxide can be measured directly or in the Suring absorbance in the UV region or visible region, or co-existence of a catalase by monitoring consumed oxygen fluorescence using Han Z reagent, formaldehyde 60 with a Sensor. dehydrogenase, formaldehyde oxidase. For example, Since Sarcosine produced by dimethylglycine oxidase can be reduced NAD (NADH) produced from NAD (nicotineamide analyzed by analyzing formaldehyde or hydrogen peroxide adenine dinucleotide) by formaldehyde dehydrogenase produced by reacting with Sarcosine oxidase by the afore reduces tetrazolium Salt to give formazan pigment in the mentioned method, or by measuring a pigment produced by presence of an electron carrier Such as diaphorase and 65 reacting Sarcosine dehydrogenase in the presence of an methylphenazinium methylsulfate, the determination electron carrier and color developing pigment. Examples of thereof affords quantitative measurement of homocysteine. the electron carrier include diaphorase, methylphenazinium US 6,867,012 B2 17 18 methylsulfate, methylene blue, and potassium ferricyanide. phosphothioate. Specifically, thiols may be dithiothreitol, Examples of the pigment include tetrazolium Salt, and dith io erythritol, 2-mer cap to e thanol, indophenol. In addition, Sarcosine dehydrogenase can be 2-mercaptomethylamine, cysteine, cyStamine, cysteine obtained directly from mammal, Pseudomona S thioglycorate, thioglycolic acid, and reduced glutathione, microorganism, or from a recombinant prepared by isolating borohydrides may be Sodium borohydride, and amalgams the gene of the enzyme and processing by genetic engineer may be Sodium amalgam. Ing. When the enzyme to be used in the present invention is Formaldehyde produced by dimethylglycine oxidase is subject to inhibition of activity by the thiol compound, the preferably measured by reacting glutathione, glutathione reagent may show degraded properties. However, dimeth dependent formaldehyde dehydrogenase and oxidized ylglycine oxidase free of an influence of thiol compound has coenzyme, and, where necessary, a reduced coenzyme of the not been reported. kind different from the oxidized coenzyme with a sample The present invention provides a novel dimethylglycine and measuring the produced or consumed compound. oxidase Stable against thiol compound. Examples of Such As the glutathione-dependent formaldehyde thiol compound include dithiothreitol, dithioerythritol, dehydrogenase, the aforementioned enzyme obtained in the 15 2-mer captoe thanol, 2-mercaptoe thane Sulfonate, present invention is most preferably used. Conventionally 2-mercaptoethylamine, cysteine, homocy Steine, known enzymes derived from mammal, higher plant, methy N-acetylcysteine, thioglycerol, thioglycolic acid, reduced lotrophic yeast, and bacteria can be also used. For example, glutathione and salts thereof. Preferably, the dimethylgly commercially available enzymes derived from Candida cine oxidase in the presence of 0.05 mmol/L dithiothreitol yeast can be used. Specific examples include FORMALDE retains at least 50% of the enzyme activity in the absence of HYDE DEHYDROGENASE (Sigma, Glutathione dithiothreitol. dependent). Preferably, the dimethylglycine oxidase of the present This enzyme may be obtained by isolating a gene encod invention is an enzyme further having the following ing the enzyme protein from a microorganism producing the physico-chemical properties. Same and allowing expression by genetic engineering, or 25 (a) action: acting on dimethylglycine in the presence of include a mutant and chemically modified enzymes pro oxygen to produce Sarcosine, formaldehyde and hydrogen cessed to have improved enzymatic properties of, for peroxide, and example, enzyme Specific activity, and Stability. b) Km value for dimethylglycine: not more than 15 mM. Examples of the coenzyme include nicotineamide adenine The dimethylglycine oxidase of the present invention is dinucleotide and its analogs (NADs), nicotineamide adenine not particularly limited as regards its derivation as long as it dinucleotide phosphate and its analogs (NADPs), thionic shows the above-mentioned properties. Preferably, it is otineamide adenine dinucleotide and its analogs (thio derived from a microorganism belonging to the genus NADs) or thionicotineamide adenine dinucleotide and its Arthrobacter, the genus Streptomyces, particularly prefer analogs (thio-NADPs), wherein NADs and NADPs may be ably derived from Arthrobacter nicotianae strain IFO14234 those that can be used as coenzyme by glutathione 35 or Streptomyces mutabilis strain IFO12800. The strains dependent formaldehyde dehydrogenase. Known examples IFO14234 and IFO12800 are available from the Institute for include nicotineamide adenine dinucleotide, acetylpyridine Fermentation, Osaka (IFO) (17-85, Juso-honmachi, adenine dinucleotide, and acetylpyridine adeninehypoxan 2-chome, Yodogawa-ku, Osaka 532-8686 Japan). thine dinucleotide, and nicotineamide hypoxanthine The dimethylglycine oxidase of the present invention may dinucleotide for NADS, and nicotineamide adenine dinucle 40 be genetically modified to improve enzymatic activity, Such otide phosphate, acetylpyridine adenine dinucleotide as Specific activity and Stability, by randomly or Site phosphate, acetylpyridine adeninehypoxanthine dinucle Specifically inducing mutation as long as resistance to the otide phosphate, and nicotineamide hypoxanthine dinucle above-mentioned thiol compound can be retained. otide phosphate for NADPs. As the thio-NADs or thio The dimethylglycine oxidase of the present invention can NADPs, those that can be used as coenzyme by glutathione 45 be obtained by isolation and purification of a cell or tissue dependent formaldehyde dehydrogenase may be used. culture capable of producing the enzyme, or by isolating a Known examples include thionicotineamide adenine gene encoding the enzyme protein by a conventional method dinucleotide, thionicotineamide hypoxanthine dinucleotide, and allowing expression thereof in a Suitable host according thionicotineamide adenine dinucleotide phosphate, and to genetic recombination techniques. Preferable embodi thionicotineamide hypoxanthine dinucleotide phosphate. 50 ment of the former is the following method The determination method of formaldehyde when con Any of the above-mentioned dimethylglycine oxidase ventionally known glutathione-dependent formaldehyde producing microorganism, Such as Arthrobacter nicotianae dehydrogenase is used may be any from among the afore strain IFO14234 or Streptomyces mutabilis strain IFO12800, mentioned methods for novel glutathione-dependent form is cultured in a nutrient medium. The nutrient medium to be aldehyde dehydrogenase of the present invention, except 55 used may be a Synthetic medium or a natural medium that those via a cycling reaction. contains carbon Source, nitrogen Source, mineral and other When the sample to be measured in the present invention necessary nutrients in Suitable amounts that the Strain to be is a biological Sample, particularly plasma or urine, the used can utilize. AS the carbon Source, for example, malic homocysteine to be contained is largely bonded with a acid, and Succinic acid can be used. AS the nitrogen Source, circulating protein Such as albumin, or in the State of a 60 for example, nitrogen-containing natural products Such as disulfide bond with cysteine or other homocysteine mol peptones, beef extract, and yeast extract, and inorganic ecule. Therefore, for the determination of the total nitrogen-containing compounds Such as ammonium homocysteine, the Sample needs to be treated with a reduc chloride, and ammonium citrate can be used. AS the mineral, ing agent or enzymatic reaction in advance to allow pro for example, potassium phosphate, Sodium phosphate, and duction of free homocysteine. 65 magnesium Sulfate can be used. The reducing agent to be used for this end may be, for The culture is generally Shaking culture or aeration example, thiols, borohydrides, amalgams, phosphine, and agitation culture. The culture temperature is about 20-about US 6,867,012 B2 19 20 40° C., preferably about 25-about 37°C., and the culture pH Homocysteine methyltransferase can be harvested from is about 5-about 9, preferably controlled to about 6-about 8. mammal, plant, and yeast (Saccharomyces). It may be AS long as the Strain in use can grow, other conditions may obtained by isolating a gene encoding the enzyme protein be employed. The culture period is generally about 1-about and allowing expression thereof in a suitable host according 7 days, dimethylglycine oxidase is generally produced and to genetic recombination techniques or may be genetically accumulated in the cell. modified in properties by spontaneous or artificial mutation. The dimethylglycine oxidase of the present invention can When homocysteine methyltransferase is used, be purified by a purification method generally used. For example, after recovery of the cells, disruption by S-adenosylmethionine is used as the other substrate, which ultraSonication, mechanical disruption with glass beads, is commercially available. disruption with French Press, or lysis with surfactant are N5-Methyltetrahydrofolate-homocysteine methyltrans applied to extract intracellular fractions. The obtained ferase is an enzyme that catalyzes the following reactions. extract is Subjected to Salting out with ammonium sulfate, L-homocysteine--N5-methyltetrahydrofolate->L-methionine--tet Sodium Sulfate, metal aggregation with magnesium chloride, rahydrofolate calcium chloride, aggregation with protamin, polyethyleneimine, ion-exchange chromatography with 15 Therefore, by measuring the produced methionine accord DEAE (diethylaminoethyl)-sepharose, or CM ing to the above-mentioned method, or by oxidizing tetrahy (carboxymethyl)-Sepharose, to purify glutathione-dependent drofolate using NADP-dependent tetrahydrofolate dehydro formaldehyde dehydrogenase. genase and measuring absorbance in the UV region or The use of betaine-homocysteine methyltransferase, dim Visible region, homocysteine can be measured. ethylglycine oxidase of the present invention and N5-Methyltetrahydrofolate-homocysteine methyltrans glutathione-dependent formaldehyde dehydrogenase ferase can be harvested from mammal or E. coli. In addition, enables detection of a trace amount of homocysteine con NADP-dependent tetrahydrofolate dehydrogenase can be tained in a Sample at a concentration of not more than 1 harvested from higher animal or plant. These enzymes may tumol/L. be obtained by isolating a gene encoding each enzyme and Methionine which is the other product of the betaine 25 allowing expression thereof in a Suitable host by genetic homocysteine methyltransferase reaction can be measured engineering or may be genetically modified in properties by using a known method. For example, there are mentioned a Spontaneous or artificial mutation. method including direct measurement based on known calo rimetric assay by nitroprusside reaction, ninhydrin reaction When this enzyme is used, N5-methyltetrahydrofolate is (e.g., J. Biol. Chem., 176, 789 (1948)) and a method includ used as the other Substrate, which is commercially available. ing measurement of ketoacid, ammonia and hydrogen per The present invention also provides a reagent kit for use Oxide produced by methionine rasemase, amino acid oxidase in the above-mentioned homocysteine determination and methionine Y-lyase. The ketoacid can be measured by, method. The reagent kit for homocysteine determination of for example, a colorimetric assay using reaction with the present invention contains at least buffer, transferase 3-methyl-2-benzothiazolonehydrazone, ammonia can be utilizing homocysteine and other compound as a pair of measured by, for example, a calorimetric assay according to 35 Substrates, said other compound and a reagent for measuring the indophenol method, fluorometric assay of a reaction a compound produced by the reaction of the enzyme. product with o-futalaldehyde, and hydrogen peroxide can be In a preferable embodiment, the reagent kit for homocys measured by the above-mentioned method. teine determination of the present invention contains buffer, Methionine rasemase is known to be produced from betaine, betaine-homocysteine methyltransferase, dimeth bacteria belonging to the genus Pseudomonas, and the genus 40 ylglycine oxidase, and a reagent for analyzing at least one of StreptpCOccus. As the amino acid oxidase, enzymes derived formaldehyde, Sarcosine or hydrogen peroxide produced by from mammal, bacteria belonging to the genus Pseudomo the reactions of the enzymes. Furthermore, it may contain naS are known. As one specifically acting on methionine, an Sarco Sine oxidase. As the betaine-homocysteine amino acid oxidase contained in Snake toxin is known. As methyltransferase, dimethylglycine oxidase and sarcosine the methionine Y-lyase, enzymes derived from the bacteria 45 oxidase, those mentioned above are preferable, but are not belonging to the genus Pseudomonas, and the genus particularly limited. The "reagent for analyzing at least one CloStridium are known, from which methionine Y-lyase can of formaldehyde, Sarcosine or hydrogen peroxide produced be isolated and purified, or can be obtained from a recom by the reactions of the enzymes' may be those based on the binant prepared by isolating a gene encoding the enzyme aforementioned principle. The reagent for analyzing form protein and processing according to genetic recombination 50 aldehyde may be formaldehyde dehydrogenase, formalde techniques. hyde oxidase, Hanz reagent, CTA reagent (J. Biol. Chem., It is known that the betaine-homocysteine methyltrans 231,813 (1958)), Purpald reagent (Anal. Biochem., 234(1), ferase can efficiently use dimethylthetin besides betaine, as 50 (1996)) or a reagent containing phenylhydrazine, potas a methyl donor to homocysteine (J. Bacteriol., 113(1), 218 Sium ferricyanide, chloroform and methanol in combination (1973)). Therefore, dimethylthetin may be added, instead of 55 and the like. The reagent for analyzing hydrogen peroxide betaine, to the enzyme as a Substrate other than may be peroxidase, hydrogen donor chromogenic reagent homocysteine, and methionine thus produced is measured and, where necessary, a coupler Such as 4-aminoantipyrine, by any of the above-mentioned methods to measure 3-methyl-2-benzothiazolinone, catalase, titanium oxide homocysteine in the sample. reagent, and potassium permanganate. The reagent for ana Homocysteine methyltransferase is an enzyme that cata 60 lyzing Sarcosine may be sarcosine oxidase, the above lyzes the following reactions. mentioned reagent for analyzing formaldehyde or hydrogen peroxide produced by the enzymatic reaction, sarcosine L-homocysteine--S-adenosylmethionine->L-methionine--S-adenos dehydrogenase, electron carrier, and color developing pig ylhomocysteine ment. Therefore, by measuring the produced methionine accord 65 In a particularly preferable embodiment, the reagent kit ing to the above-mentioned method, homocysteine can be for homocysteine determination of the present invention can measured. contain buffer, be taine, be taine-homocysteine US 6,867,012 B2 21 22 methyltransferase, dimethylglycine oxidase, glutathione,
TABLE 3 homocysteine concentration Change in absorbance creatinine (umol/L) at 405 nm. concentration Change in absorbance 25 O O (umol/L) at 405 nm. 1. O.OO24 2 O.OO62 O O 3 O.O113 1O O.O11 5 O.O226 2O O.O23 1O O.O336 40 O.042 15 O.O589 60 O.O66 2O 0.0717 8O O.084 3O O.1092 1OO O.113 40 O.1396 50 O.1720 From Table 3 and FIG. 3, it is clear that 10 umol/L of creatinine could be measured by the cycling method. 35 From Table 4 and FIG. 4, it is clear that 1 umol/L of EXAMPLE 7 homocysteine could be measured by the cycling method. This application is based on a patent application NoS. Measurement of Homocysteine Standard Solution 2000-370445 and 2001-96724 filed in Japan, the contents of AS the betaine-homocysteine methyltransferase and dim 40 which are hereby incorporated by reference. ethylglycine oxidase, those obtained in Example 2 and All of the references cited herein, including patents, Examples 3(1) were used. The activity of betaine patent applications and publications, are hereby incorpo homocysteine methyltransferase and dimethylglycine oxi rated in their entireties by reference. dase were measured in the same manner as in Example 2 and While this invention has been described with an emphasis Example 3. 45 upon preferred embodiments, it will be obvious to those of Various concentrations of aqueous L-homocystine Solu ordinary skill in the art that variations of the preferred tions were incubated in 0.5 mM dithiothreitol at 37° C. for embodiments may be used and that it is intended that the 30 min and the obtained L-homocysteine (10 uI) was used invention may be practiced otherwise than as Specifically as a sample. The sample was mixed with 20 mM PIPES described herein. Accordingly, this invention includes all buffer (200 ul, pH 7.3) containing 20 mM betaine, 1 U/ml 50 modifications encompassed within the Spirit and Scope of the betaine-homocysteine methyltransferase, 7 U/ml dimethylg invention as defined by the following claims.
SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 2 SEQ ID NO 1 LENGTH 40 TYPE DNA ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Description of Artificial Sequence: Primer
<400 SEQUENCE: 1 US 6,867,012 B2
-continued gcaatticcat atgccacccg ttgggggcaa aaaggccaag 40
<210> SEQ ID NO 2 <211& LENGTH: 40 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Primer <400 SEQUENCE: 2 atc.gc.ggatc caggctactg tdatttgaat ttttgtttitt 40
15 What is claimed is: methyltransferase, dimethylglycine oxidase and, where 1. A method for determining formaldehyde, which com necessary, Sarcosine oxidase into contact with a Sample, and prises bringing glutathione-dependent formaldehyde dehy measuring, according to the method of claim 2, formalde drogenase having an optimal pH of about 7.5 to about 8.5 hyde produced by the enzymatic reactions. and having a ratio of reactivity with thio-NAD to reactivity 11. A method for determining creatine or creatinine, with NAD of not less than 0.3, glutathione and an oxidized which comprises reacting creatine amidinohydrolase, Sar coenzyme into contact with a Sample, and analyzing a cosine oxidase, and where necessary, creatinine amidohy compound resulting from the enzymatic reaction, wherein drolase and measuring, according to the method of claim 1, the gluathione-dependent formaldehyde dehydrogenase is formaldehyde produced by the enzymatic reactions. derived from Hansenula nonfermenians IFO1473. 25 12. A method for determining creatine or creatinine, 2. A method for determining formaldehyde, which com which comprises reacting creatine amidohydrolase, Sar prises bringing glutathione-dependent formaldehyde dehy cosine oxidase, and where necessary, creatinine amidohy drogenase having a ratio of reactivity with thio-NAD to drolase and measuring, according to the method of claim 2, reactivity with NAD of not less than 0.3, glutathione, one formaldehyde produced by the enzymatic reactions. compound Selected from the group consisting of thio-NADS 13. A method for determining homocysteine, which com and thio-NADPs, and one compound selected from the prises bringing a transferase utilizing homocysteine and group consisting of reduced NADS and reduced NADPS into another compound as a pair of Substrates and said another contact with a Sample to allow cycling reaction and analyZ compound into contact with a Sample and measuring the ing changes in the amount of a compound due to the resulting compound and wherein Said transferase and Said reaction. another compound is a combination Selected from the group 3. A method for determining formaldehyde, which com 35 consisting of betaine-homocysteine methyltransferase and prises bringing glutathione-dependent formaldehyde dehy betaine, betaine-homocysteine methyltransferase and drogenase having a ratio of reactivity with thio-NAD to dimethyl the tin, homocysteine methyltransferase and reactivity with NAD of not less than 0.3, glutathione, one S-adenosylmethionine, and N5-methyltetrahydrofolate compound Selected from the group consisting of reduced homoc y Steine methyltransferase and thio-NADs and reduced thio-NADPs, and one compound 40 N5-methyltetrahydrofolate, and the resulting compound is selected from the group consisting of NADs and NADPs methionine, and wherein the betaine, betaine-homocysteine into contact with a Sample to allow cycling reaction and methyltransferase, dimethylglycine oxidase and, where analyzing changes in the amount of a compound due to the necessary, Sarcosine oxidase are brought into contact with a reaction. Sample, hydrogen peroxide produced by the enzymatic 4. The method of claim 2, wherein the amount of the 45 reactions is reacted with hydrogen donor chromogenic reduced thio-NADP or reduced thio-NAD compound is reagent and, where necessary, coupler, in the presence of analyzed. peroxidase, and the resulting pigment is measured. 5. The method of claim 1, wherein a minimum detection 14. The method of claim 13, wherein the dimethylglycine limit of the formaldehyde is not more than 1 umol/L. oxidase is an enzyme stable to a thiol compound. 6. The method of claim 2, wherein a minimum detection 50 15. The method of claim 14, wherein the thiol compound limit of the formaldehyde is not more than 1 umol/L. is at least one kind Selected from the group consisting of 7. A method for determining a biological component that dithioth reitol, dithioerythritol, 2-mercaptoethanol, produces formaldehyde as a reaction intermediate, compris 2-mercaptoethane Sulfonate, 2-mercaptoethylamine, ing measuring produced formaldehyde by the method of cysteine, homocysteine, N-acetylcysteine, thiogylcerol, claim 1. 55 thioglycolic acid, reduced glutathione and Salts thereof. 8. The method for determining a biological component 16. The method of claim 14, wherein the thiol compound that produces formaldehyde as a reaction intermediate, com is dithiothreitol. prising measuring produced formaldehyde by the method of 17. The method of claim 14, wherein the dimethylglycine claim 2. oxidase shows an enzyme activity in the presence of 0.05 9. A method for determining homocysteine, which com 60 mmol/L dithiothreitol of at least 50% of the enzyme activity prises bringing betaine, be taine-homocysteine in the absence of dithiothreitol. methyltransferase, dimethylglycine oxidase and, where 18. The method of claim 14, wherein the dimethylglycine necessary, Sarcosine oxidase into contact with a Sample, and oxidase is an enzyme having the following physico measuring, according to the method of claim 1, formalde chemical properties: action: acting on dimethylglycine in the hyde produced by the enzymatic reactions. 65 presence of oxygen to produce Sarcosine, formaldehyde and 10. A method for determining homocysteine, which com hydrogen peroxide, and Km value for dimethylglycine: not prises bringing betaine, be taine-homocysteine more than 15 mM. US 6,867,012 B2 31 32 19. The method claim 14, wherein the dimethylglycine presence of oxygen to produce Sarcosine, formaldehyde and oxidase is derived from a microorganism. hydrogen peroxide, and Km value for dimethylglycine: not 20. The method of claim 19, wherein the dimethylglycine more than 15 mM. oxidase is derived from a microorganism belonging to the 37. The method of claim 32, wherein the dimethylglycine genus Arthrobacter or the genus Streptomyces. oxidase is derived from a microorganism. 21. The method of claim 20, wherein the dimethylglycine 38. The method of claim 37, wherein the dimethylglycine oxidase is derived from Arthrobacter nicotianae IFO14234 oxidase is derived from a microorganism belonging to the or Streptomyces mutabilis IFO12800. genus Arthrobacter or the genus Streptomyces. 22. The method of claim 13, wherein the betaine, betaine 39. The method of claim 38, wherein the dimethylglycine homocysteine methyltransferase, dimethylglycine oxidase oxidase is derived from Arthrobacter nicotianae IFO 14234 and, where necessary, Sarcosine oxidase are brought in or Streptomyces mutabilis IFO12800. contact with a Sample, formaldehyde produced by the enzy 40. The method of claim 32, wherein a minimum detec matic reactions is reacted with glutathione, glutathione tion limit of the homocysteine is not more than 1 umol/L. dependent formaldehyde dehydrogenase and oxidized coen 41. An isolated glutathione-dependent formaldehyde Zyme and the resulting reduced coenzyme is measured. 15 dehydrogenase (EC 1.2.1.2) having the following physico 23. The method of claim 22, wherein the dimethylglycine chemical properties: action: acting on formaldehyde in the oxidase is an enzyme stable to a thiol compound. presence of one coenzyme Selected from the group consist 24. The method of claim 23, wherein the thiol compound ing of NADs, NADPs, thio-NADs and thio-NADPs, reduced is at least one kind Selected from the group consisting of glutathione to produce S-formylglutathione, a reduced dithiothreitol, dithioerythritol, 2-mercaptoethanol, coenzyme, a ratio of reactivity with thio-NAD to reactivity 2-mercaptoethane Sulfonate, 2-mercaptoethylamine, with NAD: not less than 0.3, optimal pH: about 7.5-about cysteine, homocysteine, N-acetylcysteine, thiogylcerol, 8.5, pH stability: about 6.0-about 9.0, and heat stability: thioglycolic acid, reduced glutathione and Salts thereof. about 40 C. or less. 25. The method of claim 23, wherein the thiol compound 42. The glutathione-dependent formaldehyde dehydroge is dithiothreitol. 25 nase of claim 41, which is derived from a microorganism. 26. The method of claim 23, wherein the dimethylglycine 43. The glutathione-dependent formaldehyde dehydroge oxidase shows an enzyme activity in the presence of 0.05 nase of claim 42, which is derived from methylotrophic mmol/L dithiothreitol of at least 50% of the enzyme activity yeast. in the absence of dithiothreitol. 44. The glutathione-dependent formaldehyde dehydroge 27. The method of claim 23, wherein the dimethylglycine nase of claim 43, which is derived from Hansenula yeast. oxidase is an enzyme having the following physico 45. The glutathione-dependent formaldehyde dehydroge chemical properties: action: acting on dimethylglycine in the nase of claim 44, which is derived from Hansenula nonfer presence of oxygen to produce Sarcosine, formaldehyde and mentans IFO14234. hydrogen peroxide, and Km value for dimethylglycine: not 46. An isolated dimethylglycine oxidase (EC 1.5.3.10) more than 15 mM. 35 having the following physico-chemical properties: action: 28. The method of claim 23, wherein the dimethylglycine acting on dimethylglycine in the presence of oxygen to oxidase is derived from a microorganism. produce Sarcosine, formaldehyde and hydrogen peroxide, an 29. The method of claim 28, wherein the dimethylglycine enzyme activity in the presence of 0.05 mmol/L dithiothrei oxidase is derived from a microorganism belonging to the tol of at least 50% of the enzyme activity in the absence of genus Arthrobacter or the genus Streptomyces. 40 dithiothreitol, and Km value for dimethylglycine: not more 30. The method of claim 29, wherein the dimethylglycine than 15 mM. oxidase is derived from Arthrobacter nicotianae IFO14234 47. The dimethylglycine oxidase of claim 46, which is or Streptomyces mutabilis IFO12800. derived from a microorganism. 31. The method of claim 13, wherein the betaine, betaine 48. The dimethylglycine oxidase of claim 47, which is homocysteine methyltransferase, dimethylglycine oxidase 45 derived from a microorganism belonging to the genus and, where necessary, Sarcosine oxidase are brought in Arthrobacter or the genus Streptomyces. contact with a Sample, formaldehyde produced by the enzy 49. The dimethylglycine oxidase of claim 48, which is matic reactions is reacted with glutathione, glutathione derived from Arthrobacter nicotianae IFO14234, or Strep dependent formaldehyde dehydrogenase and oxidized coen tomyces mutabilis IFO12800. Zyme and the resulting reduced coenzyme is measured. 50 50. A reagent kit for formaldehyde determination, which 32. The method of claim 31, wherein the dimethylglycine comprises at least buffer, glutathione, glutathione-dependent oxidase is an enzyme stable to a thiol compound. formaldehyde dehydrogenase having a ratio of reactivity 33. The method of claim 32, wherein the thiol compound with thio-NAD to reactivity with NAD of not less than 0.3 is at least one kind Selected from the group consisting of and a reagent for analyzing a compound produced by the dithiothreitol, dithioerythritol, 2-mercaptoethanol, 55 enzymatic reaction. 2-mercaptoethane Sulfonate, 2-mercaptoethylamine, 51. The reagent kit for formaldehyde determination of cysteine, homocysteine, N-acetylcysteine, thiogylcerol, claim 50, wherein the glutathione-dependent formaldehyde thioglycolic acid, reduced glutathione and Salts thereof. dehydrogenase has the following physico-chemical proper 34. The method of claim 32, wherein the thiol compound ties: action: production of S-formylglutathione and reduced is dithiothreitol. 60 coenzyme by action on formaldehyde in the presence of one 35. The method of claim 32, wherein the dimethylglycine oxidized coenzyme Selected from the group consisting of oxidase shows an enzyme activity in the presence of 0.05 NADs, NADPs, thio-NADs and thio-NADPs and reduced mmol/L dithiothreitol of at least 50% of the enzyme activity glutathione; optimal pH: about 7.5-about 8.5; pH stability: in the absence of dithiothreitol. about 6.0-about 9.0; and heat stability: about 40 C. or less. 36. The method of claim 32, wherein the dimethylglycine 65 52. A reagent kit for formaldehyde determination, which oxidase is an enzyme having the following physico comprises at least a buffer, glutathionde, glutathionde chemical properties: action: acting on dimethylglycine in the dependent formaldehyde dehydrogenase having a ratio of US 6,867,012 B2 33 34 reactivity with thio-NAD to reactivity with NAD of not less teine and another compound as a pair of Substrates and a than 0.3, one compound Selected from the group consisting reagent for analyzing a compound produced by a reaction of of thio-NADs and thio-NADPs, and one compound selected the transferease utilizing homocysteine and Said another from the group consisting of reduced NADS and reduced compound as the Substrates. NADPS. 5 63. The reagent kit for homocysteine determination of 53. The reagent kit for formaldehyde determination of claim 62, wherein the transferase and Said other compound claim 52, wherein the glutathione-dependent formaldehyde are a combination Selected from the group consisting of dehydrogenase has the following physico-chemical proper betaine-homocysteine methyltransferase and betaine, ties: action: production of S-formylglutathione and reduced be taine-homocy Steine methyltransferase and coenzyme by action on formaldehyde in the presence of one dimethyl the tin, homocysteine methyltransferase and oxidized coenzyme Selected from the group consisting of S-adenosylmethionine and N5-methyltetrahydrofolate NADs, NADPs, thio-NADs and thio-NADPs and reduced homoc y Steine methyltransferase and glutathione; optimal pH: about 7.5-about 8.5; pH stability: N5-methyltetrahydrofolate, and the produced compound is about 6.0-about 9.0; and heat stability: about 40 C. or less. methionine. 54. A reagent kit for formaldehyde determination, which 15 comprises at least a buffer, glutathionde, glutathione 64. A reagent kit for homocysteine determination, which dependent formaldehyde dehydrogenase having a ratio of comprises a buffer, betaine, betaine-homocysteine reactivity with thio-NAD to reactivity with NAD of not less methyltransferase, dimethylglycine oxidase and a reagent than 0.3, one compound Selected from the group consisting for measuring hydrogen peroxide produced by the enzy of reduced thio-NADS and reduced thio-NADPs, and one matic reactions. compound Selected from the group consisting of NADS and 65. The reagent kit of claim 64, wherein the reagent for NADPS. measuring hydrogen peroxide comprises peroxidase, and a 55. The reagent kit for formaldehyde determination of hydrogen donor chromogenic reagent. claim 54, wherein the glutathione-dependent formaldehyde 66. The reagent for of claim 64, wherein the dimethylg dehydrogenase has the following physico-chemical proper 25 lycine oxidase has the following physico-chemical proper ties: action: production of S-formylglutathione and reduced ties: action: acting on dimethylglycine in the presence of coenzyme by action on formaldehyde in the presence of one oxygen to produce Sarcosine, formaldehyde and hydrogen oxidized coenzyme Selected from the group consisting of peroxide, an enzyme activity is retained at least by 50% in NADs, NADPs, thio-NADs and thio-NADPs and reduced the presence of 0.05 mmol/L dithiothreitol relative to the glutathione; optimal pH: about 7.5- to about 8.5; pH stabil enzyme activity in the absence of dithiothreitol, and Km ity: about 6.0- to about 9.0; and heat stability: about 40 C. value for dimethylglycine: not more than 15 mM. or less. 67. A reagent kit for homocysteine determination, which 56. A reagent kit for homocysteine determination, which comprises betaine, betine-homocysteine methyltransferase, comprises, in addition to the reagent of claim 50, betaine, dimethlyglycine, oxidase and a reagent for determination of betaine-homocysteine methyltransferase, dimethylglycine 35 formaldehyde produced by the enzymatic reactions. oxidase and, where necessary, Sarcosine oxidase. 68. The reagent kit of claim 67, which comprises form 57. A reagent kit for homocysteine determination, which aldehyde dehydrogenase and oxidized coenzyme as reagents comprises, in addition to the reagent of claim 52, betaine, for determination of the formaldehyde. betaine-homocysteine methyltransferase, dimethylglycine 69. The reagent kit of claim 67, which comprises oxidase and, where necessary, Sarcosine oxidase. 40 glutathione, glutathione-dependent formaldehyde dehydro 58. A reagent kit for homocysteine determination, which genase and oxidized coenzyme as reagents for determination comprises, in addition to the reagent of claim 54, betaine, of the formaldehyde. betaine-homocysteine methyltransferase, dimethylglycine 70. The reagent kit of claim 67, wherein the dimethylg oxidase and, where necessary, Sarcosine oxidase. lycine oxidase has the following physico-chemical proper 59. A reagent kit for creatinine or creatine determination, 45 ties: action: acting on dimethylglycine in the presence of which comprises, in addition to the reagent of claim 50, oxygen to produce Sarcosine, formaldehyde and hydrogen creatine amidohydrolase, Sarcosine oxidase and, where peroxide; enzyme activity: an enzyme activity in the pres necessary, creatinine amidohydrolase. ence of 0.05 mmol/L dithiothreitol of at least 50% of the 60. A reagent kit for creatinine or creatine determination, enzyme activity in the absence of dithiothreitol; and Km which comprises, in addition to the reagent of claim 52, 50 value for dimethylglycine: not more than 15 mM. creatine amidohydrolase, Sarcosine oxidase and, where 71. The reagent kit of claim 64, further comprising necessary, creatinine amidohydrolase. Sarcosine oxidase. 61. A reagent kit for creatine or creatine determination, 72. The reagent kit of claim 64, further comprising a which comprises, in addition to the reagent of claim 54, coupler. creatine amidohydrolase, Sarcosine oxidase and, where 55 73. The reagent kit of claim 67, further comprising necessary, creatinine amidohydrolase. Sarcosine oxidase. 62. A reagent kit for homocysteine determination, which comprises at least a buffer, transferase utilizing homocys UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION
PATENT NO. : 6,867,012 B2 Page 1 of 2 DATED : March 15, 2005 INVENTOR(S) : Kishimoto et al.
It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
Column 1 Line 14, change "homocysteine and to -- homocysteine, --,
Column 2 Line 49, change “depths vein thrombosis” to -- deep-vein thrombosis --; Line 67, change "desulfrase' to -- desulfurase --; Column 3 Line 12, change “228998 A)” to -- 228998 A). --; Line 28, change “ulfrase' to -- ulfurase --; Line 50, change "creatine,” to -- creatine --, Column 13 Line 12, change “Wen” to -- When --; Column 14 Lines 22 and 31, change “antifugal' to -- antifungal --; Column 18 Line 4, change "thioglycorate' to -- thioglycolate --;
Column 19 Line 31, change “rasemase' to -- racemase --, Line 39, change “rasemase' to -- raSemase --; Line 41, change "StreptpCOccus' to -- StreptOCOccuS --;
Column 20 Line 55, delete “and the like': Column 23 Line 50, change “Epicurian Coli” to -- Escherichia coli --; Column 27 Line 46, change "L-homocystine' to --L-homocysteine --;
Column 32 Line 66, change "glutathionde, glutathionde-” to -- glutathione, glutathione --,
UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION
PATENT NO. : 6,867,012 B2 Page 2 of 2 DATED : March 15, 2005 INVENTOR(S) : Kishimoto et al.
It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
Column 33 Line 16, change "glutathionde' to -- glutathione -:
Column 34 Line 3, “transferease' to -- transferase --, and Line 32, change “betine-homocysteine' to -- betaine-homocysteine --.
Signed and Sealed this Twenty-seventh Day of December, 2005 WDJ
JON W. DUDAS Director of the United States Patent and Trademark Office UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION
PATENT NO. : 6,867,012 B2 Page 1 of 2 DATED : March 15, 2005 INVENTOR(S) : Kishimoto et al.
It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
Column 1 Line 14, change "homocysteine and to -- homocysteine, --,
Column 2 Line 49, change “depths vein thrombosis” to -- deep-vein thrombosis --; Line 67, change "desulfrase' to -- desulfurase --; Column 3 Line 12, change “228998 A)” to -- 228998 A). --; Line 28, change “ulfrase' to -- ulfurase --; Line 50, change "creatine,” to -- creatine --, Column 13 Line 12, change “Wen” to -- When --; Column 14 Lines 22 and 31, change “antifugal' to -- antifungal --; Column 18 Line 4, change "thioglycorate' to -- thioglycolate --;
Column 19 Line 31, change “rasemase' to -- racemase --, Line 39, change “rasemase' to -- raSemase --; Line 41, change "StreptpCOccus' to -- StreptOCOccuS --;
Column 20 Line 55, delete “and the like': Column 23 Line 50, change “Epicurian Coli” to -- Escherichia coli --; Column 27 Line 46, change "L-homocystine' to --L-homocysteine --;
Column 32 Line 66, change "glutathionde, glutathionde-” to -- glutathione, glutathione---,
Column 33 Line 16, change "glutathionde' to -- glutathione --, UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION
PATENT NO. : 6,867,012 B2 Page 2 of 2 DATED : March 15, 2005 INVENTOR(S) : Kishimoto et al.
It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
Column 34 Line 3, change "transferease' to -- transferase --, and Line 32, change “betine-homocysteine' to -- betaine-homocysteine --.
Signed and Sealed this Twenty-eighth Day of February, 2006 WDJ
JON W. DUDAS Director of the United States Patent and Trademark Office