Enzymatic assay method

Abstract

A method for assaying a compound of the formula ##STR1## wherein R 1 is hydroxyl or amino, or hydrogen if at least one of R 2 , R 3 , R 4 , R 5 and R 6 is hydroxyl or amino, and R 2 , R 3 , R 4 , R 5 and R 6 are hydrogen, halogen, lower alkyl, lower alkoxy, amino, substituted amino, hydroxy, carboxyl or sulfo, or R 5 and R 6 together form a ring, comprises establishing a reaction system containing the compound to be assayed and a coupler and an enzyme capable of consuming oxygen and generating a pigment in the presence of the compound and the coupler. A detectable change in the reaction system is measured, to assay the compound in question. The measurement can comprise the measurement of consumed oxygen, as by an oxygen electrode. Or the measurement can comprise the measurement of the generated pigment, as by a colorimetric assay at a specific absorption wavelength thereof.

What is claimed is: .[.1. A method for assaying a compound of the formula ##STR10## wherein R 1  is hydroxyl or amino, or hydrogen if at least one of R 2 , R 3 , R 4 , R 5  and R 6  is hydroxyl or amino, and R 2 , R 3 , R 4 , R 5  and R 6  are hydrogen, halogen, lower alkyl, lower alkoxy, amino, substituted amino, hydroxy, carboxyl, or sulfo, or R 5  and R 6  together form a ring, comprising establishing reaction system containing the compound to be assayed and a coupler and an oxidase that consumes O 2  and effects an exzymatic oxidative condensation of said compound to be assayed and said coupler, simultaneously generating a pigment, without the formation of any H 2  O 2  ; and measuring a detectable change in said reaction system to 
     
        assay said compound..]. .[.2.  An assay method according to claim 1, in which R 1  is amino, or hydrogen if at least one of R 2 , R 3 , R 4 , R 5  and R 6  is amino, and R 2 , R 3 , R 4 , R 5  and R 6  have the same meanings as before, said coupler being a compound of the formula ##STR11## wherein R 7  is hydroxyl, amino or substituted amino, and R 8 , R 9 , R 10 , R 11  and R 12  are hydrogen, halogen, lower alkyl, lower alkoxy, amino, substituted amino, hydroxyl, carboxyl or 
     
     
        sulfo, or R 11  and R 12  together form a ring..]. .[.3.  An assay method according to claim 1, in which R 1  is hydroxyl, or hydrogen if at least one of R 2 , R 3 , R 4 , R 5  and R 6  is hydroxyl, and R 2 , R 3 , R 4 , R 5  and R 6  have the same meanings as before, said coupler being a compound of the formula ##STR12## wherein R 13  is amino or substituted amino, and R 14 , R 15 , R 16 , R 17  and R 18  are hydrogen, halogen, lower alkyl, lower alkoxy, amino substituted amino, hydroxyl, carboxyl or sulfo, or R 17   
     
     
        and R 18  together form a ring..]. .[.4.  An assay method according to claim 1, wherein said oxidase is an oxidase for an aromatic ring compound having a hydroxyl or amino substituent..]. .[.5. An assay method according to claim 4, wherein said oxidase is ascorbate oxidase, laccase or tyrosinase..]. .[.6. An assay method according to claim 1, wherein said detectable change is the amount of consumed oxygen or generated pigment..]. .[.7. An assay method according to claim 6, and performing said measurement electrochemically..]. .[.8. An assay method according to 
     
     
        claim 7, and performing said measurement by an oxygen electrode..]. .[.9. An assay method according to claim 6, and performing said measurement of the amount of generated pigment by a colorimetric assay at a specific absorption wavelength thereof..]. .[.10. An assay method according to claim 1, wherein said compound to be assayed is generated from a synthetic substrate by the action of a hydrolase..]. .[.11. An assay method according to claim 10, wherein said hydrolase is a peptidase or protease and the synthetic substrate is a compound of the formula ##STR13## wherein one of R' 1  and R' 2  is R 19  CO--NH-- and the other is hydrogen, in which R 19  CO-- is an amino acid residue or peptide residue, and R 3 , R 4 , R 5  and R 6  have the same meanings 
     
     
        as before..]. .[.12.  An assay method according to claim 10, wherein said hydrolase is glycosidase and the synthetic substrate is a compound of the formula ##STR14## wherein one of R" 1  and R" 2  is (S) n  --O-- and the other is hydrogen, in which S is a monosaccharide unit and n is integer greater than 1, and R 3 , R 4 , R 5  and R 6  have the same meanings 
     
     
        as before..]. .[.13.  An assay method according to claim 10, wherein said hydrolase is a phosphatase and the synthetic substrate is a compound of the formula ##STR15## wherein one of R"' 1  and R"' 2  is ##STR16## and the other is hydrogen, in which R 20  ##STR17## or a substituted or unsubstituted glycerol residue, and R 3 , R 4 , 
     
     
        R 5  and R 6  have the same meanings as before..]. .[.14.  An assay method according to claim 10, wherein said hydrolase is an esterase or lipase, and the synthetic substrate is a compound of the formula ##STR18## wherein one of R"" 1  and R"" 2  is R 21  CO--O-- and the other is hydrogen, in which R 21  CO-- is a fatty acid residue, and R 3 , 
     
     
        R 4 , R 5  and R 6  have the same meanings as before..]. 15. A method for assaying a hydrolase, comprising the steps of: generating from a synthetic substrate by the action of said hydrolase a compound of the formula ##STR19## wherein R 1  is hydroxyl or amino, or hydrogen if at least one of R 2 , R 3 , R 4 , R 5  and R 6  is hydroxyl or amino, and R 2 , R 3 , R 4 , R 5  and R 6  are hydrogen, halogen, lower alkyl, lower alkoxy, amino, substituted amino, hydroxy, .[.carobxyl.]. .Iadd.carboxyl.Iaddend., or sulfo, or R 5  and R 6  together form a .Iadd.fused benzene .Iaddend.ring; establishing a reaction system containing .[.a.]. said compound and a coupler and an oxidase that consumes O 2  and effects an enzymatic oxidative condensation of said compound and said coupler, simultaneously generating a pigment, without the formation of any H 2  O 2  ; measuring a detectable change in said reaction system to assay said compound; and relating the assay of said compound to said hydrolase consumed in generating said compound, 
     
     
        thereby to assay said hydrolase. 16. An assay method according to claim 15, wherein the said hydrolase assay is an assay of .[.piptidase.]. 
     
     
        .Iadd.peptidase .Iaddend.or protease. 17. An assay method according to claim 15, wherein the said hydrolase assay is an assay of glycosidase. 
     
     
            An assay method according to claim 15, wherein the said hydrolase 
     
     
        assay is an assay of phosphatase. 19. An assay method according to claim 15, wherein the said hydrolase assay is an assay of esterase or lipase. .Iadd.20. An assay method according to claim 15, in which R 1  is amino, or hydrogen if at least one of R 2 , R 3 , R 4 , R 5  and R 6  is amino, and R 2 , R 3 , R 4 , R 5  and R 6  have the same meanings as before, said coupler being a compound of the formula ##STR20##.Iaddend. wherein R 7  is hydroxyl, amino or substituted amino, and R 8 , R 9 , R 10 , R 11  and R 12  are hydrogen, halogen, lower alkyl, lower alkoxy, amino, substituted amino, hydroxyl, carboxyl or sulfo, or R 11  and R 12  together form a fused benzene ring. 
     
     
        .Iadd.1.  An assay method according to claim 15, in which R 1  is hydroxyl, or hydrogen if at least one of R 2 , R 3 , R 4 , R 5   and R 6  is hydroxyl, and R 2 , R 3 , R 4 , R 5  and R 6  have the same meanings as before, said coupler being a compound of the formula ##STR21##.Iaddend. wherein R 13  is amino or substituted amino, and R 14 , R 15 , R 16 , R 17  and R 18  are hydrogen, halogen, lower alkyl, lower alkoxy, amino, substituted amino, hydroxyl, carboxyl or sulfo, or R 17  and R 18  together form a fused benzene ring. 
     
     
        .Iadd.2.  An assay method according to claim 15, wherein said oxidase is an oxidase for an aromatic ring compound having a hydroxyl or amino substituent. .Iaddend. .Iadd.23. An assay method according to claim 22, wherein said oxidase is ascorbate oxidase, lactase or tyrosinase. .Iaddend. .Iadd.24. An assay method according to claim 15, wherein said detectable change is the amount of consumed oxygen or generated pigment. .Iaddend. .Iadd.25. An assay method according to claim 24, and performing said measurement electrochemically. .Iaddend. .Iadd.26. An assay method according to claim 25, and performing said measurement by an oxygen electrode. .Iaddend. .Iadd.27. An assay method according to claim 24, and performing said measurement of the amount of generated pigment by a colorimetric assay at a specific absorption wavelength thereof. .Iaddend. .Iadd.28. An assay method according to claim 15, wherein said hydrolase is a peptidase or protease and the synthetic substrate is a compound of the formula ##STR22##.Iaddend.  wherein one of R' 1  and R' 2  is R 19  CO--NH-- and the other is hydrogen, in which R 19  CO-- is an amino acid residue or peptide residue, and R 3 , R 4 , R 5  and R 6  have the same 
     
     
        meanings as before.  .Iadd.29.  An assay method according to claim 15, wherein said hydrolase is glycosidase and the synthetic substrate is a compound of the formula ##STR23##.Iaddend. wherein one of R" 1  and R" 2  is (S) n  --O-- and the other is hydrogen, in which S is a monosaccharide unit and n is integer greater than 1, and R 3 , R 4 , R 5  and R 6  have the same meanings as before, and wherein the linkages between the monosaccharide units and between the monosaccharide unit and the phenyl ring can be 
     
     
        hydrolyzed by said glycosidase.  .Iadd.30.  An assay method according to claim 15, wherein said hydrolase is a phosphatase and the synthetic substrate is a compound of the formula ##STR24##.Iaddend. wherein one of R"' 1  and R"' 2  is ##STR25## and the other is hydrogen, in which R 20  is ##STR26## or a substituted or unsubstituted glycerol residue, and R 3 , R 4 , 
     
     
        R 5  and R 6  have the same meanings as before.  .Iadd.31.  An assay method according to claim 15, wherein said hydrolase is an esterase or lipase, and the synthetic substrate is a compound of the formula ##STR27##.Iaddend. wherein one of R"" 1  and R"" 2  is R 21  CO--O-- and the other is hydrogen, in which R 21  CO-- is a fatty acid residue, and R 3 , R 4 , R 5  and R 6  have the same meanings as before.