Determination of metal ions in solution by photoluminescence anisotropy

Abstract

Homogeneous fluorescence polarization (anisotropy) assays for detecting and quantifying metal ions in solution, based the metal-dependent binding of a fluorescent ligand to an unlabeled macromolecule, or the binding of a metal ion to a fluorescent labeled macromolecule. The metal-dependent binding of a fluorescent ligand to an unlabeled macromolecule (metallo-macromolecule) effects a measurable change in anisotropy as will the binding of metal ions to a fluorescent labeled macromolecule. Binding of the fluorescent ligand to the unlabeled macromolecule is metal dependent with the change in anisotropy being proportional to the concentration of bound metal ions. No binding of the fluorescent ligand to the macromolecule occurs in the absence of metal ions. Conversely, if the fluorescent label is first conjugated to a metallo-macromolecule and the metallo-macromolecule is subsequently stripped of its metal ion, it may then be used to transduce the binding of metal ions. Transduction is provided wherein the covalently bound fluorescent label exhibits changes in anisotropy proportional to the concentration of bound metal ions. In all methods, the change in anisotropy may be simply related to the metal ion concentration of the test solution.

What is claimed is:  
     
         1 . A composition comprising an apo-carbonic anhydrase protein and a photoluminescent molecule selected from the group consisting of dansyl aziridine, 4-chloro-7-sulfobenzofurazan, 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide and 4-nitrobenzoxadiazol-7-chloride, or a composition comprising the reaction product of an apo-carbonic anhydrase protein and a photoluminescent molecule selected from the group consisting of dansyl aziridine, 4-chloro-7-sulfobenzofurazan, 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide and 4-nitrobenzoxadiazol-7-chloride.  
     
     
         2 . A composition comprising an apo-carbonic anhydrase protein and a photoluminsecent molecule selected from the group consisting of 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide: β-mercaptoethanol adduct, dansylamide, hydroxynaphthalenesulphonamide, 2-(3-methoxy-4-ethoxyphenyl)-4-chloroquinoline-6-sulfonamide, N-(1-anthracenyl)-4-sulfonamido-benzenesulfonamide, ethyl-2-(4-sulfonamidophenyl)-4-hydroxyquinoline-6-carboxylate and N-(N′-(4′-sulfamoylglutaranily-amidoethyl))-4-amino-3,6-disulfo-1,8-naphthalimide.  
     
     
         3 . The composition of  claim 1 , wherein the photoluminescent molecule is conjugated to the apo-carbonic anhydrase protein.  
     
     
         4 . The composition of  claim 1 , wherein the apo-carbonic anhydrase protein is a human apo-carbonic anhydrase.  
     
     
         5 . The composition of  claim 2 , wherein the apo-carbonic anhydrase protein is a human apo-carbonic anhydrase.  
     
     
         6 . The composition of  claim 1 , wherein the apo-carbonic anhydrase protein is a human carbonic anhydrase II isozyme or a variant thereof having a cysteine replacement of one amino acid.  
     
     
         7 . The composition of  claim 1 , wherein the apo-carbonic anhydrase is one selected from the group consisting of carbonic anhydrase II (L198C), carbonic anhydrase II (V143C), carbonic anhydrase II (H64C).  
     
     
         8 . The composition of  claim 5 , wherein the photoluminescent molecule is conjugated to the apo-carbonic anhydrase through the cysteine replacement amino acid.  
     
     
         9 . The composition of  claim 6 , wherein the photoluminescent molecule is conjugated to the apo-carbonic anhydrase through the cysteine replacement amino acid.  
     
     
         10 . The composition of  claim 7 , wherein carbonic anhydrase II (V143C) is conjugated to dansyl aziridine.  
     
     
         11 . The composition of  claim 7 , wherein carbonic anhydrase II (L198C) is conjugated to 4-chloro-7-sulfobenzofurazan.  
     
     
         12 . The composition of  claim 7 , wherein carbonic anhydrase II (H64C) is conjugated to 7-flurobenz-2-oxa-1,3-diazole-4-sulfonamide.  
     
     
         13 . The composition of  claim 2 , wherein the photoluminescent molecule is 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide: β-mercaptoethanol adduct.  
     
     
         14 . A kit for assay of divalent metal ion concentration in a sample comprising: 
 i) an apo-carbonic anhydrase protein conjugated to a photoluminescent molecule selected from the group consisting of dansyl aziridine, 4-chloro-7-sulfobenzofuran, 7-flurorbenz-2-oxa-1,3-diazole-4-sulfonamide and nitrobenzoxadiazolyl;    ii) optionally a standard solution of at least one divalent metal ion;    iii) optionally a buffer for maintaining a concentration of free divalent metal ions in a solution; and    iv) optionally a chelating resin to prevent or remove unwanted metal contamination;    said items i), ii), iii) and iv) being packaged in a container that prevents unwanted contamination by divalent metal ions.    
     
     
         15 . The kit of  claim 13 , wherein the buffer for maintaining a concentration of free divalent metal ions is nitrilotriacetic acid.  
     
     
         16 . The kit of  claim 13 , wherein item ii) is included.  
     
     
         17 . The kit of  claim 13 , wherein item iii) is included.  
     
     
         18 . The kit of  claim 13 , wherein item iv) is included.  
     
     
         19 . The kit of  claim 13 , wherein items ii) and iii) are included.  
     
     
         20 . The kit of  claim 13 , wherein items ii) and iv) are included.  
     
     
         21 . The kit of  claim 13 , wherein items ii), iii) and iv) are included.  
     
     
         22 . The kit of  claim 13 , wherein items iii) and iv) are included.  
     
     
         23 . A kit for assay of divalent metal ion concentration in a sample comprising: 
 i) an apo-carbonic anhydrase protein;    ii) a photoluminsecent molecule selected from the group consisting of 4-aminosulfonyl[1-(4-N-(5-fluoresceinylthioureido)butyl]benzamide, 7-flurorbenz-2-oxa-1,3-diazole-4-sulfonamide: p-mercaptoethanol adduct, dansylamide, hydroxynaphthalenesulphonamide, 2- (3-methoxy-4-ethoxyphenyl)-4-chloroquinoline-6-sulfonamide, N-(1-anthracenyl)-4-sulfonamido-benzenesulfonamide, ethyl-2-(4-sulfonamidophenyl)-4-hydroxyquinoline-6-carboxylate and N-(N′-(4′-sulfamoylglutaranily-amidoethyl))-4-amino-3,6-disulfo-1,8-naphthalimide    iii) optionally a standard solution of a divalent metal ion;    iv) optionally a buffer for maintaining a concentration of free divalent metal ion in a solution; and    v) optionally a chelating resin to prevent or remove unwanted metal contamination;    said items i), ii), iii), iv) and v) being packaged in a container that prevents unwanted contamination by divalent metal ions.    
     
     
         24 . The kit of  claim 22 , wherein the buffer for maintaining a concentration of free divalent metal ion is nitrilotriacetic acid or MOPS.