Reagent and process for the determination of the activity of the enzyme gamma-glutamyl transferase

Claim 2

Process for the kinetic determination of gamma-glutamyl transferase (gamma-GT) activity by the reaction of a gamma-glutamylarylamide substrate (gamma-GT substrate) with a gamma-glutamyl acceptor and gamma-GT comprising simultaneously combining the gamma-GT substrate with an oxidation agent, gamma-GT, an aromatic amine coupling component or a phenol coupling component, and a gamma-glutamyl acceptor said combination taking place at a pH of from about 6 to 10, said gamma-GT substrate forming an arylamine which reacts with said coupling component to give a colored compound with an absorption maximum above 565 nm, and photometrically monitoring the rate of formation of said colored compound as a measure of activity of the gamma-GT.

Claim 3

The process of claim 1 wherein the oxidation agent is a ferricyanide or a peroxide/peroxidase.

Claim 4

The process of claim 1 wherein the gamma-GT substrate is a compound of the formula ##STR3## wherein X is an --NR 2 R 3 or --O--R 2 group; R 1 is hydrogen; alkoxy, alkyl or aryl containing up to 8 carbon atoms; chlorine, bromine or iodine; or --NR 2 R 3 , --SO 3 R 2 or --COOR 2 ; and R 2 and R 3 , which are the same or different, are hydrogen; or substituted or unsubstituted alkyl or aryl containing up to 8 carbon atoms with substituents selected from the group consisting of OH, alkoxy, COOH and SO 3 H.

Claim 5

A process as claimed in claim 1 wherein the coupling component is a compound of the formula ##STR4## wherein X is --NR 2 R 3 or --O--R 3 ; R 1 is hydrogen, or chlorine when R 4 and/or R 6 are also chlorine; R 2 and R 3 are individually selected from the group consisting of hydrogen; or substituted or unsubstituted alkyl, aralkyl, or aryl containing up to 8 carbon atoms, with the substituent selected from the group consisting of hydroxyl, alkoxy containing up to 5 carbon atoms, --COOH or --SO 3 H; R 4 and R 6 are individually selected from the group consisting of hydrogen, alkyl containing up to 5 carbon atoms, chlorine, bromine, iodine, --COOR 2 , --SO 3 R 2 , OR 2 and NR 2 R 3 , R 2 and R 3 having the above-given meanings; and R 5 and R 7 are individually selected from the group consisting of hydrogen or alkyl containing up to 5 carbon atoms; or R 4 and R 5 or R 6 and R 7 , together with the benzene ring form a naphthyl or anthryl structure.

Claim 6

Process according to claim 1 wherein the coupling component is 2,3-xylenol, diethylmethanilic acid, N-ethyl-N-(3'-sulphobenzene)-aniline or N-methylanthranilic acid.

Claim 7

Process for the kinetic determination of gamma-glutamyl transferase (gamma-GT) activity by the reaction of a gamma-glutamylarylamide substrate (gamma-GT substrate) with a gamma-glutamyl acceptor and gamma-GT comprising simultaneously combining the gamma-GT substrate with an oxidation agent, gamma-GT, an aromatic amine coupling component or a phenol coupling component, and a gamma-glutamyl acceptor said combination taking place at a pH of from about 6 to 10, said gamma-GT substrate forming an arylamine which reacts with said coupling component to give a colored compound with an absorption maximum above 565 nm, and photometrically monitoring the rate of formation of said colored compound as a measure of activity of the gamma-GT, the gamma-GT substrate being a compound of the formula ##STR5## wherein X is --NR 2 R 3 or --O--R 2 ; R 1 is hydrogen; alkoxy, alkyl or aryl containing up to 8 carbon atoms; chlorine, bromine or iodine; or --NR 2 R 3 , --SO 3 R 2 or --COOR 2 ; and R 2 and R 3 , which are the same or different, are hydrogen; substituted or unsubstituted alkyl or aryl containing up to 8 carbon atoms with substitutents selected from the group consisting of OH, alkoxy, COOH and SO 3 H; and the coupling component being a compound of the formula ##STR6## wherein X is --NR 2 R 3 or --O--R 3 ; R 1 is hydrogen, or chlorine when R 4 and/or R 6 are also chlorine; R 2 and R 3 are individually selected form the group consisting of hydrogen; or substituted or unsubstituted alkyl, aralkyl, or aryl containing up to 8 carbon atoms, with the substituent selected from the group consisting of hydrogen, alkyl containing up to 5 carbon atoms, chlorine, bromine, iodine, --COOR 2 , --SO 3 R 2 , OR 2 and NR 2 R 3 , R 2 and R 3 having the above-given meanings; and R 5 and R 7 are individually selected from the group consisting of hydrogen or alkyl containing up to 5 carbon atoms; or R 4 and R 5 or R 6 and R 7 , together with the benzene ring form a naphthyl or anthryl structure.

Claim 8

Process for the kinetic determination of gamma-glutamyl transferase (gamma-GT) activity by the reaction of a gamma-glutamylarylamide substrate (gamma-GT substrate) with a gamma-glutamyl acceptor and gamma-GT comprising simultaneously combining the gamma-GT substrate with an oxidation agent, gamma-GT, an aromatic amine coupling component or a phenol coupling component, and a gamma-glutamyl acceptor said combination taking place at a pH of from about 6 to 10, said gamma-GT substrate forming an arylamine which reacts with said coupling component to give a colored compound with an absorption maximum above 565 nm, and photometrically monitoring the rate of formation of said colored compound as a measure of activity of the gamma-GT, the gamma-GT substrate being a compound of the formula ##STR5## wherein X is --NR 2 R 3 or --O--R 2 ; R 1 is hydrogen; alkoxy, alkyl or aryl containing up to 8 carbon atoms; chlorine, bromine or iodine; or --NR 2 R 3 , --SO 3 R 2 or --COOR 2 ; and R 2 and R 3 , which are the same or different, are hydrogen; substituted or unsubstituted alkyl or aryl containing up to 8 carbon atoms with substitutents selected from the group consisting of OH, alkoxy, COOH and SO 3 H; and the coupling component being a compound of the formula ##STR6## wherein X is --NR 2 R 3 or --O--R 3 ; R 1 is hydrogen, or chlorine when R 4 and/or R 6 are also chlorine; R 2 and R 3 are individually selected form the group consisting of hydrogen; or substituted or unsubstituted alkyl, aralkyl, or aryl containing up to 8 carbon atoms, with the substituent selected from the group consisting of hydrogen, alkyl containing up to 5 carbon atoms, chlorine, bromine, iodine, --COOR 2 , --SO 3 R 2 , OR 2 and NR 2 R 3 , R 2 and R 3 having the above-given meanings; and R 5 and R 7 are individually selected from the group consisting of hydrogen or alkyl containing up to 5 carbon atoms; or R 4 and R 5 or R 6 and R 7 , together with the benzene ring form a naphthyl or anthryl structure.

Claim 9

The process of claim 6 wherein the gamma-GT substrate is a gamma-glutamyl derivative of aniline, naphthylamine or 2-hydroxy-4-amino-benzoic acid.

Claim 10

The process of claim 1 wherein the gamma-GT substrate is gamma-glutamyl-p-phenylenediamine-3-carboxylic acid.

Claim 11

The process of claim 8 wherein the coupling component is 2,3-xylenol, diethylmethanilic acid, N-ethyl-N-(3'-sulphobenzene)-aniline or N-methylanthranilic acid.

Claim 12

The process of claim 6 wherein the coupling component is 2,3-xylenol, diethylmethanilic acid, N-ethyl-N-(3'-sulphobenzene)-aniline or N-methylanthranilic acid.