Composition and method for determining transferase and protease activity

Claim 2

A reagent for use in a fluorometric determination of transferase and protease activity, said reagent comprises a substrate selected from the group consisting of ##STR4## wherein each of R 1 and R 2 is --OH, --NH 2 , NHCH 3 , --NHC 2 H 5 , --N(CH 3 ) 2 , --N(C 2 H 5 ) 2 , --N((CH 3 ) (C 2 H 5 ), --OCH 3 , or O(CH 2 ) n CH 3 , n is an integer of 1 through 4, and wherein R 3 is an amino acid moiety capable of being cleaved from the remainder of said substrate in the presence of a transferase or protease having activity specific to that substrate and a buffer which maintains pH at or near the optimum pH of the transferase or protease.

Claim 3

The reagent of claim 1 wherein R 3 is an amino acid moiety transferable to glycylglycine when said substrate is reacted with glycylglycine in the presence of a transferase having activity specific to said substrate.

Claim 4

The reagent of claim 2 in which each of R 1 and R 2 is --OCH 3 , said substrate being useful in the fluorometric determination of γ-glutamyl transpeptidase.

Claim 5

A reagent suitable for use in a fluorometric determination of transferase activity of γ-glutamyl transpeptidase comprising a γ-glutamyl derivative of dimethyl-5-amino isophalate, or salts thereof; an acceptor of the glutamyl moiety when said derivative is cleaved in the presence of γ-glutamyl transpeptidase; and a buffer for maintaining a pH within the range of 7.5 to 9.0.

Claim 6

A reagent suitable for use in a fluorometric determination of transferase activity of γ-glutamyl transpeptidase comprising a γ-glutamyl derivative of dimethyl-5-amino isophalate, or salts thereof; an acceptor of the glutamyl moiety when said derivative is cleaved in the presence of γ-glutamyl transpeptidase; and a buffer for maintaining a pH within the range of 7.5 to 9.0.

Claim 7

The reagent of claim 4 in which said acceptor is glycylglycine.

Claim 8

A fluorometric method for determining the activity of transferases and proteases in samples of biological fluids, comprising the steps of mixing and reacting a substrate and a sample of body fluid containing a transferase or protease capable of cleaving said substrate; then exposing the mixture to ultraviolet light having a wavelength within the range of 320 to 380 nm; and measuring the rate of change in fluorescence at a wavelength within the range of 420 to 480 nm; said substrate being selected from the group consisting of ##STR5## wherein each of R 1 and R 2 is --OH, --NH 2 , NHCH 3 , --NHC 2 H 5 , --N(CH 3 ) 2 , --N(C 2 H 5 ) 2 , --N(CH 3 ) (C 2 H 5 ), --OCH 3 , or O(CH 2 ) n CH 3 , n is an integer of 1 through 4, and wherein R 3 is an amino acid moiety capable of being cleaved from the remainder of said substrate in the presence of a transferase having activity specific to that substrate.

Claim 9

A fluorometric method for determining the activity of transferases and proteases in samples of biological fluids, comprising the steps of mixing and reacting a substrate and a sample of body fluid containing a transferase or protease capable of cleaving said substrate; then exposing the mixture to ultraviolet light having a wavelength within the range of 320 to 380 nm; and measuring the rate of change in fluorescence at a wavelength within the range of 420 to 480 nm; said substrate being selected from the group consisting of ##STR5## wherein each of R 1 and R 2 is --OH, --NH 2 , NHCH 3 , --NHC 2 H 5 , --N(CH 3 ) 2 , --N(C 2 H 5 ) 2 , --N(CH 3 ) (C 2 H 5 ), --OCH 3 , or O(CH 2 ) n CH 3 , n is an integer of 1 through 4, and wherein R 3 is an amino acid moiety capable of being cleaved from the remainder of said substrate in the presence of a transferase having activity specific to that substrate.

Claim 10

The method of claim 6 in which the reaction mixture is exposed to ultraviolet light having a wavelength of approximately 365 nm.

Claim 11

The method of claim 6 in which the change in fluorescence is measured at a wavelength of about 465 nm.

Claim 12

The method of claim 6 in which said mixing step includes mixing glycylglycine with said substrate and sample; R 3 being an amino acid moiety transferable to glycylglycine during said mixing step.

Claim 13

The method of claim 9 in which R 3 is γ-glutamyl and said transferase is γ-glutamyl transpeptidase.

Claim 14

A fluorometric method for determining the activity of γ-glutamyl transpeptidase in a sample of biological fluid, comprising the steps of mixing and reacting a γ-glutamyl derivative of 5-aminoisophthalic acid, dimethyl-5-aminoisophtholate, or salts thereof, with a γ-glutamyl acceptor, a buffer for maintaining a pH within the range of 7.5 to 9.0, and a sample of biological fluid containing γ-glutamyl transpeptidase; then exposing the mixture to ultraviolet light having a wavelength within the range of 320 to 380 nm; and measuring the rate of change in fluorescence at a wavelength within the range of 420 to 480 nm.

Claim 15

A fluorometric method for determining the activity of γ-glutamyl transpeptidase in a sample of biological fluid, comprising the steps of mixing and reacting a γ-glutamyl derivative of 5-aminoisophthalic acid, dimethyl-5-aminoisophtholate, or salts thereof, with a γ-glutamyl acceptor, a buffer for maintaining a pH within the range of 7.5 to 9.0, and a sample of biological fluid containing γ-glutamyl transpeptidase; then exposing the mixture to ultraviolet light having a wavelength within the range of 320 to 380 nm; and measuring the rate of change in fluorescence at a wavelength within the range of 420 to 480 nm.

Claim 16

The method of claim 11 in which the reaction mixture is exposed to ultraviolet light having a wavelength of approximately 365 nm.

Claim 17

The method of claim 11 in which the change in fluorescence is measured at a wavelength of about 465 nm.

Claim 18

The method of claim 11 in which said acceptor is glycylglycine.