Claims
- 1. 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.sub.1 and R.sub.2 is --OH, --NH.sub.2, NHCH.sub.3, --NHC.sub.2 H.sub.5, --N(CH.sub.3).sub.2, --N(C.sub.2 H.sub.5).sub.2, --N((CH.sub.3) (C.sub.2 H.sub.5), --OCH.sub.3, or O(CH.sub.2).sub.n CH.sub.3, n is an integer of 1 through 4, and wherein R.sub.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.
- 2. The reagent of claim 1 wherein R.sub.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.
- 3. The reagent of claim 2 in which each of R.sub.1 and R.sub.2 is --OCH.sub.3, said substrate being useful in the fluorometric determination of .gamma.-glutamyl transpeptidase.
- 4. A reagent suitable for use in a fluorometric determination of transferase activity of .gamma.-glutamyl transpeptidase comprising a .gamma.-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 .gamma.-glutamyl transpeptidase; and a buffer for maintaining a pH within the range of 7.5 to 9.0.
- 5. The reagent of claim 4 in which said acceptor is glycylglycine.
- 6. 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.sub.1 and R.sub.2 is --OH, --NH.sub.2, NHCH.sub.3, --NHC.sub.2 H.sub.5, --N(CH.sub.3).sub.2, --N(C.sub.2 H.sub.5).sub.2, --N(CH.sub.3) (C.sub.2 H.sub.5), --OCH.sub.3, or O(CH.sub.2).sub.n CH.sub.3, n is an integer of 1 through 4, and wherein R.sub.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.
- 7. The method of claim 6 in which the reaction mixture is exposed to ultraviolet light having a wavelength of approximately 365 nm.
- 8. The method of claim 6 in which the change in fluorescence is measured at a wavelength of about 465 nm.
- 9. The method of claim 6 in which said mixing step includes mixing glycylglycine with said substrate and sample; R.sub.3 being an amino acid moiety transferable to glycylglycine during said mixing step.
- 10. The method of claim 9 in which R.sub.3 is .gamma.-glutamyl and said transferase is .gamma.-glutamyl transpeptidase.
- 11. A fluorometric method for determining the activity of .gamma.-glutamyl transpeptidase in a sample of biological fluid, comprising the steps of mixing and reacting a .gamma.-glutamyl derivative of 5-aminoisophthalic acid, dimethyl-5-aminoisophtholate, or salts thereof, with a .gamma.-glutamyl acceptor, a buffer for maintaining a pH within the range of 7.5 to 9.0, and a sample of biological fluid containing .gamma.-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.
- 12. The method of claim 11 in which the reaction mixture is exposed to ultraviolet light having a wavelength of approximately 365 nm.
- 13. The method of claim 11 in which the change in fluorescence is measured at a wavelength of about 465 nm.
- 14. The method of claim 11 in which said acceptor is glycylglycine.
RELATED APPLICATION
This application is a continuation-in-part of co-pending application Ser. No. 709,720, filed July 29, 1976, now abandoned.
US Referenced Citations (7)
Non-Patent Literature Citations (2)
Entry |
Wildes et al, "Differences Between Excited States Produced Chemically and Photochemically. Ion Pairs of Excited States Derived From Luminol", J. Amer. Chem. Soc., vol. 95, No. 8, (1973) pp. 2610-2617. |
Bayley et al, "Coformational Properties of Pig-heart Cytoplasmic Aspartate Aminotransferase. Circular-Dichromism and Absorption-Steptroscopy Study of Dicarboxylic Binding", Chem. Abstracts. vol. 83, No. 15, p. 223, (1975) Abs. No. 128,197a. |
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
709720 |
Jul 1976 |
|