Non-radioactive enzyme assay

• US 5,580,747 A
• Filed: 01/21/1994
• Issued: 12/03/1996
• Est. Priority Date: 11/12/1991
• Status: Expired due to Fees

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Shultz, John W., White, Douglas H.
• Primary Examiner(s): Kight, John
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US08/185,448
• Time in Patent Office: 1,047 Days
• Field of Search: 435/24, 435/18, 435/4, 435/23, 435/21, 435/15, 435/16, 435/17, 435/810, 435/975, 436/2, 436/12, 436/13, 436/14, 436/15, 436/16, 436/17, 436/63, 436/74, 436/172, 436/800, 530/300, 530/328
• US Class Current: 435/24
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/103332 Bilirubin or uric acid standard or control
• CPC Class Codes: Y10T 436/104165 Lipid, cholesterol, or triglyceride standard or control
• CPC Class Codes: Y10T 436/104998 Glucose, ketone, nitrate standard or control
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]
• CPC Class Codes: Y10T 436/106664 Blood serum or blood plasma standard or control
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]

Abstract

The present invention is directed to the assay and purification of proteins, and particularly to the non-radioactive assay and purification of protein kinases, phosphatases and protease by incubating the enzyme with a substrate modified peptide to form a product modified peptide under conditions where the enzyme is active. The product modified peptide and substrate modified peptide are then separated, and the product modified peptide is measured. The present invention is also directed to kits and bioreagents for performing the assays.

Claims

1. A non-radioactive method of quantifying the activity of an enzyme on a peptide substrate, comprising:

a. adding a sufficient quantity of a substrate modified peptide to the enzyme to produce a product modified peptide wherein the substrate modified peptide includes a detector segment capable of being detected by non-radioactive means for detection;

b. incubating the enzyme with the substrate modified peptide under conditions where the enzyme is active for a sufficient time to form a product modified peptide;

c. separating the product modified peptide from the substrate modified peptide by agarose gel electrophoresis; and

d. measuring the quantity of the product modified peptide by non-radioactive means for detection responsive to the presence of the detector segment.

2. The method of claim 1 wherein the substrate modified peptide is a pure substrate.

3. The method of claim 1 wherein the substrate modified peptide is a crude substrate.

4. The method of claim 1 wherein the substrate modified peptide includes a detector segment selected from the group consisting of dansyl, sulforhodamine, lissamine rhodamine (rhodamine B) and fluorescein.

5. The method of claim 1 wherein the substrate modified peptide is selected from the group consisting of the following peptides:

Promega Peptide 1 (SEQ ID NO;

1), Promega Peptide 2 (SEQ ID NO;

2), Promega Peptide 3 (SEQ ID NO;

3), Promega Peptide 4 (SEQ ID NO;

4), Promega Peptide 5 (SEQ ID NO;

5), Promega Peptide 6 (SEQ ID NO;

6), Promega Peptide 7 (SEQ ID NO;

7), Promega Peptide 8 (SEQ ID NO;

8), Promega Peptide 9 (SEQ ID NO;

9), Promega Peptide 10 (SEQ ID NO;

10), Promega Peptide 11 (SEQ ID NO;

11) and analogs thereof.

6. The method of claim 1 wherein the enzyme is selected from the group consisting of kinases, phosphatases and proteases.

7. The method of claim 1 wherein the enzyme is a kinase.

8. The method of claim 7 wherein the substrate modified peptide is selected from the group consisting of Promega Peptide 1 (SEQ ID NO:

1), Promega Peptide 2 (SEQ ID NO;

2), Promega Peptide 3 (SEQ ID NO;

3), Promega Peptide 4 (SEQ ID NO;

4), Promega Peptide 5 (SEQ ID NO;

5), Promega Peptide 6 (SEQ ID NO;

6), Promega Peptide 7 (SEQ ID NO;

7), Promega Peptide 8 (SEQ ID NO;

8), Promega Peptide 11 (SEQ ID NO;

11) and analogs thereof.

9. The method of claim 7 wherein the kinase is protein kinase C.

10. The method of claim 9 wherein the substrate modified peptide is selected from the group consisting of Promega Peptide 4 (SEQ ID NO:

4), Promega Peptide 5 (SEQ ID NO;

5), Promega Peptide 6 (SEQ ID NO;

6), Promega Peptide 11 (SEQ ID NO;

11) and analogs thereof.

11. The method of claim 7 wherein the enzyme is cAMP-dependent protein kinase.

12. The method of claim 11 wherein the substrate modified peptide is Promega Peptide 1 (SEQ ID NO:

1), Promega Peptide 2 (SEQ ID NO;

2), Promega Peptide 3 (SEQ ID NO;

3), Promega Peptide 7 (SEQ ID NO;

7) and analogs thereof.

13. The method of claim 7 wherein the enzyme is tyrosine kinase.

14. The method of claim 13 wherein the substrate modified peptide is Promega Peptide 8 (SEQ ID NO:

8) and analogs thereof.

15. The method of claim 1 wherein the enzyme is a phosphatase.

16. The method of claim 15 wherein the substrate modified peptide is selected from the group consisting of Promega Peptide 1 (SEQ ID NO:

1), Promega Peptide 2 (SEQ ID NO;

2), Promega Peptide 3 (SEQ ID NO;

3), Promega Peptide 4 (SEQ ID NO;

4), Promega Peptide 5 (SEQ ID NO;

5), Promega Peptide 6 (SEQ ID NO;

6), Promega Peptide 7 (SEQ ID NO;

7), Promega Peptide 8 (SEQ ID NO;

8), [Promega Peptide 9 (SEQ ID NO;

9), Promega Peptide 10 (SEQ ID NO;

10),] Promega Peptide 11 (SEQ ID NO;

11) and analogs thereof, wherein the substrate modified peptide is derived from the products of the reaction of a protein kinase with the substrate modified peptide.

17. The method of claim 15 wherein the enzyme is alkaline phosphatase.

18. The method of claim 1 wherein the enzyme is a protease.

19. The method of claim 18 wherein the substrate modified peptide is selected from the group consisting of Promega Peptide 1 (SEQ ID NO:

1), Promega Peptide 2 (SEQ ID NO;

2), Promega Peptide 3 (SEQ ID NO;

3), Promega Peptide 4 (SEQ ID NO;

4), Promega Peptide 5 (SEQ ID NO;

5), Promega Peptide 6 (SEQ ID NO;

6), Promega Peptide 7 (SEQ ID NO;

7), Promega Peptide 8 (SEQ ID NO;

8), Promega Peptide 9 (SEQ ID NO;

9), Promega Peptide 10 (SEQ ID NO;

10), Promega Peptide 11 (SEQ ID NO;

11) and analogs thereof.

20. The method of claim 18 wherein the enzyme is HIV protease.

21. The method of claim 20 wherein the substrate modified peptide is selected from the group consisting of Promega Peptide 9, (SEQ ID NO:

9) and Promega Peptide 10 (SEQ ID NO;

10) and analogs thereof.

22. The method of claim 1 wherein the product modified peptide is measured by fluorescence.

23. The method of claim 1 wherein the product modified peptide is measured by chemiluminescence.

24. A method for detecting an enzyme in a body fluid, comprising reacting the body fluid with a sufficient amount of a substrate modified peptide containing a detector segment to enable the enzyme to form a product modified peptide and under conditions where the enzyme is active for a time sufficient to form the product modified peptide in an amount such that the product modified peptide may be detected by non-radioactive means for detection responsive to the detector segment, separating the product modified peptide from the substrate modified peptide by agarose gel electrophoresis, and measuring the amount of product peptide formed using the non-radioactive means for detection.

25. The method of claim 24 wherein the fluid is blood or lymph fluid.

26. The method of claim 24 wherein the substrate modified peptide is selected from the group consisting of the following:

Promega Peptide 1 (SEQ ID NO;

1), Promega Peptide 2 (SEQ ID NO;

2), Promega Peptide 3 (SEQ ID NO;

3), Promega Peptide 4 (SEQ ID NO;

4), Promega Peptide 5 (SEQ ID NO;

5), Promega Peptide 6 (SEQ ID NO;

6), Promega Peptide 7 (SEQ ID NO;

7), Promega Peptide 8 (SEQ ID NO;

8), Promega Peptide 9 (SEQ ID NO;

9), Promega Peptide 10 (SEQ ID NO;

10), Promega Peptide 11 (SEQ ID NO;

11) and analogs thereof.