Labeling of proteins with the fluorophore 7-amino-4-methylcoumarin (AMC) generated novel proteolytic substrates

1. A method for measuring degradation of intact proteins, the method comprising:

a) reductively attaching 7-amino-4-methylcoumarin (AMC) to a protein substrate with a reducing agent, the reducing agent being sodium cyanoborohydride (NaCNBH 3 ) to form an AMC attached protein;

b) contacting in a test solution the AMC attached protein substrate with a proteolytic enzyme that degrade the protein substrate into protein fragments; and

c) measuring fluorescence during degradation of the AMC attached protein substrate.

2. The method of claim 1 wherein the fluorescence measured in step c) is compared to a standard curve of known concentration of free AMC to quantify moles of AMC released into solution.

3. The method of claim 2 wherein the known concentration of free AMC is between 5 nM and 5 mM.

4. The method of claim 1 wherein the protein substrate is a purified proteolytic enzyme.

5. The method of claim 1 wherein the protein substrate is obtained from cell lysates or cell extracts.

6. The method of claim 1 wherein the protein substrate has a molecular weight from about 10 kDa (kilodalton) of about 300 kDa.

7. The method of claim 1 wherein the protein substrate is selected from the group consisting of bovine serum albumin (BSA), catalase, hemoglobin, and superoxide dismutase.

8. The method of claim 1 wherein the test solution has a pH from about 2 to about 11.

9. The method of claim 4 wherein the proteolytic enzyme is present in an amount from about 320 nM to about 1 mM of the test solution.

10. The method of claim 1 wherein the protein substrate is present in the test solution in an amount from about 25 ng per milliliter to about 0.5 μg per milliliter.

11. The method of claim 1 wherein greater than 50 percent of the protein fragments are smaller than 500 Da.

12. The method of claim 4 wherein the proteolytic enzyme is selected from the group consisting of pepsin, proteinase K, trypsin, and chymotrypsin.

13. The method of claim 1 wherein the fluorescence is initiated by excitation with light having a wavelength from 360 to 420 nanometers.

14. The method of claim 13 wherein the fluorescence is measured at a wavelength from about 430 to about 450 nanometers.

15. The method of claim 14 wherein the fluorescence is measured at a wavelength of about 444 nanometers.

16. The method of claim 12 wherein the fluorescence is measure at a predetermined time interval.

17. A method for measuring degradation of intact proteins, the method comprising:

a) reductively attaching coumarin derivative to a protein substrate with a reducing agent, the reducing agent being sodium cyanoborohydride (NaCNBH 3 ) the coumarin derivative having the following formula:

wherein:

R 1 , R 2 are each independently hydrogen, C 1-10 alkyl, C 1-10 alkoxy, C 3-10 amide, C 4-10 diamide, C 3-10 ester, C 4-10 diester, C 6-10 aryl, or C 6-10 heteroaryl;

R 3 , R 4 are each independently hydrogen, C 1-10 alkyl, C 1-10 alkoxy, C 2-10 carboxy, C 3-10 amide, C 4-10 diamide, C 3-10 ester, C 4-10 diester, C 6-10 aryl, or C 6-10 heteroaryl; and

R 5 , R 6 are each independently hydrogen or C 1-5 alkyl;

b) contacting in a test solution the protein substrate with a proteolytic enzyme that degrade the protein substrate into protein fragments; and

c) measuring fluorescence during degradation of the protein substrate.

18. The method of claim 17 wherein R 5 , R 6 are each independently hydrogen.

19. The method of claim 18 wherein R 1 is C 1-10 alkyl, C 1-10 alkoxy, or C 1-10 perfluoroalkyl and R 2 , R 3 , R 4 are hydrogen.