1. Developing a Fluorescamine Assay to Probe Cardiac Protein Structure Virginia Dines and Siddharth Damania 2006-2007

2. Thomas Biochemistry Lab University of Minnesota

3. Introduction Nearly 60 million Americans suffer from heart disease Cardiac muscles require calcium to flow through the membrane calcium pump to beat Used with Permission from Dr. Thomas

4. Phospholamban Oxenoid & Chou (2005), PNAS 102: 10870 – 10875 Robia, Flohr & Thomas (2005), Biochemistry 44: 4302 – 4311

5. Phospholamban

7. Spin Label

9. Phospholamban

12. Fluorescamine

15. Background Study by Udenfriend et al. (1972) showed that when fluorescamine is reacted with primary amines (in lysine and PLB), the fluorescent intensity of the fluorophor increases linearly

16. Goals Develop a fluorescamine assay

17. Goals Develop a fluorescamine assay Optimize the fluorescamine assay

18. Goals Develop a fluorescamine assay Optimize the fluorescamine assay Use the assay to assess the spin- labeling success in phospholamban

19. Fluorescamine Assay Measure the fluorescent intensity of fluorescamine bound to lysine, PLB, and labeled PLB in increasing concentrations to ultimately determine spin-labeling success rate

25. Fluorescamine Assay with Lysine

26. Fluorescamine Assay of PLB and Lysine

27. Fluorescamine Assay with Arginine

28. Spin Label Controls: DMF

29. Spin Label Controls: SUCSL in DMF

30. Fluorescamine Assay with Spin Labeled PLB

31. Subtracting Spin Label Control Shows 33% Labeled PLB

32. Fluorescamine Time Control

33. Conclusions A lysine standard curve cannot be used to quantify PLB because of structural differences

34. Conclusions A lysine standard curve cannot be used to quantify PLB because of structural differences The spin label binds to fluorescamine and forms a fluorescent compound

35. Conclusions A lysine standard curve cannot be used to quantify PLB because of structural differences The spin label binds to fluorescamine and forms a fluorescent compound Fluorescamine solution increases in fluorescence over time

36. Future Work Control the increasing fluorescence using a 96-well microplate reader http://openwetware.org/images/c/c7/Macintosh_HD-Users-nkuldell-Desktop-RTPCRplate.png

37. Future Work Apply fluorescamine assay to determine efficiency of spin-labeling PLB

38. Future Work Determine structure of pentameric phospholamban and further investigate how PLB interacts with the calcium pump

39. Acknowledgements Dr. David D. Thomas Kurt Torgersen The University of Minnesota Ms. Lois Fruen Team Research

40. Developing a Fluorescamine Assay to Probe Cardiac Protein Structure Virginia Dines and Siddharth Damania 2006-2007