Developing a Fluorescamine Assay to Probe Cardiac Protein Structure Virginia Dines and Siddharth Damania

Thomas Biochemistry Lab University of Minnesota

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

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

Phospholamban

Spin Label

Phospholamban

Fluorescamine

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

Goals Develop a fluorescamine assay

Goals Develop a fluorescamine assay Optimize the fluorescamine assay

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

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

Fluorescamine Assay with Lysine

Fluorescamine Assay of PLB and Lysine

Fluorescamine Assay with Arginine

Spin Label Controls: DMF

Spin Label Controls: SUCSL in DMF

Fluorescamine Assay with Spin Labeled PLB

Subtracting Spin Label Control Shows 33% Labeled PLB

Fluorescamine Time Control

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

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

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

Future Work Control the increasing fluorescence using a 96-well microplate reader

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

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

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

Developing a Fluorescamine Assay to Probe Cardiac Protein Structure Virginia Dines and Siddharth Damania