DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

148. Fluorescent-based Protein Kinase and Phosphatase Assays

Edward M. Davis and Wafaa Mahmoud

SymBiotech Incorporated, Wallingford, CT 06492

symbio@snet.net

With funding from a DOE SBIR Phase I grant, Number DE-FG02-99ER82901, SymBiotech is developing fluorometric-based assays for phosphorylated proteins and peptides to follow kinase and phosphatase activity. The assays employ a new reagent that eliminates the need to use radioisotopes or costly monoclonal antibodies and opens new opportunities for developing sensitive and safe diagnostic assays.

The fluorescence of pepsin (a monophosphorylated protein)was determined after treating it with different concentrations of the phospho-detecting reagent. Results showed that 3.6 nanomoles phosphoprotein yields a maximum fluorescence of about 200 fluorescent units when treated with 1 mg/mL of reagent.

To follow CAM kinase activity, an HPLC-based assay was developed as the gold standard. A fluorescent-based kinase assay is now under development. Preliminary results show that the fluorescent reagent reacts with the CAM kinase-specific peptide substrate (Autocamtide-2, Alexis Biochemicals). Work now focuses on following CAM kinase activity by monitoring the degree of peptide phosphorylation using the fluorescent-based assay.