The Electrochemical Detection of Peptide Phosphorylation Using Adenosine 5’-[Ferrocene] Triphosphate (Fc-ATP) and Src Kinase

The phosphorylation of proteins by proteins kinases plays a central role in the regulation of cell proliferation, survival, and differentiation. Any alteration in the phosphorylation state of a cellular protein will induce changes in the activities of the protein kinases, which can cause many chronic diseases including cancer and diabetes. Therefore, controlling kinases activity show the most promise for the development of anticancer agents. To regulate the activity of the protein kinase, it is important to detect the phosphorylation event. Using an electrochemical approach to detect the kinase activity has been achieved by using an electroactive reporter like ferrocene (Fc). Ferrocene- y labelled ATP serves as a sensing tool for the protein kinase catalyzed phosphorylation o f peptides. The phosphorylation involves the transfer o f the Fc-y- phosphoryl group from Fc-ATP to the peptide. Here we report the use of this method with Src kinase. Src kinase (related to Sarcoma virus) is an enzyme which belongs to the transferase family. Using this method, the kinase activity of Src was motivated by electrochemical techniques, including cyclic voltammetiy (CV), square wave voltammetry (SWV), and differential pulse voltammetry (DPV). The target peptide EGIYDVP was immobilized on a gold surface and subsequently phosphorylated in the present of the kinase. In addition, inhibitors were tested to interact with the Src kinase, which will affect the kinase activity by blocking phosphorylation of substrate peptide. Here, the following inhibitors were tested 2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7- tetrahydro-lH-indol-2-yl)methylene]-lH-indole-5-sulfonamide (SU6656), 4-amino-5- (methylphenyl)-7-(t-butyl)pyrazolo- (3,4d) pyrimidine (PP1), 4-amino-5-(4- chlorophenyl)-7-(/-butyl)pyrazolo[3,4-d]-pyrimidine (PP2), jV-benzyl-1- miaminobenzotriazole (TBB), and l-phenyl-Li/-pyrazolo[3,4-£/]pyrimidin-4-amine (PP3). The kinase kinetics was extracted from the electrochemical measurements to determine the K„, and the Vmaxvalue