Surface-plasmon-resonance-based biosensor with immobilized bisubstrate analog inhibitor for the determination of affinities of ATP- and protein-competitive ligands of cAMP-dependent protein kinase

• Published in: Analytical biochemistry 2007-03, Vol.362 (2), p.268-277
• Main Authors: Viht, Kaido, Schweinsberg, Sonja, Lust, Marje, Vaasa, Angela, Raidaru, Gerda, Lavogina, Darja, Uri, Asko, Herberg, Friedrich W.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Binding, Competitive; Biosensing Techniques - methods; Bisubstrate inhibitor; cAMP-dependent protein kinase; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Inhibitors - metabolism; Enzymes, Immobilized - metabolism; Ligands; Models, Biological; Protein Binding; Proteins - metabolism; Solution competition assay; Substrate Specificity; Surface plasmon resonance; Surface Plasmon Resonance - methods
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2006.12.041

Interactions between adenosine–oligoarginine conjugates (ARC), bisubstrate analog inhibitors of protein kinases, and catalytic subunits of cAMP-dependent protein kinase (cAPK Cα) were characterized with surface-plasmon-resonance-based biosensors. ARC-704 bound to the immobilized kinase with subnanomolar affinity. The immobilization of ARC-704 to the chip surface via streptavidin–biotin complex yielded a high-affinity surface ( K D = 16 nM). The bisubstrate character of ARC-704 was demonstrated with various ligands targeted to ATP-binding pocket (ATP and inhibitors H89 and H1152P) and protein-substrate-binding domain of Cα (RIIα and GST–PKIα) in competition assays. The experiments performed on surfaces with different immobilization levels of ARC-704 produced similar results. The closeness of the obtained affinities of the tested compounds to the inhibitory potencies and affinities of the compounds measured with other methods demonstrates the applicability of the chip with the immobilized biligand inhibitor for the characterization of both ATP- and substrate protein-competitive ligands of basophilic protein kinases.