Synthesis and development of proteasome and kinase fluorescent assays for quantitation of enzyme activity

Enzyme activity is a major component of signal transduction. However, many current methods of enzyme detection, such as fluorophore-labeled antibodies or western blotting, quantitate enzyme concentration and lack the ability to determinate activity levels. This can lead to imprecise depictions of signal transduction pathways. For example, the cyclic-AMP dependent protein kinase (PKA) is ubiquitously expressed, but activity is highly regulated. Near the mitochondria, PKA activity is responsible for the activation of apoptotic pathways and as a consequence is highly regulated. For a complete understanding of apoptotic signal transduction, it is necessary to precisely define PKA activity. Gaining an increased understanding of enzyme activity is the goal of this thesis.;In pursuit of this goal, we have developed chemical tools for the quantitation of PKA activity, as well as the activities of the proteasome. An assay for PKA activity was developed termed "deep quench" using primarily electrostatic interactions between a positively charged fluorescently-labeled PKA substrate and a negatively charged non-fluorescent quenching dye. Upon phosphorylation of the fluorescently-labeled peptide substrate by PKA, interaction between the positively charged residues of the substrate and the negatively charged dye weaken, causing a lower affinity for quenching dye. Since the quenching dye has an absorbance near the emission of the PKA-substrate fluorophore, the energy transfer between the fluorophore and dye weakens as well, producing extraordinary fluorescent enhancements (up to 150-fold).;Using the "deep quench" system, suborganelle mitochondrial PKA activity was revealed as a relative matrix/intermembrane space/outer membrane (85:6:9) distribution of PKA in bovine heart mitochondria. While this sensor works beautifully in vitro, applying the system in an intracellular fashion has proven difficult due to the system's bimolecular nature. We have attempted to solve this problem by covalently linking the non-fluorescent dye carboxy Acid Blue 40 (cAB40) to the fluorescently labeled PKA substrate. However, due to the FRET mechanism of fluorescent quenching, no fluorescent increase was observed upon substrate phosphorylation. To solve this problem, the cAB40 and fluorescent PKA substrate were attached to 100 nm silica nanoparticles. Upon phosphorylation, a 2.2-fold fluorescent increase was observed. cAB40 was more completely characterized and found to quench fluorophores with a wide range of spectral properties. In further studies, Trypsin substrates were assembled, as well as a photolabile cassette, which furnished up to 110-fold enhancements in fluorescence. After demonstrating the utility of the cAB40 quenching dye, the technology was applied to biosensors for the Caspase-like (proteasome β1-subunit), Trypsin-like (proteasome β2-subunit), and Chymotrypsin-like (proteasome β5-subunit) activities of the proteasome. The specificity for the Caspase-like (proteasome β1-subunit) and Chymotrypsin-like (proteasome β5-subunit) activities of the proteasome was demonstrated. Overall this body of work demonstrates the utility of the "deep quench" PKA and proteasome sensors towards the goal of quantifying enzyme activity.