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A Dithio-Coupled Kinase and ATPase Assay

Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI

Phani Kumar Pullela

Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI

Daniel S. Sem

Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI

Kinases and ATPases produce adenosine diphosphate (ADP) as a common product, so an assay that detects ADP would provide a universal means for activity-based screening of enzymes in these families. Because it is known that most kinases accept ATPßS (sulfur on the ß-phosphorous) as a substrate in place of adenosine triphosphate (ATP), the authors have developed a continuous assay using this substrate, with detection of the ADPßS product using dithio reagents. Such an assay is possible because dithio groups react selectively with ADPßS and not with ATPßS. Thiol detection was done using both Ellman’s reagent (DTNB) and a recently developed fluorescent dithio reagent, DSSA. Therefore, the assay can be run in both absorbance and fluorescence detection modes. The assay was used to perform steady-state kinetic analyses of both hexokinase and myosin ATPase. It was also used to demonstrate the diastereoselectivity of hexokinase (R) and myosin ATPase (S) for the isomers of ATPßS, consistent with previous results. When run in fluorescence mode using a plate reader, an average Z' value of 0.54 was obtained, suggesting the assay is appropriate for high-throughput screening.

Key Words: kinase • dithio • high throughput • continuous assay • chemical proteomics • DSSA

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				T. Chiku, P. K. Pullela, and D. S. Sem Thiol reactive dyes as probes for kinase assays FASEB J, April 1, 2007; 21(5): A263 - A263.