A Dithio-Coupled Kinase and ATPase Assay

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

^* To whom correspondence should be addressed. E-mail: Daniel.Sem{at}mu.edu.

	Abstract

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 ATPS (sulfur on the -phosphorous) as a substrate in place of adenosine triphosphate (ATP), the authors have developed a continuous assay using this substrate, with detection of the ADPS product using dithio reagents. Such an assay is possible because dithio groups react selectively with ADPS and not with ATPS. 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 ATPS, 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

First published on August 30, 2006, doi:10.1177/1087057106292142

Journal of Biomolecular Screening 2006;11:844.

A more recent version of this article appeared on October 1, 2006


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