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Europium Tetracycline as a Luminescent Probe for Nucleoside Phosphates and Its Application to the Determination of Kinase Activity
Author(s) -
Schäferling Michael,
Wolfbeis Otto S.
Publication year - 2007
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.200601509
Subject(s) - chemistry , pyrophosphate , gtp' , nucleoside diphosphate kinase , adenylate kinase , europium , guanosine , nucleoside , enzyme , quenching (fluorescence) , kinase , adenosine triphosphate , adenosine monophosphate , biochemistry , phosphorylation , adenosine , fluorescence , organic chemistry , physics , quantum mechanics , ion
The determination of enzyme activities and the screening of enzyme regulators is a major task in clinical chemistry and drug development. A broad variety of enzymatic reactions is associated with the consumption of adenosine triphosphate (ATP), including, in particular, phosphorylation reactions catalyzed by kinases, formation of adenosine cyclic monophosphate (cAMP) by adenylate cyclases, and ATP decomposition by ATPase. We have studied the effect of a series of adenosine (ATP, ADP, AMP, cAMP) and guanosine (GTP, GDP) phosphoric esters, and of pyrophosphate (PP) on the fluorescence emission of the europium tetracycline (EuTC) complex. We found that these compounds have strongly different quenching effects on the luminescence emission of EuTC. The triphosphates ATP and GTP behave as strong quenchers in reducing the fluorescence intensity of EuTC to 25 % of its initial value by formation of a ternary 1:1:1 complex. All other phosphate esters showed a weak quenching effect only. The applicability of this fluorescent probe to the determination of the activity of phosphorylation enzymes is demonstrated by means of creatine kinase as a model for non‐membrane‐bound kinases. In contrast to other methods, this approach does not require the use of radioactively labeled ATP substrates, additional enzymes, or of rather complex immunoassays.