Fluorescent coumarin-labeled nucleotides to measure ADP release from actomyosin

Several coumarin-labeled nucleotides have been synthesized, based on 2 ' (3 ')-O-(2-aminoethyl)carbamoyl-ATP (edaATP). The fluorescent coumarins coupl ed with the free amino group are 7-diethylaminocoumarin-3-carboxylic acid ( to give deac-edaATP), coumarin 343 (but-edaATP) and 7-ethylamino-8-bromocou marin-3-carboxylic acid (mbc-edaATP). The carbamoyl linkage of these nucleo tide analogs undergoes interconversion between 2 '- and 3 ' -hydroxyl attac hment very slowly, so that the 2 '- and 3 ' -isomers were separated and sto red with minimal equilibration. 3 ' -Deac-edaADP had fluorescence excitatio n and emission maxima at 430 nm and 477 nm, with a fluorescence quantum yie ld of 0.012. The equivalent data for 3 ' -but-edaADP are 445 nm, 494 nm, an d 0.51, respectively, and for 3 ' -mbc-edaADP, 405 nm, 464 nm, and 0.62. Th e interaction with skeletal myosin subfragment 1 was measured in the absenc e and presence of actin. In each case the fluorescence was decreased when b ound to subfragment 1, 3-fold for 3 ' -deac-edaADP, 7-fold for 3 ' -but-eda ADP, and 11-fold for 3 ' -mbc-edaADP. Steady-state ATPase measurements and the kinetics of binding and release of nucleotides were similar to those re ported for the natural nucleotide. Large fluorescence changes could be obse rved for the release of these analogs from actomyosin subfragment 1, enabli ng a direct measurement of the kinetics of this process. In the case of 3 ' -deac-edaADP a rate constant of 474 s(-1) was measured (at pH 7.0, 20 degr eesC, and low ionic strength).