A new approach to time-resolved studies of ATP-requiring biological systems; laser flash photolysis of caged ATP.

Abstract

2-Nitrobenzyl derivatives have been used for several years as photolabile protecting groups in synthetic organic chemistry. Recently, P3-1-(2-nitro phenylethyladenosine 5'-triphosphate "caged ATP" was synthesized and its photolysis was shown to generate ATP in situ. This and related reactions have great potential for structural and kinetic studies of both intact and soluble biological systems and it is thus important to define the kinetic characteristics of the photolytic reaction. Caged ATP (2.5 mM) was photolyzed at 347 nm by a single 30-nsec pulse from a frequency-doubled ruby laser of 25 mJ energy to generate 500 microM ATP. The kinetics of the overall reaction were determined by monitoring the kinetics of ATP-induced dissociation of actomyosin, a reaction of known kinetic characteristics. Release of 500 microM ATP was found to be controlled by a process having a rate constant of 2.2 X 10(9) [H+] sec-1 at 22 degrees C at pH 5.8-9.5, which corresponds to 220 sec-1 at pH 7. This process is believed to be the breakdown of an aci-nitro compound, which was identified on the basis of its spectral properties and the photochromicity of related 2-nitrobenzyl compounds.