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Hydroperoxides. Although the hydroperoxides have been isolated as a mixture of isomers, complete characterization was not possible because of decomposition. Strong evidence in support of their structure comes from NMR analysis: 'H NMR, 8.15, 8.10, and 7.92 ppm (br s) consistent with hydroperoxidic protons; 6.76, 6.30, 5.82, 5.50, and 5.32 ppm (presumably vinylic protons with very complex splitting patterns); 4.80 and 4.54 ppm (allylic protons a to oxygen); 1.73 and 1.26 ppm (alkyl protons, multiplicities could not be established because of overlapping peaks). 13C NMR, peaks are also consistent with the assigned structure; 138-126 ppm (substituted olefinic carbons), 119.7 and 119.4 ppm (terminal alkene carbons); 87-80 ppm (oxygen-substituted allylic carbons); 30-18 ppm (alkyl carbons). Appropriate vinyl-vinyl, vinyl-allyl, and vinyl-alkyl couplings appear in the 2-D COSY ( -) NMR at -20 °C.

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 .M 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 IAM ATP was found to be controlled by a process having a rate constant of 2.2 X log0H+J sec-1 at 220C at pH 5.8-9.5, which corresponds to 220 sec-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. Photodissociation of carbon monoxide and dioxygen from hemoglobin (1, 2) is an important tool for analysis of hemoglobin functions. It would be valuable if a similar approach could be developed to encompass biological reactions in general. In principle, it should be possible to generate photochemically reactants such as metabolites from biologically inert photolyzable compounds. This would allow us to study intact macromolecular systems in which limitations arise because of the difficulty of introducing reactants to their sites of reaction in a time shorter than the characteristic time of the reaction under investigation.First steps toward this goal of photochemical deprotection have been reviewed by Knowles (3). These include the generation of inorganic phosphate from 3-nitrophenyl phosphate by Havinga et al. (4) (5) and Engels and Schlaeger (6), who described the synthesis of photolabile amino acid, peptide, and cyclic AMP precursors and suggested possible applications of their methods. Alternatively, inhibitors can be photogenerated, as has been shown by Lester et al. (7) in their studies of ion-channel blockade mechanisms. These ideas were taken further by Kaplan et al. (8), who synthesized a y-phosphate ester of ATP, P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (I), which they called caged ATP. They showed that caged ATP was inert toward the sodium pump, could be incorporated into vesicles and, on continuous irradiation (on the time scale of seconds), formed ATP. In the work described here, the amount and rate of ATP generation after laser flash photolysis of caged ATP was measured to determine the potential of caged ATP as an ATP precursor for use in kinetic studies of intact biological systems. It is shown that the energy per pulse from a frequency doubled ruby laser generates ATP in sufficient concentration for biological studies. By using a well ...

SUMMARY1. A technique was developed to generate 2-8°C step temperature perturbations (T-jumps) in single muscle fibres to study the thermodynamics of muscle contraction. A solid-state pulsed holmium laser emitting at 2-065 usm heated the fibre and surrounding solution in approximately 150 /is. The signal from a 100 ,um thermocouple fed back to a heating wire maintained the elevated temperature after the laser pulse.2. Tension of glycerol-extracted muscle fibres from rabbit psoas muscle did not change significantly following T-jumps when the fibre was relaxed.3. In rigor, tension decreased abruptly on heating indicating normal (not rubberlike) thermoelasticity. The thermoelastic coefficient (negative ratio of relative length change to relative temperature change) of the fibre was estimated to be -0-021 at sarcomere lengths of 2-5-2-8 ,um. Rigor tension was constant after the temperature step and returned to the original value on recooling.4. In maximal Ca2+ activation, tension transients initiated by T-jumps had several phases. An immediate tension decrease suggests that thermoelasticity during contraction is similar to that in rigor. Active tension then recovered to the value before the T-jump with an apparent rate constant of approximately 400 s-1 (at 10-20°C). This rate constant did not have an appreciable dependence on the final temperature. Finally, tension increased exponentially to a new higher level with a rate constant of approximately 20 s-5 at 20 'C. This rate constant increased with temperature with a Q10 of 1-4.5. At submaximal Ca2+ activation the tension rise was followed by a decay to below the value before the T-jump. This decline was expected from the temperature dependence of steady pCa-tension curves. The final tension decline occurred on the 1-5 s time scale.6. The value and amplitude dependence of the rate constant for the quick recovery following T-jumps were similar to those of the quick recovery following length steps during active contractions. The enthalpy change associated with the quick tension

Photolabile precursors of ATP are needed to investigate mechanisms of muscle contraction and its regulation and ion conductance in sensory neurons within intact biological systems.