METHODS OF INVESTIGATION OF THE STRUCTURE AND ACTION MECHANISMS OF ACTIVE SITES OFENZYMES - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 13

experiment, and measuring the rate of increase of emitted fluorescence, it has been
possible to monitor the process of the sensitised cis-trans photoisomerisation (Mekler
and Likhtenshtein, 1986). The cis -stilbene concentration, which is proportional to
fluorescence intensity, approaches the photostationary level exponentially with the rate
constant

where and the rate constants for the triplet-triplet energy transfer from a
sensitiser to trans and cis -stilbenes respectively, and the fractions of the trans and
cis -stilbene molecules respectively that undergo the photoisomerisation after encounters
with the triplet sensitiser and and the sensitiser's triplet lifetime and
phosphorescence quantum yield respectively. Eq. (1.6) permits the calculation of the
experimental rate constant with the use of regular fluorescence
technique if all other constants from this equation are measured independently or
calibrated in a model system with these known values.
Due to the relatively long lifetime of the sensitiser triplet state and the possibility of
integrating data on the stilbene photoisomerisation, the apparent characteristic time of
the method can reach hundreds of seconds. This unique property of the cascade system
and, therefor triplet-photochrome technique, allows the investigation of slow diffusion
processes, including encounters of proteins in membranes using very low concentrations
of both the triplet and photochrome probes.
An additional step in the cascade reaction scheme is the quenching of the sensitizer
triplet state with relatively low-concentration radicals (Fig. 1.5) (Papper et al., 1999,
2000; Papper and Likhtenshtein, 2001). The entire investigated reaction that is shown in
Fig. 1.5 is the sequence of the four kinetic processes and serves as a basis for the spin-
triplet-photochrome labeling technique. This technique combines the three types of
biophysical probes: stilbene photochrome probe, triplet probe and stable nitroxide-
radical spin probe, which depresses the sensitiser exited triplet state.
Solving the kinetics equation based on the total cascade reaction with the consequent
quenching by radicals, and taking into account the steady-state approximations, one can
calculate a product of the quenching rate constant kq and the sensitizer excited triplet
state life time and the kq value if is known. The quenching radical concentration
in the vicinity of the probes can be determined using appropriate calibration.
Eventually, this method allows quantitative measuring of the translational diffusion
of proteins modified with these three labels in solution and in biomembranes. The
minimal approximate volume of a sample available for the fluorescence measurement
(using a regular commercial spectrofluorimeter) in this method is about when the

total concentration of fluorophores is close to 0.01 and the local concentration of

radicals is about 10

Experimental data
The fluorescence-photochrome technique was first applied to studying molecular
dynamics of a stilbene fluorescence-photochrome molecule, SITC, attached covalently