METHODS OF INVESTIGATION OF THE STRUCTURE AND ACTION MECHANISMS OF ACTIVE SITES OFENZYMES - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 7

can also be independently derived from the analysis of the temperature (T)
dependencies of the relaxation shift using the following equation:

where (T) is the relaxation shift in the steady-state fluorescence spectra and is
the fluorescence life time.Gradual increase of temperature results in the gradual
decrease of the The experimental (T)- T dependence can be used for the
estimation of in each temperature if is known. In real systems (viscose liquids,
polymers, proteins, membranes, etc.) there is, as a rule, a set of values, relaxation
energy and entropy activation, and other parameters. Analysis of relaxation shifts in such
systems requires special approaches. For instance, if one assumes a Gaussian distribution
over the free activation energies of the reorientation of surrounding particles it is
possible to find an expression to relate the energy activation of relaxation in the
distribution maximum

to the second moment of the distribution curve

where (T) is the experimental value of apparent energy activation derived from the
Arrhenius plot, log (T) -1/T. Eq. 1.4 allows the estimation of and
plotting (T) versus 1/T.
Nano- and picosecond time resolved fluorescence technique is used for monitoring
the dynamic Stokes shift of a tryptophane, Trp31, in cytidine monophosphate kinase
from E. coli in a water-glycerol mixture at temperatures, ranging from 293 to 230 K
(Vincent at al., 2000). This residue is located at the opposite site of the nucleotide-
binding sites, in a partially hydrophobic region, but not very far from the protein surface.
It is shown that the emission maximum of the fluorophore fluorescence is shifted to 320
nm by decreasing the temperature to 230-240 K with the time constant about 100 ps. In
the temperature range 293-232 K, the relaxation shift kinetics exhibit multiphase
behavior with time constants ranging from 100 ps to several nanoseconds with a total
amplitude between 130 and
The dynamics of the fluorescence relaxation shift of the dual fluorescence-nitroxide
probe

incorporated into the hydrophobic binding site of human serum albumin (HAS) was
monitored indirectly ( Rubtsova et. al., 1993, Fogel et al., 1994; Likhtenshtein et al.,