METHODS OF INVESTIGATION OF THE STRUCTURE AND ACTION MECHANISMS OF ACTIVE SITES OFENZYMES - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 14

to the terminal amino group of sperm-whale myoglobin (Likhtenshtein et. al., 1993). The
same myoglobin residue was also labeled with a spin label, 4-iodoacetamide-TEMPO.
Kinetics of the stilbene trans-cis photoisomerisation and the rotational diffusion
frequency of nitroxide radicals were monitored by fluorescence and ESR techniques,
respectively. These data on the probes in a bound state were compared with data
obtained in 60%-ethylene glycol/water solution. The values of and for labels
bound to myoglobin were found several times less than those values for the free labels
indicating that microviscosity in the vicinity of the labels attached to myoglobin is
higher than that in the bulk solution.
The triplet-photochrome labeling method has been used to study very rare encounters
in a system containing the Erythrosin B sensitiser and SITC photochrome probe (Mekler
and Likhtenshtein, 1986). Both types of the molecules were covalently bound to
chymotrypsin. The photoisomerisation kinetics was monitored by fluorescence decay of
the trans- S TS . The rate constants of the triplet-triplet energy transfer between

Erythrosin B and SITS (at room temperature and pH 7) were found
and It should be emphasized that the concentration of the triplet
sensitiser attached to the protein did not exceed M in those experiments, and the
collision frequencies were close to 10 which are 8-9 orders of magnitude less than
those measured with the regular luminescence or ESR techniques.
The triplet-photochrome labeling technique was first used to follow the protein-
protein dynamic contacts in biomembranes (Mekler and Umarova, 1988). SITS and
Erythrosin-NCS (ERITC) were bound covalently to ATPase. Triplet-triplet
energy transfer from the light-excited triplet ERITC to SITS initiated the cis-trans
photoisomerisation of cis -SITS. The photoisomerisation kinetics of SITS was recorded
with a regular spectrofluorimeter. The apparent rate constant of triplet-triplet energy
transfer from ERITC to

I

-SITS was found to be (at 25 °C). The
value of the triplet-triplet energy transfer between unbound ERITC and SITS was
measured in solution to be The drop of in the case of labels bound to
ATPase is a result of the increased media viscosity and steric factors.

cis

1.1.5. ELECTRON SPIN RESONANCE (ESR)

In the past 18 years new electron spin resonance technology similar to that of nuclear
magnetic resonance (NMR) has been developed (Freed, 2000; Eaton and Eaton, 2000).
These technologies include two-dimensional Furier transform ESR (2D FT ESR),
multiple quantum ESR, high-frequency (high-field) ESR, and low frequency ESR
imaging. Such developments have significantly improved method application in
investigation molecular structure and dynamics of biological objects.

High-field-high-frequency ESR.
Accordingly, the electron magnetic resonance condition is: