SOME PROTEIN PROPERTIES IMPORTANT FOR ENZYME CATALYSIS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 137

introduced (Sergeev et al., 1976). This was accompanied by a change of spin dipole-
dipole interaction of the labels with water protons, which was monitored by the rate of
the proton spin relaxation by NMR.
The direct experimental evidence of the nanosecond intramolecular mobility of a
protein matrix at ambient temperatures was obtained using spin and Mössbauer labels
and probes (Likhtenshtein, 1976a, b, 1979a, b; Likhenshtein et al., 1969; Frolov et al.,
1973, 1974, 1977; Belonogova et al., 1978), employing the phenomenon of fluorescence
quenching of the buried tryptophane residues (Lackovicz and Weber, 1973; Munro et al.,
1979), and NMR (Wutrich, 1986). To illustrate, a hydrophobic aromatic derivative of a
nitroxide radical was embedded in the human serum albumin binding site and the
mobility of the spin probe was traced by ESR spectroscopy. The apparent correlation
frequency of the probe, which is essentially faster than macromolecular
tumbling, was not found to be dependent on viscosity and, therefore, was attributed to
the local mobility of the label. The apparent energy kcal/mole) and entropy
activation were determined. Thus, it was concluded that probe
mobility follows the mobility of the flexible walls of the protein binding site with a
similar frequency.
This conclusion was strongly supported by the investigation of the mobility of
Mössbauer atoms which were attached as a metal-complex to the surface of an
HSA globule and incorporated as a polynuclear serum-iron cluster within the globule.
The experiment showed a sharp decrease in NGR spectra intensity (f) at temperatures
exceeding 210 K. Such a change is caused by an anharmonic vibration of the Mössbauer
atom whose correlation frequency is higher than and whose amplitude is A > 0.4

Å at T > 210 K. The serum albumin intramolecular mobility in a nanosecond temporary

region at ambient temperatures was confirmed later by a series of independent dynamical
methods such as spin and fluorescence labeling, tryptophane fluorescence and proton
NMR (Likhtenshtein and Kotelnikov, 1983; Krynichny et al., 1985; Likhtenshtein,
1988a, b, 1993; Likhtenshtein et al., 1993; Vogel et al., 1994; Likhtenshtein et al., 2000)
Recently, the dynamics of the HSA binding site around the dansyl moiety of the dual
fluorophore-nitroxide probe was monitored indirectly by the temperature dependent
relaxation shift max (T) and directly using the picosecond fluorescent time-resolved
technique (see Fig. 1.4. in Section 1.1.3.) (Rubtsova et al., 1993; Fogel et al., 1994;
Lozinsky et al., 2000; 2002; Likhtenshtein, et al., 2000). Both methods showed that the
relaxation of the protein groups in the vicinity of the excited chromophore occurs with a
rate constant of approximately Polarization fluorescence technique experiments
showed rotational mobility of the probe fluorophore fragment with the correlation
2-(2'-Hydroxyphenyl)-methloxazole (PMO), a proton-transfer
fluoresecent dye was used as a probe for the study of HAS hydrophobic binding site
dynamics (Zhong et al., 2000). The observed dynamics indicated that the binding
structure is rigid and the local motions of the probe are nearly “frozen” in the
femtosecond-to-nanosecond time scale. The probe intramolecular twisting of the two
heterocycles rings was slowed down in the protein hydrophobic pocket. Measurement of
the fluorescence dynamic Stokes shift in single tryptophane of cytidine monophosphate
kinase, located in the protein hydrophobic pocket, showed multiphase dynamic processes