[Analysis of log-normal components of fluorescence spectra of prodan and acrylodan bound to proteins]. / Analiz log-normal'nykh komponent spektrov fluorestsentsii sviazannykh s belkami prodana i akrilodana.

Steady-state fluorescence spectra of prodan and acrylodan covalently bound to cystein residue of Lys-Cys-Phe tripeptide in solvents of different polarity were analyzed. It was shown that the shape of spectral bands is well described by a log-normal function. Linear relations between three shape-determining parameters of the log-normal function (namely, the positions of spectral maximum and two half-maximum amplitudes) were revealed and evaluated for both fluorophores. This finding enabled us to present the shape of spectral bands of these fluorophores in any environment as analytical log-normal functions depending on only two parameters, the maximum position and the peak amplitude. The empirical uniparametric log-normal curve was used for the analysis of composite fluorescence spectra of prodan bound to bovine serum albumin and acrylodan covalently attached to actin or subfragment 1 of myosin.

[Assignment of a component of serine proteinase fluorescence spectrum to a cluster of tryptophan residues]. / Otnesenie komponent spektra fluorestsentsii serinovykh proteaz k klasteram ostatkov triptofana.

The two log-normal spectral components in, alpha-chymotrypsin, trypsinogen and beta-trypsin fluorescence and the single component of chymotrypsinogen A emission were analyzed using characteristics of microenvironment of atoms of indolic fluorophores of individual tryptophan residues in the three-dimensional structures of the proteins. Tryptophan residues (eight ones in chymotrypsinogen A and alpha-chymotrypsin and four ones in trypsinogen and beta-trypsin) in these homologous proteins form three clusters with resonance excitation energy exchange between fluorophores within each of them. In all the proteins, Trp51 and 141 determine the shorter-wavelength spectral components (ca. 330 nm) and Trp215 and 237 emit as longer-wavelength ones (ca. 340 nm). Excited states of fluorophores of Trp27, 29, 207 (cluster A) and 172 (in cluster B) are deactivated by the near disulfide bonds. The longer-wavelength component is quenched in chymotrypsinogen emission due probably to the formation of an electron trap including Asn91 amide and several water molecules. The contribution of longer-wavelength component in trypsin spectrum increases comparing with that of trypsinogen due to some little changes in localization and a huge rise of mobility of Met180 sulfur atom nearly Trp215 in trypsin.

[Assignment of a component of protein fluorescence spectra to tryptophan residues by their three-dimensional microoenvironmental properties]. / Otnesenie komponent spektra fluorestsentsii belka k ostatkam triptofanapo svoistvam ikh mikrookryzheniia v trekhmernoi strukture.

Parameters of fluorescence of three single-tryptophan-containing proteins and of two log-normal components of proteinase K (2 tryptophans) were analyzed in relation to the microenvironment characteristics of indolic atoms in crystal structures of the proteins. For this purpose, it was constructed a system of microenvironment description including accessibility of the atoms to the bulk and bound water; the density, polarity and mobility of environment within radii of 5.5 and 7.5 A from each indolic atom; and the existence of eventual partners in hydrogen bonding with excited fluorophore. The analysis showed that, in the cases of the most shorter-wavelength emission bands (those structured at 308 nm for azurin and at 316 nm for L-asparaginase), as well as of the monomer melittin band at 350 nm, the microenvironment characteristics well agreed to those predicted in the model of discrete states of tryptophan in proteins [1,3,7] and can be used for assignment of protein fluorescence spectral components to individual tryptophan residues. However, differences of the microenvironment parameters included in the system are little discernible for the component bands of proteinase K emission at ca. 330 and 340 nm. In order to reliably assign such components of tryptophan fluorescence, it seems to be sufficient to take into account some additional structural characteristics, which could be revealed in a comprehensive analysis of a great number of proteins possessing such spectral components.