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Analysis of the proteolytic degradation of the native protein structure carried out by the comparison of the temperature dependence of the hydrogen exchange and proteolytic splitting rates of the hen egg-white lysozyme and human Hb and apoHb. Acceleration of the burst-like (all or none) proteolytic degradation in the high temperature range is provided by the intensification of the global fluctuations with overall unfolding revealed by hydrogen exchange. For Hb and apoHb the rate of burst-like proteolytic degradation and hydrogen exchange weakly depends on temperature in the range, where hydrogen exchange reveals only local fluctuations of the native protein structure. The splitting of the two proteins proceeds by the selfaccelerated burst-like mechanism with the initial rate-limiting single cleavage owing to the local fluctuation of the native structure. The local fluctuations play important role also upon the intracellular burst-like degradation of native proteins.

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Structural transitions in the tetrameric melittin from bee venom in 2 M KCl induced by variations of pH (from 0.7 to 12.0) and temperature (from 2 to 95 degrees C) have been studied. The pH and temperature ranges of structural changes and the zones of emission quenching of discerning tryptophan classes were revealed. The analysis of the temperature dependence of monotonic changes of spectral maximum positions and relative fluorescence yields allowed one to discriminate the zone of structural transitions in the tetramer from that of temperature quenching due to the thermal activation of fluorophore collisions with neighboring quenching groups in protein. Based on the new and earlier published results, some advantages and modes of using the method of component analysis of protein tryptophan spectra were summarized to determine the main characteristics of physicochemical transitions in proteins.

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The oligomerization of melittin with increasing ionic strength and protein concentration was investigated using the methods of decomposition of its tryptophan fluorescence spectra into "elementary" log-normal components. At high ionic strength (up to 2 M KCl), the emission spectra of tetrameric melittin are well described as the sum of two log-normal components, suggesting the presence of tryptophan residues in two sorts of environment with greatly differing polarity. Measurements of fluorescence spectra by iodide showed that these two spectral components possess different Stern-Volmer constants, that is, the tryptophans emitting them have different solvent accessibility, which does not correlate with the crystallographic structure of tetrameric melittin. Moreover, in the oligomerization transition induced by ionic strength, the tetrameric intermediate is formed, which has log-normal spectral components with relative contributions differing from those in 2 M KCl.

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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.

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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.

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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.

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The novel algorithm QUENCH is described which resolves fluorescence spectra onto two components differing in the accessibility (the Stern-Volmer constants) for small quenchers. In contrast to the known algorithms, the QUENCH first estimates Stern-Volmer constants using the total arrow of spectra measured at different quencher concentrations and, then, calculates contributions of each component into the emission intensities at every wavelength value. The component spectra, resolved with QUENCH from the tryptophan fluorescence of ribosomal S7 protein, were very similar to the log-normal components revealed using the SIMS program [16]. The QUENCH algorithm may be of use in the component analysis of fluorescence spectra of any system containing fluorophores with differing accessibilities for quenchers.

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An algorithm of decomposition of protein tryptophan spectra into components was developed. The spectral shape of components is described by a uniparametric log-normal function. Rise of certainty and accuracy of resolution of widely overlapping smooth spectral components (a typical uncorrect reverse problem) was achieved using several regularizing factors: (i) the set of experimental spectra used were measured at several quencher concentrations; (ii) the functional being minimized, along with the root mean square residuals of intensities, the term depending on the obedience to the Stern-Volmer law; (iii) an extra information is used--the number of experimental values greatly exceeds the number of parameters to be estimated. The minimum of functional is determined by a consecutive setting of all possible combinations of component spectral maxima values, which allows to avoid sticking in the local minima of noisy functional. The real experimental noise restricts the decomposition into not more than three components. The decomposition error does not exceed the experimental one. The algorithm functioning is illustrated by resolution of tryptophan fluorescence spectra of papain into one, two, and three components.

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To elucidate the details of pH-induced conformational transformation of ricin [I] in the region surrounding tryptophan residues, we studied parameters of fluorescence of the native toxin and its isolated A- and B-subunits at pH 4.0, 5.0 and 7.4. The studies were carried out using resolution of fluorescence spectra according to different degree of tryptophan accessibility to ionic (iodide) and non-ionic organic (acrylamide) quenchers. Application of the new method allowed to reveal three classes of tryptophan residues differing in their accessibility to quenchers alpha-residues are accessible neither to ions nor to organic molecules; beta-residues are accessible only to organic molecules; while surface gamma-residues are accessible to both types of quenchers. The fluorescence spectra were assessed for each class of tryptophan residues. The major part of them was shown to be localized in apolar rigid microenvironment. Fluorescence of ricin and especially of its isolated subunits proved to be strongly dependent on the pH value. At pH less than 5 the structure of B-chain loosens, this process being reflected by an increase in accessibility of tryptophan residues to quenchers. In acidic solution at least one out of seven tryptophan residues in the ricin molecule undergoes conformational transformation. Positive charge prevails in the regions surrounding quencher-accessible tryptophan residues. Binding of lactose leads to a slight compactization of the toxin structure that causes, in its turn, short-wave shifts of the fluorescence spectra and reduction of Stern-Volmer constants for intraglobular tryptophan residues.

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In order to investigate the effects of temperature and ionic strength on the N-B-transition and the alkaline denaturation of the human serum albumin, the pH-dependences of fluorescence position and relative yield of Trp-24 and of protein bound dye ANS were measured. The measurements were carried out at temperatures from 10 to 45 degrees C and ionic strengths (NaCl) from 0.001 to 0.2. The pH-induced structural transitions have different realization in environments of tryptophanyl and tightly bound ANS. The alkaline denaturation does not change the Trp-214 fluorescence. The N-B-transition gives rise to the slight polarity and/or mobility lowering in the Trp-214 environment (the shorter-wave-length spectral shift). Increase in the temperature and ionic strength induces the shift of the transition midpoint from ca. 8 to 8.7 and reduces the spectral shift amplitude. At low ionic strengths, the new structural transition in the Trp-214 environment is observed at pH change from 6.7 to 5.7. This transition is not observable using ANS fluorescence. The N-B-transition is accompanied by an enhancement and longer-wavelength shift of the ANS fluorescence spectra. The transition midpoint is independent of temperature, but is shifted to lower pH values at a decrease of ionic strength value. At ionic strengths less than or equal to 0.01 the shorter-wavelength spectral shift is seen at pH from 7.5 to 9, which seems to reflect the disulfide B-A-isomerisation. The alkaline denaturation gives rise to the sharp quenching of ANS fluorescence, probably due to the ANS binding site decomposition.(ABSTRACT TRUNCATED AT 250 WORDS)

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Using fluorescence parameters of tryptophanyl and bound ANS, the acid-induced structural transitions of defatted monomeric human serum albumin were measured as pH-dependences from 6 to 2.5 in the wide range of temperature (10 to 45 degrees C) and ionic strength (from 0.001 to 0.2 M NaCl or 0.067 M Na2SO4). Temperature rise and decrease in ionic strength value result in the splitting of the N-F-transition onto two stages, N-F1 and F1-F2. The N-F1-transition is accompanied by the blue shift of tryptophanyl and ANS fluorescence spectra and increase in the ANS emission yield. The F1-F2-stage is manifested in an additional blue spectral shift and a sharp drop of the ANS emission yield, which is shown to be due to the lowering of albumin affinity for the dye. In the acidic-extension stage (F2-E), the spectra undergo a red shift which means that the nanosecond dipole relaxation of protein groups and bound water becomes faster. In the F2 from, the albumin affinity for ANS is significantly lowered; the association constant of the primary binding site is lower by an order of quantity and two secondary sites are practically disappeared. The complex effect of temperature, ionic strength and pH changes on the properties of ANS-binding sites is considered as a model of possible control influences of these factors upon the albumin transport of amphiphilic anions in organism.

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In order to investigate effects of temperature in the physiological range (from 10 to 50 degrees C) on structural, physical and functional properties of the N-form of human serum albumin (HSA), the temperature dependences of fluorescence parameters of Trp-214 residue of HSA and of the specifically bound dye ANS, as well as of association constants of ANS binding in the primary and secondary binding sites on HSA molecule were measured. The temperature-induced changes of these properties of HSA are essentially dependent on pH (7.0 or 5,6) and ionic strength (0.001-0.008 or 0.2 M NaCl). At pH 7.0 and 0.2 M NaCl the environment of Trp-214 remained invariant at temperature changes between 10 and 50 degrees C. On the other hand, the affinity to ANS of a primary binding site doubled and that of secondary ones halved. These affinity changes seem to be due, are least partly, to the heating-induced dissociation of Cl-ions, which are inhibitors of the primary dye binding. By lowering pH (to 5.6) and ionic strength the temperature-induced changes in the Trp-214 environment were observed. The changes are interpreted as indole group transition into the buried region, inaccesible to water (the "closing" of a structural slit). The affinity of secondary binding sites of ANS was halved.

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The effect of ionic strength on the 1-anilino-8- naphtalene sulfonate (ANS) binding sites of the N-form of human serum albumin (HSA) was studied by means of the protein and ligand fluorescence. The parameters of the binding of ANS to HSA (the number of sites and the binding constants) were determined by two methods: by measuring the ANS fluorescence either (i) at increasing protein concentrations and constant ANS concentration, or (ii) at increasing ANS concentration and two constant protein concentrations (9.6 and 2.53 microM). An increase in the NaCl concentration results in a monotonous decrease of the ANS fluorescence yield of HSA-ANS complex, which could be interpreted in terms of two different effects: first, the direct collisional quenching interaction of Cl-ions with bound ANS located on the surface of HSA and, second, the ca. 10 per cent decrease of the number of bound ANS molecules due to the lowering of the ANS-HSA association constant values. The fluorimetric titration showed that at low ionic strength (0.008 M NaCl) HSA molecule has one strong ( lgKaI = 6.75-7.25) and two secondary sites with lower affinity ( lgKaII = 6.35). The increase in the NaCl concentration results in a decrease of the affinity for both kinds of binding sites (in 0.2 NaCl lgKaI = 6.3-6.7; lgKaII = 5.6-5.9). In contrast with NaCl, Na2SO4 induces only a limited decrease of the ANS fluorescence occurring within a narrow concentration range (from ca. 0.02 to 0.05 M Na2SO4, e. i. at ionic strengths from 0.07 to 0.15) and which can be described as a cooperative interaction of six SO4(2)-ions with a HSA molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

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The photoexcitation of Trp, Tyr or Phe chromophore in a protein is analogous to the instant (10(-15) to 10(-14) s) introduction of a local probe carrying strong electronic and protonic donor and acceptor moieties and, moreover, having a significant dipole moment (4 to 11 D). Depending on the protein structure mobility during the excited state lifetime, the intra-macromolecular complexes (the exciplexes) with polar groups can be formed and some reversible charge transfer processes, which qaench the fluorescence, can take place with greater or lesser probability. The shifts of Trp fluorescence spectra from 307 to 316 nm (due to the 1:1 exciplex formation) and to 325-330 nm (2:1 exciplex) are typical for many proteins. An additional spectral shift up to 350 nm is caused by the reorientational relaxation of a protein matrix dipoles during the nanosecond excitation lifetimes. The bathochromic fluorescence of several proteins evidences the slow (times much longer than 10 ns) reorientation mobility in these cases. The fluorescence quenching rates by proteic groups and extrinsic small quenchers is linearly related to the diffusion coefficient of a surrounding solvent. Analysis of such relationships allows, in principle, to evaluate the diffusional internal friction inside a protein and parameters of co-operativity and anisotropy of the mobility. Some sources of possible misinterpretations of Trp fluorescence depolarisation as a measure of the rotational mobility of indole.

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Lifetimes of phenylalanine, tyrosine and tryptophan self-fluorescence of three Ca2+-binding proteins (parvalbumins pI 4.47 and 3.95 and bovine alpha-lactalbumin) in the Ca2+-saturated state and without Ca2+ were measured on a device functioning in a channel of synchrotron radiation of the Lebedev Physical Institute electron accelerator C-60 with a single photon counting system. The decay curve of phenylalanine fluorescence of Ca2+-saturated parvalbumin pI 4.47 is two-exponential, which results from the presence of two subsystems of phenylalanine residues in this protein. Radiation of these subsystems is almost independent of one another. Detachment of Ca2+ from protein disturbs these subsystems. In case of tyrosine fluorescence of carp parvalbumin pI 3.95 a change in the quantum yield value of the stationary fluorescence induced by elimination of Ca2+ proceeds without a change of fluorescence lifetime. This seems to be related to the existence of static quenching of fluorescence in this case at the expense of complex formation between the chromophore and some adjacent quenching groups. Detachment of Ca2+ from alpha-lactalbumin induces conformational changes in its structure. The latter result in a transition of a number of tryptophane residues from its interior to the surface of the globule which is reflected in an increase of fluorescence quantum yield duration. It is concluded that in Ca2+-saturated alpha-lactalbumin some tryptophane residues are located near the quenching groups (dynamic quenching), most likely the disulfide bridges.

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Titration of metal-freed bovine alpha-lactalbumin with Mg2+ ions causes a two-stepped decrease in the fluorescence quantum yield and a pronounced spectral shift towards shorter wavelengths. It seems to reflect conformational changes induced by the binding of two Mg2+ ions to the protein molecule which results in a transfer of some tryptophan residues from the protein surface into an interior part of the protein in rigid unpolar environment. The Mg2+ association constants evaluated from the fluorimetric Mg2+-titration are 2x10(3) and 2x10(2) M-1. Mg2+ ions in millimolar concentrations almost do not influence the binding of Ca2+ ions to protein. It is assumed that Ca2+ and Mg2+ ions are bound to different sites on alpha-lactalbumin. Mono-calcium, mono-magnesium, bi-magnesium and apo-forms of alpha-lactalbumin are distinct in their fluorescence properties which suggests the difference in the conformation of these forms. The binding of Ca2+ and Mg2+ ions to alpha-lactalbumin has to modulate its function.

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Binding of Ca2+ ion to bovine alpha-lactalbumin molecule causes a conformational change reflected in an almost two-fold decrease of the fluorescence quantum yield and a rather pronounced spectral shift towards shorter wavelengths (by ca. 20 nm). The changes could be interpreted as a result of a transfer of highly exposed tryptophan residue(s) into a rigid internal part of the protein molecule containing effective quenching groups (probably, vicinal disulphide bridges). The Ca2+ binding constant evaluated from EGTA--and pH-titrations is (3-6) X 10(8) M-1. The results of the spectrofluorometric pH-titration of alpha-lactalbumin in the presence of various Ca2+ concentrations suggest that the well-known acid conformational change in alpha-lactalbumin is due in fact to a competitive replacement of the bound Ca2+ by three H+ ions (pK = 5.0 +/- 0.1) in the Ca2+ binding site.

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Properties of tryptophane and tyrosine fluorescence of intact and iodinated thyroglobulin from normal human thyroid and nodular euthyroid goiter were studied. It has been shown that practically all (95%) tryptophane residues in "normal" thyroglobulin are in the inner regions of the globule. In "pathological" thyroglobulin in the regions inaccessible for water there are located only 68% of trypthophanyls. After iodination only in "pathological" thyroglobulin redistribution of tryptophan residues takes place on the surface and inside the globule. For bovine thyroglobulin shifts of tryptophane fluorescence spectra to the long wave region were observed, as well as a fall of the quantum yield at pH below 5 and above 11, which is in accord with acid and alkaline denaturation. A conformation transition was observed in the pH region 6--7 which is accompanied by a change in the efficiency of excitation energy transfer from tryptophan to iodoamino acids.

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The binding of Ca+ to whiting parvalbumin has been studied by intrinsic (tryptophan) protein fluorescence. It has been shown that parvalbumin can exist in three states, which are interpreted as the protein without Ca2+ (P), with one bound Ca2+ (PC) and with two bound Ca2+ (CPC). The spectra of the states have been identified and the protein fluorescence spectra at different Ca2+ concentration have been decomposed on the components corresponding to these states. Relative populations of the P, PC and CPC states have been plotted against Ca2+ concentration. On the basis of a scheme of the successive Ca2+ binding; P+Ck1 in equilibrium PC; PC+Ck2 in equilibrium CPC, corresponding theoretical dependences have been computed and fitted to the experimental ones. The fitted equilibrium constants K1 and K2, are 5.10(8) and 6.10(6)M-1, correspondingly. The schemes are in a good agreement with the experimental data on EGTA-titration of Ca2+-saturated parvalbumin and on pH-titration of parvalbumin in the presence of EGTA.

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