A new procedure is described for using fluorescence-quenching data of tryptophan residues in proteins to resolve their fluorescence emission spectra. In this concept the Stern-Volmer quenching plot is determined at each particular emission wavelength and itterative non-linear least-squares fitting procedure allowed to resolve the steady-state emission spectra into components. The resolved components, attributed to each of tryptophan residue, can be characterized by different accessibility to the quencher. The ability to resolve fluorescence emission spectra can be improved by using different kinds of efficient quenchers, which can selectively quench the emission of exposed or both exposed and buried fluorophores. The method was used to decompose emission fluorescence spectra in two-tryptophan-containing proteins; horse liver dehydrogenase, sperm whale apomyoglobin and metalloprotease from Staphylococcus aureus. The resolved spectra of alcohol dehydrogenase and metalloprotease are in excellent agreement with those previously obtained by single-photon counting or phase methods. The method presented here is technically simple and does not require expensive instrumentation.The intrinsic fluorescence of tryptophan residues in proteins has been widely used in the analysis the dynamics and conformational perturbation in these macromolecules. In native proteins these residues can occupy different locations where they can be influenced by the distinct environment, characterized by a particular set of physico-chemical conditions. In consequence the fluorescence contributions of particular tryptophan residues in multitryptophan-containing proteins can vary over a wide range [l]. Interpretation of the intrinsic fluorescence data of proteins often requires the resolution of the fluorescence properties. The resolution of fluorescence emission in proteins and its correlation with an individual tryptophan residue is still very difficult to achieve, since even single-tryptophan proteins can exhibit multiexponential decay kinetics [l].In principle, when dealing with a protein containing more than one tryptophan residue, steady-state fluorescence emission spectra can be resolved into components by the timedomain [2 -61 or frequence-domain method [7 -101. Using single-photon counting techniques and the analysis of fluorescence decay curves as a function of emission wavelengths, Wahl et al. [3, 41 first resolved complex fluorescence spectra of two-tryptophan-containing proteins such as Escherichia cofi lac repressor protein [3] and phosphoglycerate kinase [4]. Following a similar method, Ross et al. [5] resolved the emission spectrum of alcohol liver dehydrogenase into two tryptophan residues. Using the phase-resolved fluorescence method, it has been shown recently that the spectrum of twotryptophan-containing metalloprotease from Staphylococcus aureus can be decomposed into two components, which have been attributed to each of the tryptophan residues [9].Correspondence to Z. Wasylewski, Zaklad Biochemii, Instytut Biologii Molekula...

The dependence of the fluorescence emission maximum of the tryptophan residues in several twotryptophan-containing proteins (horse liver alcohol dehydrogenase, yeast 3-phosphoglycerate kinase, Staphylococcus aureus metalloprotease and bee venom phospholipase A,) on the excitation wavelengths has been studied. Using fluorescence-resolved spectroscopy, we have dissected the contributions of particular tryptophan residues located in different parts of the protein molecule. The results demonstrate that dipolar structural relaxation can occur in the environment of tryptophan residues buried within protein molecules. The observed spectral shifts upon red-edge excitation of these residues can depend on temperature or ligand binding, as demonstrated in case ofmetalloprotease and alcohol dehydrogenase. No spectral shifts upon red-edge excitation have been observed for tryptophan residues totally exposed to the rapidly relaxing aqueous solvent.The intrinsic fluorescence emission of tryptophan residues in proteins provides a sensitive probe of the dynamic properties of their microenvironment. A variety of fluorescence methods, including time-and phase-resolved spectroscopy, have been used in such studies. Some information about protein dynamics can be obtained on the basis of steady-state fluorescence measurements. The steady-state collisional quenching of tryptophan residues buried inside a protein molecule reveals some information about the protein dynamics, since a contact between a fluorophore and a quencher is required in quenching reactions. Other dynamic properties of proteins, which can also be deduced from steady-state measurements, are associated with the phenomenon called the rededge excitation effect [I]. In this phenomenon the tryptophan fluorescence emission spectra can shift to the longer wavelengths as the excitation wavelength is increased toward the red side of the tryptophan absorption.Recently Demchenko [2] has shown that, in several singletryptophan-containing proteins, the red-edge effect is particularly visible in proteins which show fluorescence emission maximum at about 325 -340 nm. In the case of short-wavelength-emitting proteins like azurin, parvalbumin and ribonuclease T I and proteins emitting at a long wavelength (about 350 nm), like melittin in water, no spectral shifts upon the rededge excitation have been observed [2]. This might be due to the fact that in azurin, parvalbumin and ribonuclease TI the single tryptophan residues are located in a non-polar environment or due to the absence of dipole relaxation processes in the tryptophan residue's surroundings.On the other hand, in proteins like melittin (at low ionic strength) the single tryptophan residue is totally exposed to the rapidly relaxing aqueous solvent [2]. Such an explanation remains in agreement with a number of observations indicating that excitation-dependent shifts of fluorescence emission can be easily detected for polar fluorophores in a polar viscous solvent [l].The fluorescence emission of most proteins is heterogeneous s...

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The interactions between polymorphonuclear cells (PMN), Staphylococcus saprophyticus cells and rabbit antibodies against Staphylococcus aureus V8 serine proteinase or normal rabbit serum proteins were investigated. The effect of opsonization on phagocytosis due to human peripheral polymorphonuclear cells was measured. The results were as follows: phagocytosis index values were relatively increased after the incubation of PMN cells with anti-serine proteinase gamma-globulin serum fraction, anti-serine proteinase IgG, non-immunized rabbit serum or with complement.