Influence of aluminum and iron chlorides on the parameters of zigzag patterns on films dried from BSA solutions.

The relationships between the structural and aggregational state of bovine serum albumin (BSA) and the specific length and total number of zigzag pattern segments of the film textures formed upon drying biopolymer solutions with aluminum and iron chlorides have been shown. To obtain films, saline solutions of BSA were dried in a glass cuvette under thermostatically controlled conditions. It is shown that the formation of zigzag structures is sensitive to the influence of aluminum chlorides Al3+ and iron chlorides Fe3+ and depend on the concentration of AlCl3 and FeCl3. This may be due to a change in the charge and size of BSA particles and due to a change in conformation or a violation of the structure of BSA. These factors, in turn, affect the hydration of the solution components and the structural state of free water in solution, which presumably also affects the formation of zigzag structures. It is established that the analysis of the specific length and the number of segments of zigzag patterns makes it possible to evaluate changes in the state of biopolymers in the initial solution during structural changes and aggregation.

Aminofluoresceins Versus Fluorescein: Ascertained New Unusual Features of Tautomerism and Dissociation of Hydroxyxanthene Dyes in Solution.

Within the course of this spectroscopic research, we revealed novel features of the protolytic behavior, which extend the knowledge of the chemistry of xanthene dyes and rationalize the utilization of these compounds. In addition to the well-known tautomerism of the molecular form, H2R, of fluorescein dyes, new aspects of tautomeric transformation of anions are disclosed. First, for the dyes bearing the substituents in the phthalic acid residue, 4'- and 5'-aminofluoresceins and 4'-fluorescein isothiocyanate, the monoanion HR- exists in non-hydrogen-bond donor solvents not only as a tautomer with the ionized carboxylic and nonionized OH group but also as a "phenolate" ion with a nonionized COOH group. Such state of HR- ions is typical for dyes bearing halogen atoms or NO2 groups in the xanthene moiety but was not observed until now in the case of substitution in the phthalic residue. Second, the possibility of the existence of the HR- species in DMSO in the form of colorless lactone is deduced for the 5'-aminofluorescein using the visible and infrared spectra. This results in a dramatic difference in medium effects. For instance, whereas for fluorescein in DMSO, the inversion of the stepwise ionization constants takes place and the Ka1/Ka2 value equals 0.08, the same ratio for 5'-aminofluorescein is as high as ∼800. In addition, the pKa values of sulfonefluorescein, erythrosin, methyl ether of fluorescein, and phenol red were obtained to verify the acidity scale in DMSO and to support the detailed scheme of protolytic equilibria of fluorescein dyes.

Aminofluoresceins Versus Fluorescein: Peculiarity of Fluorescence.

In this article, we examined the fluorescent properties of 4'- and 5'-aminofluorescein, unsubstituted fluorescein, and its 4'-nitro derivative in a set of solvent systems. Fluorescence lifetimes, quantum yields, time-resolved fluorescence spectra, and quantum chemical calculations allowed clarifying the reasons of the emitting properties in this dye series. In water, the dianions R2- of aminofluoresceins are practically nonfluorescent; in alcohols, the quantum yields are low. In dimethylsulfoxide (DMSO), acetonitrile, and other non-hydrogen bond donor solvents, the bright fluorescence of R2- ions is quenched either on adding small amounts of water which hydrate the carboxylate group or under conditions of protonation of this group (COO- → COOH). The last observation is possible owing to the peculiarities of the tautomerism of the 5'-aminofluorescein monoanion, HR-, which exists in DMSO as an equilibrium mixture of a colorless lactone and colored "phenolate" tautomer with an ionized xanthene moiety and unionized carboxylic group. In contrast, the R2- anion of 4'-nitrofluorescein demonstrates spectral behavior different from that of the amino derivatives. It practically does not emit in aprotic solvents; however, in alcohols or water media, its quantum yield increases to some extent. Such changing spectral properties are explained in terms of the excited-state interfragmental charge transfer.

Ionization and tautomerism of methyl fluorescein and related dyes.

The protolytic equilibrium of methyl ether of fluorescein is studied in water, aqueous ethanol, and in other solvents. The constants of the two-step dissociation are determined by spectrophotometry. In water, the fractions of the zwitterionic, quinonoid, and lactonic tautomes are correspondingly 11%, 6%, and 83%, as deduced from the UV-visible spectra. Corresponding study of the ionization of the methyl ether ester of fluorescein, fluorescein ethyl ester, and sulfonefluorescein allows testing the correction of the attribution of the microscopic dissociation constants of methoxy fluorescein. The results of nuclear magnetic resonance and infrared spectroscopy, as well as the X-ray analysis confirm the predomination of the lactonic structure of the molecular species in solid state and in DMSO. Contrary to it, the spectroscopic studies in both hydrogen-donor bond (HDB) and non-HBD solvents confirm that the presence of lactonic monoanion is atypical for the dye under study and, with high probability, also for the mother compound fluorescein.