The interaction of collagen with the hydrophobic fluorescent probe-2-p-toluidinylnaphthalene-6-sulfonate.

Three kinds of fluorescence enhancement result from the interaction of 2-p-toluidinylnaphthalene-6-sulfonate and calf-skin collagen. They are negatively cooperative, independent, and highly cooperative fluorescence enhancement. In the independent region at pH 3.7, the binding number is about 36 moles of 2-p-toluidinylnaphthalene-6-sulfonate per mole of tropocollagen with a binding constant of 2.0 × 10⁴ M^?1; with ΔG = ?5.7 kcal/mole, ΔH = ?4.0 kcal/mole, and ΔS = 6 e.u. The pH dependence of fluorescence of native collagen shows that the deprotonated forms of the β and γ carboxyl groups of aspartic and glutamic acid decrease the intensity, possibly by charge repulsion of the negatively charged sulfonate group of 2-p-toluidinylnaphthalene-6-sulfonate. The positive charge of lysine is found to be unimportant in the interaction of 2-p-toluidinylnaphthalene-6-sulfonate with collagen. Fluorescence enhancement is caused mainly by the hydrophobic interactions of 2-p-toluidinylnaphthalene-6-sulfonate and collagen. Salt bridge formation between basic and acidic side chains in very low salt concentration may be detectable by 2-p-toluidinylnaphthalene-6-sulfonate fluorescence.     

L-phenylalanyl-tRNA synthetase of E. coli. Active site specific binding of 2-p-toluidinylnaphthalene-6-sulfonate

2-p-Toluidinylnaphthalene-6-sulfonate was found to be suitable as an active site directed indicator. Results of titration and inhibition experiments are consistent with binding of the dye to the site of L-phenylalanine at the active site. An analytical procedure is described to evaluate the dissociation constant of the enzyme-L-phenylalanine complex under catalytic conditions.     

Interaction of amylose and other α-glucans with hydrophobic fluorescent probe (2-p-toluidinylnaphthalene-6-sulfonate)

Fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was enhanced in the presence of maltooligosaccharides, amylose, and other α-glucans. The dependence of relative TNS fluorescence intensity per glucose unit on chain length of oligosaccharides was examined. The values of binding constant and thermodynamic parameters, assuming the 1:1 complex for TNS-amylose (number-average degree of polymerization, DP_N = 17), were determined by the fluorescence titration. The values of thermodynamic parameters for 1:1 complex formation of TNS-α- and β-cyclodextrins were also determined and compared with those of TNS-amylose (DP_N = 17). The fluorescence intensity of TNS in the presence of amylose (DP_N = 600) decreased by the action of glucoamylase and taka-amylase A. The fluorescence of TNS-amylose (DP_N = 17) system increased with the increased ionic strength. In the presence of pullulan, TNS fluorescence was also enhanced and decreased by the action of pullulanase. Amylopectin enhanced TNS fluorescence rather more strongly than amylose (DP_N = 17) at the same concentration. In the presence of dextran, the fluorescence of TNS was scarcely enhanced. The degree of fluorescence enhancement of TNS in the presence of α-glucans seems to reflect the structures of α-glucans in solution, since TNS fluorescence is enhanced in the hydrophobic environment or by the disturbance of free intramolecular rotation.     

Determination of beta-amylase activity by a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate flow-injection analysis (2, 6-TNS-FIA) method, using amylose and amylopectin as substrates

2-p-Toluidinylnaphthalene-6-sulfonate (2,6-TNS) is a compound which is barely fluorescent in pure water but whose fluorescence can be strongly enhanced if the environment becomes hydrophobic, i.e. by the addition of suitable substrates such as proteins or 1, 4-alpha-D-glucans. The enhancement of fluorescence results from the formation of a 2,6-TNS/substrate complex. For linear and ramified 1, 4-alpha-D-glucans, the fluorescence intensities of the complexes depend linearly on their concentrations but nonlinearly on their average molecular weights (AMW). Thus, the fluorescence detector acts simultaneously as a linear detector concerning the concentration of 1,4-alpha-D-glucan and as a nonlinear mass-selective detector concerning its AMW. These properties have been used for the development of a fluorimetric 2,6-TNS-FIA methodology for the determination of beta-amylase activity, using amylose and amylopectin as substrates. The experimental data points, corresponding to the concentration of "detectable" substrate vs depolymerization time, were fitted using a two-parameter exponential decay curve, and the depolymerization rates at time zero were calculated. The depolymerization rates at time zero vs the corresponding initial substrate concentrations were fitted using the Michaelis-Menten hyperbola and the enzymic constants k(3) and K(m) for amylose (5.93 x 10(-3) g/microKat. min and 1.49 g/L, respectively) and for amylopectin (7.40 x 10(-3) g/microKat+. min and 1.65 g/L, respectively) were determined.     

Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.     

Motions studies of the human alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and polarization of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues.

Dynamics studies on tryptophan residues of human alpha 1-acid glycoprotein (orosomucoid) and of 2-p-toluidinylnaphthalene-6-sulfonate bound to the protein are performed. Excitation at the red edge of the absorption spectrum of the tryptophan does not lead to a shift of the fluorescence emission maximum of the fluorophore. This reveals that Trp residues present motions with respect to their microenvironment. This is confirmed by polarization studies as a function of temperature. Excitation at the red edge of the absorption spectrum of TNS leads to an important shift (15 nm) of the fluorescence emission maximum of the probe. This reveals that emission of TNS occurs before relaxation of the amino-acids dipole occurs. Emission from a non-relaxed state means that TNS molecules are bound tightly to the protein, a result confirmed by polarization studies.     

Bile acid and sterol solubilization in 2-hydroxypropyl-beta-cyclodextrin.

The use of 2-hydroxypropyl-beta-cyclodextrin has made it possible to prepare stable aqueous solutions of cholesterol, 26-hydroxycholesterol, 7 alpha-hydroxycholesterol, and monohydroxy bile acids such as lithocholic and 3 beta-hydroxy-5-cholenoic acids. These solutions are suitable for cell culture studies and for parenteral administration to animals.     

Proton magnetic resonance study of complex formation between N6-methyladenosine and polyuridylic acid

The interaction between N₆-methyladenosine and polyuridylic acid in D₂O solution at neutral pD has been studied as a function of temperature and N₆-methyladenosine concentration by proton magnetic resonance spectroscopy. A rigid double-stranded 1:1 complex is formed below ～10°C, involving hydrogen-bonded N₆-methyladenine:uracil base-pairing and stacking of the adenine bases. This complex is less stable than the 1:2 complex formed between adenosine and polyU, and involves a more rapid exchange of the monomer between free and polymer-bound environments.     

Crystallographic study of the complex between sulfite and bakers' yeast flavocytochrome b2

The complex between Saccharomyces cerevisiae flavocytochrome b2 and the sulfite anion has been analyzed by x-ray diffraction. A map of the difference in electron density between the complex and the native protein has been computed. One positive peak of electron density is visible at the active site of each of the two subunits in the asymmetric unit, very close to the N-5 of the flavin. The molecular fragment SO3(2-) can account for the shape of this difference in electron density. A third peak is visible in the subunit containing pyruvate, the reaction product. It is a peak of negative electron density localized at the position where the pyruvate usually is in the native form. These results are interpreted on the basis of the mechanism defined in solution for the reaction between flavins and sulfite.     

Inclusion complexes of pyrimethamine in 2-hydroxypropyl-beta-cyclodextrin: characterization, phase solubility and molecular modelling

The inclusion complexation of pyrimethamine in 2-hydroxypropyl-beta-cyclodextrin has been investigated by 2D (1)H NMR, FTIR and UV/visible spectroscopy and also by molecular modelling methods (AM1, PM3, MM3). From the phase-solubility diagram a linear increase was observed in pyrimethamine aqueous solubility in the presence of 2-hydroxypropyl-beta-cyclodextrin, evidencing the formation of a soluble inclusion complex. According to the continuous variation method (Job's plot) applied to fluorescence measurements, a 1:1 stoichiometry has been proposed for the complex. Concerning the structure of the complex, a Cl-in orientation of pyrimethamine in the 2-hydroxypropyl-beta-cyclodextrin cavity has been proposed from the theoretical calculations, being confirmed by two-dimensional (1)H NMR spectroscopy (ROESY). The thermal behaviour has also been studied, providing complementary evidences of complex formation.