muramidase and dansyl-chloride

In a previous work, we reported that contrary to native calcium-loaded alpha-lactalbumin (holo alpha-LA), calcium-depleted form (apo alpha-LA) has the ability to self-assemble with lysozyme (LYS) to form different supramolecular structures in temperature-dependent manner. In this study, we examine what happens at molecular scale using fluorescence techniques. Fluorescence anisotropy coupled with fluorescence lifetime measurements provides a means to measure intermolecular interactions. We showed that LYS interacts with both apo alpha-LA and holo alpha-LA to form oligomers, assumed to be heterodimers, at 10 degrees C and 45 degrees C. The dissociation constants for dimerization were found to be in the muM range and increased significantly with increasing ionic strength from 39 to 124 mM. Although the binding constants of holo alpha-LA-LYS and apo alpha-LA-LYS complexes were of the same order of magnitude, the shape or conformation of formed heterodimers differed as assessed by fluorescence parameters in particular correlation time calculations. Such conformation differences could explain why holo alpha-LA-LYS complexes are trapped as heterodimers while the apo alpha-LA-LYS complexes have the ability to further self-assemble into various supramolecular structures.

Topics: Animals; Apoproteins; Cattle; Chickens; Dansyl Compounds; Fluorescence Polarization; Kinetics; Lactalbumin; Muramidase; Osmolar Concentration; Protein Binding; Protein Conformation; Protein Multimerization; Spectrometry, Fluorescence; Temperature

The onset of hen egg white lysozyme aggregation on exposure to alkaline pH of 12.2 and subsequent slow growth of soluble lysozyme aggregates (at 298 K) was directly monitored by steady-state and time-resolved fluorescence anisotropy of covalently attached dansyl probe over a period of 24 h. The rotational correlation time accounting for tumbling of lysozyme in solution (40 microM) increased from approximately 3.6 ns (in pH 7) to approximately 40ns on exposure to pH 12.2 over a period of 6 h and remained stable thereafter. The growth of aggregates was strongly concentration dependent, irreversible after 60 min and inhibited by the presence of 0.9 M l-arginine in the medium. The day old aggregates were resistant to denaturation by 6 M guanidine.HCl. Our results reveal slow segmental motion of the dansyl probe in day old aggregates in the absence of L-arginine (0.9 M), but a much faster motion in its presence, when growth of aggregates is halted.

Topics: Animals; Anisotropy; Dansyl Compounds; Fluorescence Polarization; Hydrogen-Ion Concentration; Muramidase; Protein Structure, Quaternary; Time Factors

A method for the quantitative determination of lysine, histidine and tyrosine in foods based on pre-column derivatization with 5-dimethylaminonaphthalene-1-sulfonyl chloride (DnsCl) and reversed-phase liquid chromatography has been developed. Derivatization conditions, including DnsCl concentration, time, temperature, and buffer solution were studied. To establish the reliability of the proposed liquid chromatographic (LC) method, the precision and accuracy of the analyses were evaluated using samples of casein and lysozyme.

Topics: Caseins; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Dansyl Compounds; Food Analysis; Histidine; Hydrogen-Ion Concentration; Lysine; Muramidase; Sensitivity and Specificity; Tyrosine

The binding of lysozyme (LZM) to bacterial lipopolysaccharide (LPS) inhibited the biological activities of LPS as well as the enzymic activity of LZM. The mode of binding has been characterized by using dansylated LZM and enzyme inhibition. The binding of LPS to LZM significantly increased the fluorescence intensity (Fl-intensity) of the danyl group and was found to be time-dependent; the complex was produced gradually and became stabilized within 20 min at 37 degrees, 10 min at 50 degrees, and 1 min at 70 degrees. The maximum level of binding was also dependent on the reaction temperature, and more complex was formed at higher temperatures. Complexation was strongly dependent on the salt concentration and was not observed at greater than 0.5M NaCl. From collected evidence of the Fl-intensities of various dansyl derivatives and amphiphiles, it is concluded that LZM interacts with LPS by multiple binding-modes, the first being strongly related to the enzyme inhibition, the second being close to the Fl-intensity, and the third being dependent on the inhibition of immunopharmacological activities. For the amphiphiles used in this study, sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-propanesulfonate (CHAPSO), decansulfonic acid, and cardiolipin have binding modes similar to that of LPS.

Topics: Affinity Labels; Animals; Dansyl Compounds; Detergents; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Lipopolysaccharides; Muramidase; Osmolar Concentration; Spectrometry, Fluorescence; Temperature

Peptide fragments corresponding to the signal sequence of chicken lysozyme, labelled with the fluorescent 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group have been synthesized. The emission characteristics and fluorescence polarization of the dansyl group have been used to study the interaction of signal sequence fragments with liposomes. The peptide fragments bind to liposomes and are associated with the hydrophobic core of the bilayer.

Topics: Dansyl Compounds; Fluorescence Polarization; Fluorescent Dyes; Liposomes; Muramidase; Oligopeptides; Peptide Fragments; Peptides; Protein Sorting Signals; Spectrometry, Fluorescence