Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes

After synthesis of transmembrane proteins (TMPs), they are transferred and inserted into plasma membranes to play biological functions. Crucially, orientation of TMPs in membranes determines whether they have biological activities. In cellular environments, a number of cofactors, such as translocon, can assist TMPs to be inserted into membranes in defined orientations. During in vitro reconstitution of TMPs with mimic membranes, both insertion and orientation of TMPs are primarily determined by interactions with the membrane. Yet the knowledge is limited, hindering the in vitro applications of TMPs. Here, we take Bacteriorhodopsin (bR) as a model TMP, using fluorescence quenching titration experiment to identify orientation of bR in mimic membranes, examining effects of a number of factors, including lipid composition, pH value, ionic strength and membrane curvature. The most effective determinant is the lipid type, which modulates insertion and orientation of bR in membranes by changing the membrane surface charge and the membrane fluidity. Both the pH value and the ionic strength play secondary roles by tuning the nature of the electrostatic interaction. The membrane curvature was found to have a minor effect on orientation of bR in membranes. By comparing orientations of bR in folded and unfolded states, no obvious change was observed, informing that nascent proteins could be inserted into membranes in defined orientations before folding into the native state inside the membrane.

Comparison of pendimethalin binding properties of serum albumins from various mammalian species

Background To investigate the interaction of pendimethalin (PM), a commonly used herbicide, with various mammalian serum albumins. Methods The interactions of PM with serum albumins of bovine (BSA), sheep (SSA), porcine (PSA) and rabbit (RbSA) were studied using fluorescence quenching titration and site marker displacement experiments. Results A comparison of the PM-induced quenching of the fluorescence of these albumins with that published for human serum albumin (HSA) showed similarity between BSA and HSA. The PM binding affinity of these albumins was found to follow the order: SSA>BSA>RbSA>PSA. Warfarin (WFN) displacement results also suggested similar displacing action of PM on WFN-BSA complex, when compared to the published results on WFN-HSA complex. Conclusion The results suggested close similarity between BSA and HSA in terms of PM binding characteristics and hence bovine can be selected as a suitable animal model for further toxicological studies of PM.

Effect of Composting on Dissolved Organic Matter in Animal Manure and Its Binding with Cu

The agricultural application of raw animal manure introduces large amounts of dissolved organic matter (DOM) into soil and would increase transport of heavy metals such as Cu which are widely present in animal manure. The purpose of this research was to evaluate the evolution of DOM from pig and cattle manures during composting through excitation-emission matrix (EEM) fluorescence spectroscopy and the binding ability of DOM toward copper (Cu) ions with the aid of fluorescence quenching titration. The excitation-emission matrix spectra indicated that tyrosine-like, tryptophan-like, and soluble microbial byproduct-like fluorescence decreased significantly, while humic-like and fulvic-like fluorescence increased and became the main peaks in composted manure DOM. Fluorescence quenching titration showed that the complexing capacities of pig and cattle manure DOM decreased after composting. Correlation analysis confirmed that complexing capacity of DOM positively and significantly correlates with tyrosine-like and soluble microbial byproduct-like materials which mostly degraded after composting. These results would suggest that the ability of manure DOM to complex with Cu is inhibited as a result of reduced protein-like materials after composting.

Comparison of Mn2+, Co2+, and Cu2+ binding to fulvic acid as measured by fluorescence quenching

The binding of Cu2+ to soil-derived fulvic acid (SFA) measured by fluorescence quenching titration is much stronger than Co2+ and Mn2+ which are similar. The conditional stability constants from curve fitting at pH 6 are 1.1 × 105 for Cu2+, 5.1 × 103 for Co2+, and 4.2 × 103 for Mn2+. The slight advantage in strength for Co2+ over Mn2+ is also demonstrated by the binding curves at pH 6 and 7. These two metal ions not only tend to bind more weakly but also bind to fewer sites, giving complexing capacity values of about 1.5 × 10−6 M compared to 2.0 × 10−5 for Cu2+ at pH 6. This lower binding capacity may be due to outer sphere complexation that does not allow access to some sites complexed by inner sphere binding Cu2+. Scattering experiments demonstrate that Cu2+ is more effective at precipitating and aggregating SFA than Co2+ and Mn2+. Cu2+ probably neutralizes the negative charges on SFA molecules allowing larger hydrophobic aggregates to form and precipitate.