muramidase and perfluorooctanoic-acid

A droplet (digital) microfluidic device has been developed that enables complete protein sample preparation for MALDI-MS analysis. Protein solution dispensing, disulfide bond reduction and alkylation, tryptic digestion, sample crystallization, and mass spectrometric analysis are all performed on a single device without the need for any ex situ sample purification. Fluorinated solvents are used as an alternative to surfactants to facilitate droplet movement and limit protein adsorption onto the device surface. The fluorinated solvent is removed by evaporation and so does not interfere with the MALDI-MS analysis. Adding a small amount of perfluorooctanoic acid to the MALDI matrix solution improves the yield, quality and consistency of the protein-matrix co-crystals, reducing the need for extensive 'sweet spot' searching and improving the spectral signal-to-noise ratio. These innovations are demonstrated in the complete processing and MALDI-MS analysis of lysozyme and cytochrome c. Because all of the sample processing steps and analysis can be performed on a single digital microfluidic device without the need for ex situ sample handling, higher throughput can be obtained in proteomics applications. More generally, the results presented here suggest that fluorinated liquids could also be used to minimize protein adsorption and improve crystallization in other types of lab-on-a-chip devices and applications.

Topics: Caprylates; Crystallization; Cytochromes c; Fluorocarbons; Halogenation; Microfluidic Analytical Techniques; Muramidase; Peptides; Signal-To-Noise Ratio; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surface-Active Agents; Trypsin

Using equilibrium dialysis, isothermal titration calorimetry (ITC) and circular dichroism (CD), the interactions of perfluorooctanoic acid (PFOA) and lysozyme were investigated under normal human physiological conditions, i.e., at pH 4.40, 6.00 and 7.40 at 37 degrees C in 0.15 M electrolyte. A simple and rapid spectrophotometric method was developed for determining PFOA concentrations. Interactions between PFOA and lysozyme were found to result from non-specific non-covalent bonds-F/N and F/O affinity, ion-pair attraction, hydrogen bond, hydrophobic interaction and van der Waals force-and were affected by chemical adsorption to monolayers. The results indicated that binding of PFOA altered the secondary structure and activity of lysozyme. This work provides a useful experimental strategy for research into the enzyme toxicity of organic chemicals, e.g., food additives and organic contaminants, and it may help to elucidate the molecular toxicology of human health risks.

Topics: Animals; Caprylates; Chickens; Enzyme Inhibitors; Fluorocarbons; Hydrogen-Ion Concentration; Kinetics; Muramidase; Protein Binding; Protein Structure, Secondary; Thermodynamics

The interactions of a fluorinated surfactant, sodium perfluorooctanoate, with lysozyme, have been investigated by a combination of UV absorbance, electrical conductivity and dynamic light scattering to detect and to characterize the conformational transitions of lysozyme. By using difference spectroscopy, the transition was followed as a function of surfactant concentration, and the data were analyzed to obtain the Gibbs energy of the transition in water (DeltaGw(o)) and in a hydrophobic environment (DeltaGh(o)) for saturated protein-surfactant complexes. Electrical conductivity was used to determine the critical micelle concentration of the surfactant in the presence of different lysozyme concentration. From these results, the average number of surfactant monomer per protein molecule was calculated. Finally, dynamic light scattering show that only changes in the secondary structure of the protein can be observed.

Topics: Caprylates; Chemistry, Physical; Conductometry; Fluorocarbons; Muramidase; Protein Conformation; Solutions; Water