muramidase and n-hexane

Detection of native proteins, particularly directly from raw biological samples, has been a challenging task for mass spectrometry. In this study, we demonstrated that solid-substrate electrospray ionization mass spectrometry with nonpolar solvents such as n-hexane could allow detection of native proteins and protein complexes directly from raw biological samples. Mechanistic study revealed that the process involved rapid vaporization of the nonpolar solvent, temperature reduction of substrate surface, condensation of water from the ambient air, and spray ionization of the condensed water with analytes under the electric field. The fine spray with water at low temperature allowed the technique to detect native proteins, even directly from viscous samples (e.g., egg white) and solid samples (e.g., bone marrow). This study sheds new insight into the sampling and ionization process of mass spectrometry and provides a technique of great potential for characterization of proteins.

Topics: Hexanes; Muramidase; Myoglobin; Solvents; Spectrometry, Mass, Electrospray Ionization; Volatilization; Water

Refractive index matching was used to create optically transparent polyaphrons to enable proteins adsorbed to the aphron surface to be characterized. Due to the significant light scattering created by polyaphrons, refractive index matching allowed for representative circular dichroism (CD) spectra and acceptable structural characterization. The method utilized n-hexane as the solvent phase, a mixture of glycerol and phosphate buffer (30% [w/v]) as the aqueous phase, and the non-ionic surfactants, Laureth-4 and Kolliphor P-188. Deconvolution of CD spectra revealed that the immobilized protein adapted its native conformation, showing that the adsorbed protein interacted only with the bound water layer ("soapy shell") of the aphron. Isothermal calorimetry further demonstrated that non-ionic surfactant interactions were virtually non-existent, even at the high concentrations used (5% [w/v]), proving that non-ionic surfactants can preserve protein conformation.

Topics: Animals; Buffers; Cattle; Chickens; Chymotrypsin; Circular Dichroism; Glycerol; Hexanes; Immobilized Proteins; Muramidase; Ovalbumin; Polidocanol; Polyethylene Glycols; Protein Conformation; Refractometry; Serum Albumin, Bovine; Solutions; Solvents

The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reversed micelles in n-hexane. The reversed micelles formed by the mixture of Span 85 and CB-Span 85 conjugate were extensively characterized in water content, hydrodynamic radius, and aggregation number. The results show that the water content and hydrodynamic radius of the reversed micelles were significantly increased by the introduction of CB ligands (CB-Span 85 conjugate), and the reversed micelles with CB-Span 85 conjugate had a wider aggregation number distribution than the Span 85 reversed micelles. Using lysozyme as a model protein, protein solubilization by the reversed micelles was investigated. Lysozyme solubilization increased significantly with the coupled CB concentration, indicating that the extraction was based upon the affinity interactions between lysozyme molecules and the CB ligand. High solubilization of lysozyme was obtained by the affinity-based reversed micelles of 62.7 mmol/L Span 85 with coupled CB higher than 0.25 mmol/L. Lysozyme recovery was carried out using a stripping solution of high ionic strength. The recovered lysozyme exhibited an activity equivalent to the native lysozyme and its secondary structure was also unchanged. The results indicate that the reversed micellar system would find potential application in protein separation.

Topics: Hexanes; Hexoses; Hydrogen-Ion Concentration; Ligands; Micelles; Muramidase; Osmolar Concentration; Sensitivity and Specificity; Solubility; Spectrophotometry, Ultraviolet; Triazines; Water