Changes in peptic digestibility of bovine beta-lactoglobulin as a result of food processing studied by capillary electrophoresis and immunochemical methods.

Digestion studies constitute a functional tool for allergen characterisation. This strategy for investigating allergenic proteins relates to the observation of increased proteolytic resistance of some proteins recognised to exhibit allergenic potential. beta-Lactoglobulin (betaLG) is one of the major whey proteins, a potent milk allergen and shows a high stability against peptic hydrolysis in its native form. In order to study the impact of milk fermentation process on its digestibility, two complementary analytical methods were applied: capillary zone electrophoresis (CZE) to quantitatively study proteolytic degradation of betaLG isolated from different fermented bovine milk products, and enzyme linked immunosorbent assay (ELISA) to assess differences in immunoreactivity. betaLG, isolated from either raw or pasteurised cow's milk (CM), as expected, showed only minimal digestibility (less than 10% in 2 h). However, when raw milk or pasteurised milk was fermented, the rate of peptic digestion of the protein significantly increased (up to 45% in 2 h). In accordance with changes in digestibility, the immunochemical response for all fermented samples was lower than that of non-fermented references. Raw and pasteurised milk "naturally" fermented in our laboratory only resulted in a slight reduction (betaLG detected is still in the range of milligrams per gram sample), whereas the industrially manufactured sour milk as well as the "Acidophilus milk" reflected a remarkably lower level of immunoreactivity (55-56 microg/g sample).

Binding of fluorescent dye to genomic RNA inside intact human rhinovirus after viral capsid penetration investigated by capillary electrophoresis.

RiboGreen is used for concentration measurements of RNA. Upon binding to the RNA, an approximately 1000-fold increase in sensitivity in comparison with the UV absorbance of the free polynucleotide is observed. In the present work, we demonstrate that this dye can penetrate in a time- and temperature-dependent manner the intact viral capsids of human rhinovirus serotypes 2 and 14, where it forms a fluorescent complex with the viral RNA. Capillary electrophoresis with laser-induced fluorescence detection of virus incubated with RiboGreen shows that the electrophoretic mobility of the viruses remained unchanged upon dye-binding. As shown for human rhinovirus serotype 2, its native conformation was conserved, since it still bound a recombinant soluble receptor fragment derived from the very low density lipoprotein receptor. The labeled RNA was released by heat-induced uncoating of the virus, and the RNA-dye complex could be directly detected if degradation was prevented with an RNase inhibitor. This in vitro labeling of viral RNA encased within a protein shell demonstrates the virion's dynamic nature that temporarily allows access of a low-molecular-mass compound to the otherwise protected RNA. It might be of great value for experiments requiring fluorescent viral particles with an unmodified surface, such as investigations of endocytosis and viral uncoating on the single molecule level.

Kinetics of thermal denaturation of human rhinoviruses in the presence of anti-viral capsid binders analyzed by capillary electrophoresis.

In vivo, the icosahedral capsid of human rhinoviruses undergoes well-defined transitions during the infection pathway. Native virus, sedimenting at 150S, is converted to subviral particles with a sedimentation coefficient of 135S, which have lost the innermost capsid protein VP4. Upon release of the genomic RNA empty 80S capsids remain. Similar structural modifications are observed in vitro upon exposure to low pH and/or elevated temperature. Virions are stabilized against these transitions by various antiviral compounds, which bind to a hydrophobic pocket in the capsid protein VP1. Using capillary electrophoresis the kinetics of viral decay in the presence of such hydrophobic drugs was investigated. Assuming first-order kinetics, the increase of the time constant reflects the extent of stabilization. Exposure of the virions to 55 degrees C after presaturation with the antivirals increased the time constants (as compared to native virus) by a factor of 8-30, from a few minutes to several ten minutes. Denaturation of the stabilized capsid gave rise to heterogeneous material rather than to defined subviral particles. This was confirmed by electron microscopy and indicates that the structural modification of the virus follows a kinetically well-defined pathway which is disturbed by the drugs resulting in disorganized disruption of the virion.