Determination of Kinetic Parameters within a Single Nonisothermal On-Flow Experiment by Nanoliter NMR Spectroscopy.

Conventional methods to determine the kinetic parameters for a certain reaction require multiple, separate isothermal experiments, resulting in time- and material-consuming processes. Here, an approach to determine the kinetic information within a single nonisothermal on-flow experiment is presented, consuming less than 10 µmol of reagents and having a total measuring time of typically 10 min. This approach makes use of a microfluidic NMR chip hyphenated to a continuous-flow microreactor and is based on the capabilities of the NMR chip to analyze subnanomole quantities of material in the 25 nL detection volume. Importantly, useful data are acquired from the microreactor platform in specific isothermal and nonisothermal frames. A model fitting the experimental data enables rapid determination of kinetic parameters, as demonstrated for a library of isoxazole and pyrazole derivatives.

Net charge affects morphology and visual properties of ovalbumin aggregates.

The effect of ovalbumin net charge on aggregate morphology and visual properties was investigated using chromatography, electrophoresis, electron microscopy, and turbidity measurements. A range of differently charged ovalbumin variants (net charge ranging from -1 to -26 at pH 7) was produced using chemical engineering. With increasing net charge, the degree of branching and flexibility of the aggregates decreased. The turbidity of the solutions reflected the aggregate morphology that was observed with transmission electron microscopy. Increasing the stiffness of the aggregates transformed the solutions from turbid to transparent. Artificially shielding the introduced net charge by introducing salt in the solution resulted in an aggregate morphology that was similar to that for low-net-charge variants. The morphology of heat-induced aggregates and the visual appearance of the solutions were significantly affected by net charge. We also found that the morphology of ovalbumin aggregates can be rapidly probed by high-throughput turbidity experiments.

Heat-induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics.

The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly pure ovalbumin, the decrease in nondenatured native protein showed first-order dependence. The activation energy obtained with different techniques varied between 430 and 490 kJ*mole(-1). First-order behavior was studied in detail using differential scanning calorimetry. The calorimetric traces were irreversible and highly scan rate-dependent. The shape of the thermograms as well as the scan rate dependence can be explained by assuming that the thermal denaturation takes place according to a simplified kinetic process where N is the native state, D is denatured (or another final state) and k a first-order kinetic constant that changes with temperature, according to the Arrhenius equation. A kinetic model for the temperature-induced denaturation and aggregation of ovalbumin is presented. Commercially obtained ovalbumin was found to contain an intermediate-stable fraction (IS) of about 20% that was unable to form aggregates. The denaturation of this fraction did not satisfy first-order kinetics.