Renaturation of formyltetrahydrofolate synthetase from urea and guanidinium chloride solutions

Formyltetrahydrofolate synthetase from Clostridium cylindrosporum and Clostridium acidi-urici was denatured in 6 M urea and 4 M guanidinium chloride. Viscometric, fluorimetric and ultracentrifugal measurements were used to determine that the protein is completely unfolded under these conditions. The polypeptide chains refold upon dilution of the denaturant-protein solutions to give final concentrations of 0.5 M urea or 0.1 M guanidinium chloride. In the presence of NH4+, but not in its absence, the refolded proteins associate to produce the catalytically active tetramer. Refolding and reassociation were followed by measuring changes in protein fluorescence and by determination of sedimentation constants. Under most conditions 80% of the enzymic activity is recovered.     

Performance of large-size superconducting coil in 0.21T MRI system

A high-temperature superconductor (HTS) was used on magnetic resonance imaging (MRI) receiver coils to improve image quality because of its intrinsic low electrical resistivity. Typical HTS coils are surface coils made of HTS thin-film wafers. Their applications are severely limited by the field of view (FOV) of the surface coil configuration, and the improvement in image quality by HTS coil is also reduced as the ratio of sample noise to coil noise increases. Therefore, previous HTS coils are usually used to image small in vitro samples, small animals, or peripheral human anatomies. We used large-size HTS coils (2.5-, 3.5-, and 5.5-in mean diameter) to enhance the FOV and we evaluated their performance through phantom and human MR images. Comparisons were made among HTS surface coils, copper surface coils, and cool copper surface coils in terms of the signal-to-noise ratio (SNR) and sensitivity profile of the images. A theoretical model prediction was also used to compare against the experimental result. We then selected several human body parts, including the wrist, feet, and head, to illustrate the advantage of HTS coil over copper coil when used in human imaging. The results show an SNR gain of 200% for 5.5-in HTS coil versus same size copper coils, while for 2.5- and 3.5-in coils it is 250%. We also address the various factors that affect the performance of large size HTS coils, including the coil-to-sample spacing due to cryogenic probe and the coil-loading effect.     

One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection

Miniaturized electrophoresis was successfully coupled with native fluorescence detection for direct analysis of proteins in one- and two-dimensional separations. The detection setup was based on direct observation of the UV-induced fluorescence of proteins using a CCD camera and a Hg (Xe) lamp for sample excitation. Protein mixtures were readily separated by size on a 1-cm segment of the one-dimensional gel in 8 min, and a detection limit of 0.04 ng per band was achieved. The dynamic range of the system was larger than 2 orders of magnitude. Miniaturized slab gel electrophoresis was performed on a special holder designed to couple isoelectric focusing with SDS-PAGE. Two-dimensional separation, including rehydration of IEF strip and fluorescence detection was completed in 2.5 h. Approximately 200 protein spots from Escherichia coli were detected on a 1 cm(2) area. A detection limit of 0.1 microg of total protein was achieved. The operation should be amenable to total automation.     

Nanoliter-volume selective sampling of peptides based on isoelectric points for MALDI-MS

A simple way to selectively isolate peptides based on their isoelectric points (pI) for MALDI mass spectral analysis is described. An applied voltage was used to electromigrate peptides into a capillary. The capillary was modified with a zwitterionic surfactant, 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), to suppress the electroosmotic flow (EOF) during injection. Hence, either the cationic or the anionic peptides were introduced, depending on the voltage polarity. By controlling the pH, selective loading of peptides was performed to isolate trace components from a mixture. The injected sample plugs were subsequently spotted in nanoliter volumes for MALDI-MS analysis. No significant sample losses resulting from selective sampling were detected. Low attomole-level detection of peptides (adrenocorticotropic hormone fragment 18-39, pI 4.25) was achieved from a mixture containing other peptides (angiotensin I, pI 6.92, and bradykinin, pI 12.00) at 100 000-fold higher concentrations.