PubMed · 3046429
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H G Barth, W E Barber, C H Lochmüller, R E Majors, F E Regnier. 1988-06-15. Column liquid chromatography.. https://doi.org/10.1021/ac00163a025
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Urea-induced effects in clostridial glutamate dehydrogenase (GDH, EC 1.4.1.2) were studied by spectrophotometry, circular dichroism, FPLC, affinity chromatography and PAGE. Denaturation of enzyme occurred over a narrow range of urea concentrations (2.5-3.5 M), accompanied by inactivation of enzyme with a similar rate constant. The contribution of instantaneous inhibition by urea was also ascertained. FPLC studies of urea-treated GDH gave no evidence for dissociated oligomeric fragments of the hexamer in the presence of subdenaturing concentrations of urea. Likewise a mixture of fully 5,5'-dithiobis-(2-nitrobenzoic acid)-modified GDH hexamers and unmodified enzyme in 2 M urea failed to give rise to hybrid molecules. Exposure of unmodified GDH to high concentrations of urea led to the dissociation of hexamers to denatured monomers followed by association to form non-specific high-M(r) aggregates. This conclusion was confirmed by native gradient PAGE experiments. Various specific ligands stabilized the enzyme against urea-induced inactivation, succinate and 2-oxoglutarate being particularly effective. This protection of the native state was enhanced in ternary complexes, and the complex most resistant to urea-induced inactivation was the productive ternary complex GDH-NADH-2-oxoglutarate. Native gradient PAGE experiments indicate that these protecting ligands preserve the native hexameric structure of GDH.
Myelin basic protein (MBP) occurs in multiple forms. Three of these isoforms from human MBP (HMBP) have been highly purified. HMBP, component 1 (18.5 kDa HMBP-1), was purified by ion-exchange chromatography at pH 10.6 in 2 M urea. During this ion-exchange chromatography, a fraction (Fraction 3), which contained HMBP component 3 (monophosphorylated or deamidated 18.5 kDa) and 17.2 kDa HMBP, was collected and further purified by fast protein liquid chromatography, which separated 17.2 kDa HMBP and HMBP component 3. When the latter was subjected to limited thrombic digestion, all of HMBP component 3 not phosphorylated at theonine 98 was cleaved. This digestion mixture was separated on Sephadex, and yielded pure component 3, monophosphorylated at theonine 98 (HMBP 3pT98), for which phosphate analysis yielded approximately 1 mole P/mole protein, and NMR showed only one phosphorylation site present. Circular dichroism (CD) studies were carried out on dilute solutions of HMBP-1 (18.5 kDa), 17.2 kDa HMBP, and HMBP3pT98 (phosphorylated 18.5 kDa). The CD spectrum of HMBP-1 was similar to that reported for rabbit MBP-1 and bovine MBP-1, but the spectra of 17.2 kDa HMBP and HMBP 3pT98 were distinctly different from HMBP-1. When analyzed by best-fit computations, 17.2 kDa HMBP showed about a 9% increase of ordered structure, and a greater increase, about 12%, was estimated for HMBP3pT98, attributable to beta-structure and beta turn.
The potential of ion chromatography to serve as a new reference method principle for the determination of total sodium and potassium in human serum was investigated. Sample pretreatment consisted of acidic dilution and filtration and detection was based on conductivity. Methods for the separate and simultaneous determination of both analytes were investigated. Further, the influence of calibration (using either a single-point calibration or a standard curve) on method imprecision, inaccuracy and analysis time was examined. The best method performance was achieved by separate analysis using single-point calibration with bracketing analysis scheme. For this variant, the mean total coefficient of variation for sodium and potassium was 1.0% and the mean method bias was -0.2% for sodium and 2% for potassium, as determined with three control materials from the National Institute of Standards and Technology. Our results are comparable to those of reference methods based on flame atomic emission spectrometry. Therefore, we consider ion chromatography as a valuable reference methodology for the determination of total sodium potassium in human serum.