Photodegradation products of bilirubin studied by high pressure liquid chromatography, gel permeation chromatography, nuclear magnetic resonance and mass spectrometry.
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Neutrophil elastases are serine proteinases released during acute and chronic inflammatory states. We have developed a novel isolation method for neutrophil elastase, involving conventional gel chromatography followed by adsorption of protein at low ionic strength on a high-performance liquid chromatography gel permeation column. The bound elastase is then eluted by application of higher ionic strength. This adsorption step at low ionic strength, a step to be avoided in most purification methods, was used to advantage here to allow isolation of homogeneous material. This purification procedure should be useful for quick, simple bulk preparation of the enzyme.
Detergent gel chromatography was successfully applied for the separation of protein subunits and lipid micelles of [14C]Vincristine-treated and sodium dodecyl sulphate-solubilized liver cell plasma membranes. The elution profiles of solubilized membranes depended on the pore size of the commercial agarose and agarose-polyacrylamide gels used. It was found that most of the membrane-bound Vincristine was associated with the solubilized lipid micelles and only a very small proportion was bound to protein subunits.
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Analytical gel chromatography has been used to examine self-association of bovine neurophysins I and II under several sets of conditions. The data provide no evidence for associated species larger than the dimer. Association constants and Stokes radii of both monomer and dimer are very similar for both proteins in both 0.1 M KOAc, 0.16 M KCl and 0.1 M KPO4, 0.16 M KCl at pH 5.6 and 25 degrees C. The average values derived for the Stokes radii of the monomer and dimer under these conditions are 14.5 +/- 0.7 and 23.0 +/- 0.4 A, respectively. These results confirm the conclusion of Rholam and Nicolas [(1981) Biochemistry 20, 5837-5843] that the monomer and, to a lesser extent, the dimer are highly assymmetric. The Stokes radius of the monomer calculated by Rholam and Nicolas (op cit.) is approximately 30% larger than the value derived here. This discrepancy is probably the result of end-on penetration of the gel by elongated molecules [Y. Nozaki, N. M. Schechter, J. A. Reynolds, and C. Tanford (1976) Biochemistry 15, 3884-3890]. In contrast to Tellam and Winzor [(1980) Arch. Biochem. Biophys. 201, 20-24], it was found that neurophysin II does not exist solely as the dimer in 0.1 M KPO4, pH 5.6, although substitution of 0.1 M KPO4 for 0.1 M KOAc does increase the association constant by a factor of seven. Addition of 1.4 M LiCl at pH 8.1 also increases the association constant sevenfold, as well as increasing the Stokes radius of the monomer approximately 20%. The effects of ionic strength are consistent with the conclusion of Nicolas et al. [(1978) J. Biol. Chem 253, 2633-2639] that formation of the dimer depends upon hydrophobic bonding.
Lipophilic gel chromatography using Sephadex LH-20 helps in separating alpha-, beta- + -gamma, and delta-tocopherol and also in separation of their oxidation products e.g. the tocopherolquinones or other oxidation products. This preseparation can help to overcome analytical problems due to the complexity of synthesis mixtures of tocopherol oxidation procedures as well as in separation of complex physiological matrices. Determination of the preseparated tocopherols, tocopherolquinonesand tocopherolhydroquinones can then be achieved by means of GC-MS measurement of the free substances or their trimethylsilyl derivatives.
High performance gel chromatography (HPGC) was used to separate lipoproteins on the basis of their size and to generate lipoprotein profiles for plasma collected from patients with different lipoprotein phenotypes. These profiles provided a direct measurement of low density lipoprotein (LDL)-cholesterol which was more precise than LDL-cholesterol values calculated by the Friedewald equation. In addition, LDL-cholesterol concentrations were obtained in patients with combined hyperlipidemia in whom LDL-cholesterol could not be accurately calculated by the Friedewald equation. The response of LDL-cholesterol to the drug gemfibrozil was reliably monitored and in addition changes in LDL particle size could be assessed from the LDL apolipoprotein B/cholesterol ratio. HPGC also assisted in the diagnosis of type III hyperlipidemia by revealing a characteristic lipoprotein profile. HPGC-derived lipoprotein profiles provided additional useful clinical information for combined hyperlipidemia (Fredrickson lipoprotein phenotypes IIb, III).
The gel chromatographic patterns of a monomer-dimer-tetramer system under kinetic control have been studied by computer simulation. In no case do the derivative curves of the concentration profiles exhibit more than bimodality in these systems and in some cases are found to exist as a single, almost symmetric, peak. The monomer-dimer reaction affects the dimer-tetramer reaction only slightly and the same is true for the effect of the dimer-tetramer reaction on that of the monomer-dimer. All systems can be fit, to a first approximation, by an empirical formula which suggests any reaction with a first order half life within one and one half or two orders of the transport time will be under kinetic control.
A method for purifying platelet membrane glycoproteins IIb and IIIa to homogeneity has been developed. The procedure involves high-pressure gel filtration chromatography using a TSK-4000SW column in the presence of sodium dodecyl sulfate. This technique is capable of rapidly preparing milligram quantities of each glycoprotein with greater than 90% recovery. The use of this technique should aid in defining the structural and functional properties of GPIIb and GPIIIa.
Using gel chromatography two different 99mTc-PPi complexes (complex I and II) were observed under various pH-values. In neutral medium complex II was the main product while in acidic or alkaline media complexes I and II were formed. Our results related to the organ distribution in mice revealed that complex II is a bone-seeking agent whereas complex I concentrates in the kidneys.
The combination of lipophilic gel chromatography and high-resolution glass capillary chromatography has been shown to be an important means of identification and quantification of steroid urinary metabolites. Some problems in the use of capillary columns, such as coating with polar and apolar stationary phases, column performance, thermal stability and injection systems have been investigated and this type of chromatography has been used in the analysis of 33 dihydroxysteroids. Gel chromatography is a method complementary to high-efficiency gas chromatography. The steroids are eluted according to the number of hydroxyl groups in the molecules.
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Combination of gel chromatography on Sephadex and polyacrylamide gel electrophoresis was used to obtain molecular weight- and electrophoretic mobility-uniform fractions of humic acids from soil of different types (chernozem, sod-podzol, grey forest, and red soils). Extinction and color (E4/E6) coefficients, as well as weight distribution (%) were studied for all humic acids and their fractions. The fractions differed by the weight distribution (%). Sod-podzol and red soils had twice the amount of high-molecular and half the amount of low molecular HA fractions as compared to chernozem and grey forest soil.
Information on molecular composition of human albumin preparations is important for characterization of these protein mixtures. Permeation chromatography in gel columns is best suited for separative analysis with respect to time consumption and experimental and instrumental expenditure. Standardization, precision and reproducibility of this quality control method have been especially considered in our investigations and the best conditions for practical performance and evaluation have been established. Results will be discussed in comparison to those of sedimentation analysis in the ultracentrifuge.
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