Fractionation of human low density lipoprotein by column chromatography.

We have devised a method to fractionate low density lipoprotein (LDL) into subspecies by means of column chromatography. DEAE-agarose columns, 2.6 X 60 cm, were loaded with LDL (25-45 mg LDL protein) and eluted with a 0.045-0.13 M NaCl gradient. The LDL eluted over a volume of 900 ml. Specific portions of the eluted LDL, reapplied to a column identical with the original, reelute at about the same point. Altering the NaCl concentration of the elution fluid changed the elution volume. The cholesterol-protein ratio of the LDL subfractions was progressively lower in fractions eluting at higher NaCl concentrations. These results indicate the LDL is not a homogenous lipoprotein species but consists of subfractions which differ in at least charge and cholesterol content.     

Fractionation of glycopeptides by affinity column chromatography on concanavalin A-sepharose.

Using [3H]-labeled oligosaccharides, we found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column. This finding is also applicable to N-[14C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which has been purified by paper electrophoresis, were further separated into two fractions by chromatography on a concanavalin A-Sepharose column.     

Enzymically iodinated human salivary proteins. Fractionation and characterization by column chromatography and electrofocusing.

Human salivary proteins were enzymically iodinated by the 125I-lactoperoxidase system. The proteins were than subjected to DEAE-cellulose column chromatography, preparative column electrofocusing and thin-layer polyacrylamide-gel electrofocusing. The radioactivity in the resolved protein pools and bands was determined. Results show that salivary proteins differ in their susceptibility to iodination carried out by this enzymic method. Two major iodine-binding protein fractions were discovered: one behaved like serum albumin on electrofocusing and was most susceptible to iodination by lactoperoxidase, and other had pI characteristics similar to those of salivary amylase. The physiological significance of the iodination of salivary proteins, which can also take place in vivo, is discussed.     

Fractionation of Escherichia coli isoaccepting tRNA species by sepharose 4B column chromatography.

We have determined the elution profile on Sepharose 4B chromatographic column ofthe tRNA isoaccepting species of all 20 amino acids from Escherichia coli MRE 600. Further chromatography on a reversed phase column (RPC-5) is sufficient, in some cases, for a complete purification.     

Fractionation of simian virus 40 DNA fragments by RPC-5 column chromatography

This report describes several modifications of the original radioenzymatic assay for serotonin (4) which increase the sensitivity of the assay 10-fold as well as enhance its reliability. Serotonin is converted to [³H]melatonin, in two steps. First, serotonin is acetylated to N-acetylserotonin by acetic anhydride. The N-acetylserotonin is then incubated with hydroxyindole-O-methyltransferase and S-[methyl-³H]adenosyl methionine and is converted to [³H]melatonin. The radioactive melatonin is extracted with toluene-isoamyl alcohol (7:3), dried, reconstituted, isolated by one-dimensional, silica gel, thin-layer chromatography, and counted in a liquid scintillation counter. The assay is specific and sensitive to approximately 5 pg of serotonin and thus can be used to measure serotonin levels in single brain nuclei or microliter quantities of biological fluids. The assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

Fractionation of heparin by affinity chromatography on covalently-bound human alpha-thrombin

Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of $\text{N-α-tosyl-L-glycyl-L-prolyl-L-arginine-}\text{p}\text{-nitroanilide}$ hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.     

Fractionation by size of casein micelles on controlled-pore glass

Casein micelles have been separated from skim milk by chromatography on CPG-10 3000 glass beads. Fractionation of the micelles according to size has been demonstrated. Polyacrylamide gel electrophoresis of urea treated micelles reveals that different relative amounts of the major casein components occur in the various micelle fractions. No discernible dissociation of the micelles into monomeric caseins has been observed.     

Fractionation of human milk oligosaccharides by high-performance liquid chromatography

An ion-exchange chromatographic system was used to isolate several human milk oligosaccharides, the elution being carried out with a linear gradient of a sodium borate buffer. Lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, II and III, lacto-N-difucohexaose I and 2'-alpha-fucosyllactose can be separated by this method.     

Fractionation of dG homooligonucleotides using RPC-5 column chromatography at high pH.

dG oligomers, prepared by partial acid hydrolysis of dG_n, were fractionated in large quantity according to chain length by RPC-5 column chromatography at high pH (0.1 m NaOH). Resolution of oligonucleotides up to approximately the 30-mer was achieved. The oligomers were characterized by partial chemical digestion and analysis on 20% polyacrylamide-urea gels.