Chromatographic fractionation of Aspergillus endotoxins

Summary Crude preparations of the endotoxins extracted from the mycelia ofAspergillus fumigatus andAspergillus flavus, after preliminary concentration by ammonium sulfate, have been fractionated by column chromatography on DEAE-cellulose. Although the biological activity of the chromatographed preparations was not limited to a single fraction, examination of the most active fractions by starch gel electrophoresis showed no bands common to the two nephrotoxins.The highest hemolytic and toxic activities of the fumigatus toxin were found in different fractions of the chromatographed material, and starch gel electrophoresis showed no bands common to these two fractions.The molecular weight of the flavus toxin has been estimated to be in the range of 32,000 to 34,000 as judged by the results of ultracentrifugation and microelectrodialysis in starch gels of the most toxic fractions.Both of the toxins have been shown to contain small amounts of hexosamine and larger amounts of non-amino sugars.Presented in part at the 46th Annual Meeting of the American Society of Biological Chemists, Atlantic City, New Jersey, April, 1962.     

Chromatographic fractionation of histones on Bio-Gel P.

Chromatography of histones on columns of polyacrylamide beads (Bio-Gel P) was studied to determine what factors influence separations. The fractionation achieved on eluting with dilute HCl depends on the HCl concentration, the temperature, and a variable characteristic of the gel which is independent of its pore size and mesh. Somatic histone will be eluted in three, four, or five separate fractions, depending on these variables. Retardations of histones that apparently result from interactions between their charged groups and those of the gel have a strong influence on resolution between different types. Certain histones are retarded more than others when the HCl concentration or temperature is lowered or if the gel has been hydrolyzed. Increases in retardation lead to resolution of more fractions; but if too extreme, coelution of the most retarded fractions will occur.     

Chromatographic fractionation of Escherichia coli transfer RNA on a new support, naphthoyl-Sepharose.

The noncharged naphthoyl-Sepharose CL-6B has been prepared. Escherichia coli tRNA binds to this new adsorbent in 0.75 M ammonium sulphate at neutral pH at room temperature. Using a negative salt gradient, the tRNAs are eluted in a defined order. The chromatographic pattern is clearly different from those of other commonly used tRNA separation techniques.     

Acid-labile amino acid precursors in the Murchison meteorite. 1: Chromatographic fractionation.

The amino acid content of a hot water extract of the Murchison meteorite can be increased by over 100 per cent by subjecting the extract to acid hydrolysis. The acid-labile compounds in the extract that account for this increase were fractionated by column chromatography on a cation exchange resin. Seventy mole per cent behaved as neutral or acidic compounds and were eluted from the column with an initial water wash. The remaining 30 mole per cent (basic precursors) were retained on the column and were eluted with the free amino acids by aqueous NH4OH. The acid-labile amino acid precursors in the water eluate could be retained and further fractionated on an anion exchange column, indicating that they are acidic compounds.     

The significance of cytochrome c redistribution during the subcellular fractionation of rat liver

1. The redistribution of mitochondrial cytochrome c during homogenization and subcellular fractionation of the liver was studied. Chromatographically homogeneous (14)C-labelled cytochrome c was added in different amounts to liver suspensions immediately before homogenization and the adsorption of radioactivity was determined in cytochrome c fractions extracted at pH4.0, first with water and then with 0.15m-sodium chloride. 2. The soluble cytochrome c remaining in the cell sap after subcellular fractionation was 7% of the calculated amount of cytochrome c passing through a soluble form during the whole process. The total amount of cytochrome c released in a soluble form and subsequently redistributed was 25-30% of the total liver cytochrome c. 3. In the standard microsomal fraction the cytochrome c extracted with water originated entirely from redistribution whereas that extracted with 0.15m-sodium chloride was 80% endogenous. In the mitochondrial fraction both cytochrome c pools were truly endogenous, so that practically none of the mitochondrial cytochrome c released to the soluble cell sap was readsorbed by the mitochondria. 4. These results support our former hypothesis that the cytochrome c extracted with 0.15m-sodium chloride at pH4.0 from the standard microsomes represents the cytochrome c newly synthesized in situ, since it does not originate from redistribution. However, the microsomal pool extracted with water cannot be an intermediate in the postulated transfer of cytochrome c from the microsomal particles to the mitochondria, since this pool arises from redistribution of mitochondrial cytochrome c.