Phospholipids of Clostridium butyricum. IV. Analysis of the positional isomers of monounsaturated and cyclopropane fatty acids and alk-1'-enyl ethers by capillary column chromatography

The positional isomers of the cyclopropane fatty acids of Clostridium butyricum phospholipids have been analyzed by capillary column gas-liquid chromatography. Greater than 95% of the methylenehexadecanoic acids was the 9,10 isomer. On the other hand, 60-70% of the hexadecenoic acid precursors was the Delta(7) isomer, and the remainder was the Delta(9) isomer. Of the methyleneoctadecanoic acids 75-80% was the 11,12 isomer, with the remainder being the 9,10 isomer. There were approximately equal amounts of the Delta(9)- and Delta(11)-octadecenoic acids in the phospholipids. This study reveals a surprisingly strong specificity of the cyclopropane synthetase for the (n-7) series of monoenoic fatty acids. An analysis by capillary column chromatography of the monoenoic and cyclopropane aldehyde dimethylacetals derived from the plasmalogens (1-alk-1'-enyl-2-acyl-glycero-phosphatides) of C. butyricum revealed the presence of the same positional isomeric mixtures of the 16- and 18-carbon monoenoic residues in approximately the same ratios as were found in the fatty acids. In the formation of the cyclopropane alk-1'-enyl ethers there was also specificity for the (n-7) series, but it was not as strong as that seen in the fatty acids. The ratio of the 7,8 isomer to the 9,10 isomer was higher in the methyl-enehexadecanals than in the corresponding fatty acids. This paper extends the use of Golay capillary columns to the analysis of the positional isomers of plasmalogen aldehydes as their dimethylacetal derivatives.     

THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na₂HPO₄. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied.