ANALYSIS OF RICIN TOXIN PREPARATIONS FOR CARBOHYDRATE AND FATTY ACID ABUNDANCE AND ISOTOPE RATIO INFORMATION

Wunschel, David S. ; Kreuzer-Martin, Helen W. ; Antolick, Kathryn C. ; Colburn, Heather A. ; Moran, James J. ; Melville, Angela M.

关键词: ABUNDANCE;    ARABINOSE;    CARBOHYDRATES;    CARBOXYLIC ACIDS;    CASTOR;    CASTOR OIL;    DERIVATIZATION;    GLUCOSAMINE;    ISOTOPE RATIO;    MANNOSE;    PRECIPITATION;    PROTEINS;    PURIFICATION;    QUADRUPOLES;    REMOVAL;    SEEDS;    SPECTROSCOPY;    STABLE ISOTOPES;    TOXINS;    XYLOSE;   

DOI  :  10.2172/1000835
RP-ID  :  PNNL-19064
PID  :  OSTI ID: 1000835
Others  :  Other: 400904120
Others  :  TRN: US201101%%668

美国|英语

来源: SciTech Connect

【 摘 要 】

This report describes method development and preliminary evaluation for analyzing castor samples for signatures of purifying ricin. Ricin purification from the source castor seeds is essentially a problem of protein purification using common biochemical methods. Indications of protein purification will likely manifest themselves as removal of the non-protein fractions of the seed. Two major, non-protein, types of biochemical constituents in the seed are the castor oil and various carbohydrates. The oil comprises roughly half the seed weight while the carbohydrate component comprises roughly half of the remaining “mash” left after oil and hull removal. Different castor oil and carbohydrate components can serve as indicators of specific toxin processing steps. Ricinoleic acid is a relatively unique fatty acid in nature and is the most abundant component of castor oil. The loss of ricinoleic acid indicates a step to remove oil from the seeds. The relative amounts of carbohydrates and carbohydrate-like compounds, including arabinose, xylose, myo-inositol fucose, rhamnose, glucosamine and mannose detected in the sample can also indicate specific processing steps. For instance, the differential loss of arabinose relative to mannose and N-acetyl glucosamine indicates enrichment for the protein fraction of the seed using protein precipitation. The methods developed in this project center on fatty acid and carbohydrate extraction from castor samples followed by derivatization to permit analysis by gas chromatography-mass spectrometry (GC-MS). Method descriptions herein include: the source and preparation of castor materials used for method evaluation, the equipment and description of procedure required for chemical derivatization, and the instrument parameters used in the analysis. Two types of derivatization methods describe analysis of carbohydrates and one procedure for analysis of fatty acids. Two types of GC-MS analysis is included in the method development, one employing a quadrupole MS system for compound identification and an isotope ratio MS for measuring the stable isotope ratios of deuterium and hydrogen (D/H) in fatty acids. Finally, the method for analyzing the compound abundance data is included. This study indicates that removal of ricinoleic acid is a conserved consequence of each processing step we tested. Furthermore, the stable isotope D/H ratio of ricinoleic acid distinguished between two of the three castor seed sources. Concentrations of arabinose, xylose, mannose, glucosamine and myo-inositol differentiated between crude or acetone extracted samples and samples produced by protein precipitation. Taken together these data illustrate the ability to distinguish between processes used to purify a ricin sample as well as potentially the source seeds.