Abstract
A rapid and sensitive spectrophotometric assay for free fatty acids using rhodamine 6G dye base in benzene as a fatty acid indicator is described. The procedure involves no phase separations or transfers, making it much more convenient and sensitive than previously published colorimetric methods of fatty acid determination. The assay was designed to follow hydrolysis of fatty acids from spinach chloroplast membrane galactolipids by bean leaf galactolipid lipase. Assay procedure consists of ( 1) hydrolysis of fatty acids from spinach galactolipids, ( 2) extraction of unesterified fatty acids into petroleum ether, and ( 3) determination of the fatty acid concentration in an aliquot of the petroleum ether phase by the rhodamine 6 G method. This assay is sensitive to 10 nmoles of fatty acid and has a linear standard curve up to 125 nmoles of fatty acid. The millimolar extinction coefficient for linolenate, under optimal conditions, is approximately 27 cm −1. In addition to previously reported interferences by acetone, alcohols, and mineral acids, we found negligible to moderate interference by methyl stearate, phosphatidylcholine, cholesterol, partially purified spinach galactolipids, and spinach subchloroplast particle pigments. These interferences could be corrected for by running appropriate blanks and zero times.
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