Formation and structure of SaOhyA products.
A, an example illustrating the LC-MS method for separating and detecting hydroxy-fatty acids in SaOhyA assays (Table 1). The experiments shown are samples from an assay containing 18:1(9Z) in the presence (vermilion trace) or absence (blue trace) of SaOhyA (2.5 μg). There was almost complete conversion of 18:1(9Z) to h18:0 in this experiment. B, mass spectrum of h18:0 product peak from an SaOhyA assay containing 18:1(9Z). The molecular ion (m/z = 299.1) corresponds to the addition of water, and the m/z = 185.1 fragment was diagnostic for hydroxylation at carbon-10 as indicated in the fragmentation diagram (inset). C, mass spectrum of h18:1 from an SaOhyA assay containing 18:2(9Z,12Z). The same m/z = 185.1 peak was diagnostic for hydroxylation at carbon-10 in this fatty acid as indicated in the fragmentation diagram (inset). Chromatographic conditions and MS parameters are detailed under “Experimental procedures.”