Reversed-phase high-performance liquid chromatography of oat proteins: application of cultivar comparison and analysis of the effect of wet processing.
Lapvetelainen A. | Bietz J.A. | Huebner F.R.
Oat protein fractions were characterized by reversed-phase high-performance liquid chromatography (RP-HPLC). Salt-soluble, alcohol-soluble, and alkali-soluble protein fractions were extracted with 1.0 M NaCl, 52% ethanol, and 1% sodium dodecyl sulfate (SDS) in 0.05 M borate buffer (pH 10), respectively. RP-HPLC analysis conditions were first optimized for column performance, concentration of ion-pairing reagent (trifluoroacetic acid [TFA]), protein reductive state, and elution temperature. These analysis conditions were used to characterize five Finnish oat cultivars (Puhti, Ryhti, Veli, Nasta, and Virma). In addition, effects of processing on oat protein composition were analyzed in high-protein oat flour and steamed oat groats derived from the oat starch process. Wet processing only slightly influenced RP-HPLC separation profiles of protein fractions. The greatest difference between high-protein oat flour and groats was the amount of salt-soluble components eluting during the first 15 min. Prolamin patterns of Puhti, Ryhti, and Virma clearly differentiate these cultivars. Prolamin patterns of cultivars Veli and Nasta were similar, half the genome in these cultivars is from the same parent. For all cultivars, RP-HPLC separations of salt- and alkali-soluble proteins were similar. However, quantities of some components differed, particularly those in the alkali-soluble fraction. RP-HPLC reproducibility was generally good, although replicate alcohol extractions revealed some components not consistently present. These were probably due to the extractant (52% ethanol). Other trials suggested that 70% ethanol may be a more reliable oat prolamin extractant for RP-HPLC analysis. These results emphasize the importance of thoroughly optimizing RP-HPLC analysis conditions for protein characterization.
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