Characterization of oat proteins and aggregates using asymmetric flow field-flow fractionation

J. Ray Runyon, Lars Nilsson, Johan Alftrén, Björn Bergenståhl

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Abstract

The soluble proteins and protein aggregates in Belinda oats were characterized using asymmetric flow field-flow fractionation (AF4) coupled with online UV–vis spectroscopy and multiangle light-scattering detection (MALS). Fractions from the AF4 separation were collected and further characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The AF4 fractogram of the oat extracts revealed three peaks which were determined to be monomeric forms of soluble proteins, globulin aggregates, and β-glucan, respectively. The early eluting monomeric proteins ranged in molar mass (MM) between 5 and 90 kg/mol and in hydrodynamic diameter (D h) from 1.6 to 13 nm. The MM at peak maximum of the globulin aggregate peak was found to be ∼300 kg/mol and the D h was measured to be ∼20 nm. SDS-PAGE of the collected fraction across this peak revealed two bands with MM of 37 and 27 kg/mol which correspond to the α and β subunits of globulin indicating the elution of globulin aggregates. A third peak at long retention time was determined to be β-glucan through treatment of the oat extract with β-glucanase and by injection of β-glucan standards. The amount of soluble protein was measured to be 83.1 ± 2.3 wt.%, and the amount of albumin proteins was measured to be 17.6 ± 5.7 wt.% of the total protein in the oats. The results for Belinda oat extracts show that the AF4-MALS/UV platform is capable of characterizing the physicochemical properties such as MM and hydrodynamic size distribution of proteins and protein aggregates within a complicated food matrix environment and without the need to generate protein isolates.
Original languageEnglish
JournalAnalytical and Bioanalytical Chemistry
Volume405
Issue number21
Pages (from-to)6649-6655
ISSN1618-2642
DOIs
Publication statusPublished - 2013
Externally publishedYes

Keywords

  • Protein aggregation
  • Light scattering
  • Field-flow fractionation
  • Oats

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