Flow field-flow fractionation of wheat proteins

The use of flow field-flow fractionation (FFF) for separation and size characterisation of wheat proteins was examined. Optimum resolution for a range of wheat protein extractability fractions from defatted Katepwa red spring wheat flour was attained with 0.05 M acetic acid containing 0.002% FL-70 as eluant using a channel flow of 1.0 ml/min and a cross flow of 5.0 ml/min for the 127 lan thick channel. Very small samples (approximately 1 mu g of protein) could be characterised. Salt extractable proteins (albumin and globulin) showed two major peaks and a poorly resolved third peak with elution times corresponding to Stokes diameters (d) of 4.5, 7.2 and 10.9 nm while 70% ethanol extractable proteins (gliadin) showed a major peak (d = 7.4 nm) and an incompletely resolved second peak (d = 9.3 nm). Acetic acid extractable glutenin showed major peaks at elution times corresponding to Stokes diameters of 7.4, 9.9 and 16.9 nm, as well as an early eluting poorly resolved peak (d = 5.8 nm). High molecular weight polymeric glutenin extracted from the acetic acid unextractable protein by sonication in dilute acetic acid or HCl showed a poorly resolved early eluting minor peak containing smaller diameter proteins followed by a broad peak containing proteins ranging in Stokes diameter from about 11 nm to well over 36 nm. Reduction of these proteins with DTT resulted in fractograms showing two peaks with diameters of 6.7 and 11.4 nm, corresponding to low M(r) and high M(r) glutenin subunits, respectively. On the basis of these results, flow FFF appears to offer promise as a rapid procedure for the fractionation and size characterisation of very small amounts of wheat proteins. Its use for studying the larger polymeric wheat proteins may be particularly advantageous since, unlike gel filtration, electrophoresis and size-exclusion HPLC techniques, resolution is not impeded by an exclusion limit. (C) 1996 Academic Press Limited