Sequential Extraction and Quantitative Recovery of Gliadins, Glutenins, and Other Proteins from Small Samples of Wheat Flour (1)
The major flour protein types are the water-soluble albumins, the salt-soluble globulins, the alcohol-soluble gliadin monomers, and the high and low molecular mass glutenin subunits. Several reports describe methods to solubilize flour proteins and then directly analyze them by SDS-PAGE, acid PAGE, or HPLC but do not discuss how to quantitatively recover the proteins from the solvent.
Procedures for sequentially extracting and recovering protein fractions from small flour samples, ranging from 100 mg to 2 g, were tested and compared. The protein patterns in an SDS-PAGE gel illustrate the outcome of the two methods.
Analysis of the proteins obtained by method 1 further confirms the cross-contamination of flour protein fractions. The gliadin 1 fraction included 1B ω-gliadins that eluted in peaks 5 and 7, 1D ω-gliadins (1Dω) in peak 10, and 1A ω-gliadins (1Aω) in peaks 15-19. Nongliadin trace components include HMM-GS in peaks 7-20 and LMM- albumins in fractions 7-14. The main proteins were the five HMM-GS in peaks 7-12 and over 14 different LMM-GS protein bands in peaks 14-21. The main LMM-GS band in peak 15 is the abundant chromosome 1B- encoded LMM-GS with N-terminal sequence of SHIPGL. Gliadins were also extracted by water or 1.5 M NaCl. Most a- and γ-gliadins were then extracted by 50% 1-propanol, many ω-gliadins were only extracted along with the glutenins.
The albumin/ globulin 2 fraction contained little gliadin and encompassed approximately 10% of the total flour protein. Proteins in peaks 11-12 were grain softness proteins, in peaks 14-16, alpha amylase-0.19 inhibitor proteins, and in peaks 17-19, CM3-type alpha-amylase trypsin inhibitors. Several small peaks were combined in lane 20, which contained a-gliadins, and peak 21 contained γ-gliadin. Peak 22 contained a protein related to an avenin storage protein from oats. The 1B1 and 1B2 ω-gliadins were prominent in peaks 2 and 3, the 1D ω-gliadins in peak 6, 1A ω-gliadins in peaks 8-10, and many R- and γ-gliadins in peaks 15-26. Minor traces of HMM-GS were present in peaks 6-13, and minor albumins or globulins were present throughout, including HMM-albumins in fractions 26-30. Trypsin digestion is the first step in the most common techniques for obtaining protein identifications by mass spectrometry, but gliadins and glutenins are somewhat resistant to trypsin digestion.
The gliadin 2 fraction comprised approximately 40% of total flour protein. The albumin/globulin 2 fraction comprised approximately 10% of total flour protein. The glutenin 2 fraction comprised approximately 48% of total flour protein. A similar estimate of 48% was obtained by N analysis of method 2 pellet 1. The sum of the albumin/globulin 2, gliadin 2, and glutenin 2 fractions accounted for 98% of total flour protein.
