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Isolation of cadmium-binding components from proteins of flaxseed (Linum usitatissimum L.) Lei, Bo

Abstract

Flaxseed contains the toxic heavy metal cadmium (Cd) at concentrations often exceeding the recommended maximum dietary intake limit. The objectives of this study were to investigate the distribution of Cd-binding components in flaxseed, and to establish a protocol for separating Cd-binding components from storage proteins. The results indicated that over 80% of Cd in flaxseed (NorMan cultivar) was extracted with proteins into Tris buffer. Fractionation of protein extracts by ion exchange chromatography yielded a major Cd-binding fraction eluting at 0.1 M NaCl with over 50% of the eluted Cd and less than 8% of the eluted proteins, while the major storage proteins were eluted into the 0.25 M NaCl fraction containing over 60% of the eluted protein and less than 25% of the eluted Cd. The Cd level in flaxseed was strongly influenced by growing location. Similar trends in percent distribution of Cd and protein for 15 flaxseed samples (5 cultivars grown in 3 locations) suggest that this fractionation protocol could form the basis for developing an industrial process to produce flaxseed proteins with low Cd content for use as food ingredients. Further separation of the 0.1 M NaCl eluted major Cd-binding fraction by size exclusion chromatography resulted in three peaks. The major components in the first two peaks were a 19 kDa protein and a 14 kDa protein, while 0.6-0.9 kDa constituents in the third peak were comprised of unusual amino acids or organic acids and a 649.7 Da component tentatively identified as Cd-(γ-Glu-Cys)₂ Gly. The second and third peaks bound over 40% of the Cd contained in the flaxseeds. This study also demonstrated in vitro protective effects of flaxseed components against toxicity by Cd and H₂O₂. The protein extract and the major Cd-binding fraction at a concentration of 110 μg/mL reduced Cd (300 μM) toxicity in THP-1 cell culture by 14% and 44%, respectively, and reduced H₂O₂ (0.06%) toxicity by 48% and 89%, respectively. The protein extract was also found to promote THP-1 cell growth in a dose dependent manner. Further investigations should be conducted to explore the underlying mechanisms and potential applications of flaxseed constituents.

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