Isolation and subunit composition of 11 S globulin of linseed (Linum usitatissimum L.)

Abstract

Early reports on linseed proteins are mostly concerned with solubilization of total proteins from oil-free meal by different solvents and their isolation [6, 81, paper electrophoretic characterization and amino acid composition [7]. Classification of proteins in defatted linseed meal, based on their solubilities in various solvents, has shown the salt-soluble protein to be a major fraction in linseed [3, 91. Employing different physicochemical techniques, it has been demonstrated that total proteins of linseed consist of at least 3 components of which the major one, apparently, is a high molecular weight globulin [Z, 41. MADHUSUDHAN et al. [5] isolated the major fraction of linseed proteins and reported its physicochemical characteristics. Here, we report the isolation of linseed globulin by a different procedure and its characterization by sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation analysis. Material and methodsSeeds, purchased locally, were milled and defatted by repeated extractions, alternately, with diethyl ether and acetone at 4 C . Total proteins were extracted from the oil-free meal, following desolventization at room temperature and passing through a 0.25 mm screen, with phosphate buffer containing I .O M NaCl using meal :solvent ratio of 1 : 20. The clear extract, obtained by repeated centrifugations (3000 x g for I5 min) at 20 C, was diluted with 5.5 times its volume ofdistilled water and kept at 4 'C for several hours. The cryoprecipitated globulin was separated by centrifuging at 4 C and dissolved in a small volume of the extractant. The process of precipitation and centrifugation was repeated twice. Finally, the isolated protein was dissolved in the extractant buffer to which sodium azide was added at a concentration of 0.02"/,. This sample was called "crude globulin".The crude globulin fraction was eluted through a Sephadex (3-200 column using phosphate buffer as described elsewhere (21. The ultraviolet absorption spectrum of the eluate at 280 nm showed a peak with a prominent shoulder. The eluate fraction corresponding to the peak were pooled. concentrated by ultrafiltration using a PM-10 membrane with a cut-off limit of 10000 daltons and rechromatographed to obtain a somewhat symmetric peak. The eluate volume under this peak was diluted in the ratio 3 : 5 with distilled water and concentrated 10-fold by ultrafiltration using the same membrane. This was referred to as "purified globulin".Electrophoresis was carried out on 8.79< polyacrylamide disc gels ( 5 x 0.5 cm. length x diameter) using 0.5 "6 SDS -0.05 M Tris-glycine (pH 8.3) buffer system [2]. The relative intensity of the stained bands on polyacrylamide gel was determined by scanning the cylindrical gels with a Hitachi dual wavelength chromatoscanner. Peak areas were determined by integration from the densitometric scans of the PAGE gels and the relative proportions of the major subunits were calculated.The sedimentation coefficient (s;,,,) of purified globulin was determined according to BEHL...

Effect of extraction conditions on composition, surface activity and rheological properties of protein isolates from flaxseed (Linum usitativissimum L)

“…Linin is a typical 11S globulin with a molecular weight of 250 000–300 000 and an isoelectric point at pH 4.75 26. 29 A low‐molecular‐weight protein fraction that might correspond to the 2S albumins30 could not be detected in the isolates used. Despite this similar protein composition, FI contains about four times as much pentosans, whereas FM is nearly free of phytic acid.…”

Purification, Crystallization and Preliminary X-ray Characterization of Prunin-1, a Major Component of the Almond (Prunus dulcis) Allergen Amandin

“…Prunin-1 shares the properties of being highly water soluble and readily cold-precipitable with other globular proteins of the 11S family, such as legumin of peas (49), arachin of peanuts (50), glycinin of soybeans (51), and the major globulins of linseed (52) and coconut (53). In this work, amandin was isolated from defatted almond flour by water extraction and cryoprecipitation and Pru1 was further purified by anion exchange, hydrophobic interaction, and size exclusion chromatography.…”

Anti-Cancer Properties of Flaxseed Proteome

“…Among flaxseed proteins, globulin takes precedence as the principal component, its size being reported to be in the range of 252-298 kDa (for 11-12 S Globulins). Comprising about 3% α-helical and 17% β-structures [83][84][85], globulins, akin to albumins, have not undergone extensive examination for their anti-cancer attributes. Nevertheless, some evidence indicates the potential anti-cancer properties of globulins.…”