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The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3s subunit forming a 7s intermediate. At a level of 75 -80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place.The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 x cm2 . s p l a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.Legumin is one of the main storage proteins in the seeds of pea (Pisum sativum L.). It belongs to the group of the socalled l l S globulins, having sedimentation coefficients between lls and 14S, and molecular masses between 300 kDa and 400 kDa [l]. These proteins possess an oligomeric structure characterized by an arrangement of six subunits (3s components) [2 -41. The molecular mass of pea legumin was reported by different authors to be in the range 330 -410 kDa [l, 5, 61. By means of small-angle X-ray scattering, a value of 359 f 25 kDa was obtained [7]. Disulphide-bonded polypeptide pairs with molecular mass 54 kDa constitute most of the 3s subunit of the legumin [8]. In addition, polypeptide pairs varying in molecular mass over 35-58 kDa have been observed which associate in various ways to give rise to different molecular forms of legumin IS].Due to very similar quaternary structures [9, lo], the 11s plant proteins show an analogous behaviour dissociating into subunits [l 11. Therefore, the decay of oligomeric structure by an increase of negative charge caused by succinylation effects similar changes in the structure of different 11s proteins. Although there are specific differences in the dissociation behaviour of the peanut [12], sunflower seed [13] and rape seed [14] 11s globulins, a step-by-step decay of the native structure, and a marked change in conformation at a critical step of succinylation, were observed in each case.The present paper deals with the effect of succinylation on the oligomeric structure of the 11s globulin (legumin) from pea. Structural changes of the protein were followed using ultracentrifugation, viscometry, circular dichroism and fluorescence spectroscopy. The physicochemical properties of the dissociation products, including molecular mass and shape, were investigated by a combination of hydrodynamic methods, including dynamic light scattering. MATERIALS AND METHODSLegumin was isolated and purified according to Gueguen et al. [15]. Lyophilized preparations containing small amounts of aggregated material were purified additionally by gel filtration on a ...

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An isolation procedure for the 12 S rapeseed globulin is described which includes precipitation by dialysis, purification using gel chromatography on Sephadex G-200, and ion-exchange chromatography on DEAE-Sephadex A-50. The isolated globulin represents a neutral protein with an isoelectric point at pH 7.25--determined by isoelectric focusing--and a relation of the acidic to basic amino acid residues (epsilon Glu, Asp--Amide ammonia: epsilon Arg, Lys, His) of 1.0. As in other storage globulins high contents of glutamic (19%) and aspartic (10%) acid and a low content of sulphur containing amino acids are characteristic for the amino acid composition. Amongst the basic amino acids arginine has the highest percentage (7%). Opposite to results of other authors the sugar content of the globulin is low (0.5%). From the amino acid composition an average hydrophobicity according to Bigelow was calculated which amounts to be 1041 cal/res. (4.36 kJ/res.).

An improved procedure for the isolation and purification of the 11 S globulin from sunflower seeds (helianthinin) is described, including a combined purification by gel chromatography and ionexchange chromatoThe protein has a sedimentation constant of szo, = 12.8 x lo-'' s, a STOKES radius of 57 A and a diffusion constant of 3.76 x lo-' cm2 s-' (the last two derived from gel chromatographic analysis). Hence it follows a molecular weight of M, = 305000. The isoelectric point determined by isoelectric focusing lies at pH 4.7.High contents of glutamic (26%) and aspartic (14%) acid and arginine (9.7 %) as well as a low content of sulphur containing amino acids are characteristic for the amino acid composition. 59 % of the acidic amino acids are present in an amidated form. The globulin contains 12 disulphide bridges per molecule. graphy.Helianthinin is the main storage protein in sunflower seed [l]. It represents a typical 11 S globulin with a molecular weight near 300000, dissociation properties depending on pH and ionic strength, a low content of a-helix, a relatively high amount of p-conformation, and a high content of arginine, glutamic and aspartic acid (2-51. In the present paper an improved purification for the globulin combining gel filtration and ion-exchange chromatography, the corrected amino acid composition, the content of amide-nitrogen and SS/SHgroups, and some physico-chemical properties of the globulin are described. Material and Methods Protein isolationGround dehulled sunflower seeds were defatted with diethyl ether and extracted with a 5% solution of sodium chloride in a cold room. The protein was precipitated with cold water and twice chromatographed on Sephadex G-200 as described in a previous paper [6].