Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation

Abstract

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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