Detailed Physicochemical Characterization of the 2S Storage Protein from Rape (Brassica napus L.) ISABELLE SCHMIDT, ² DENIS RENARD, ² DAVID RONDEAU, # PASCAL RICHOMME, # YVES POPINEAU, § AND MONIQUE AUGUSTIN-VIGUIER AXELOS* ,² Unite ´ de Physico-Chimie des Macromole ´cules and Unite ´ de Recherches sur les Prote ´ines Ve ´ge ´tales et leurs Interactions, INRA, Rue de la Ge ´raudie `re, 44316 Nantes Cedex 03, France; and Service Commun d’Analyses Spectroscopiques, Universite ´ d’Angers, 2 Boulevard Lavoisier, 49045 Angers, France Chromatographic, chemical, and spectroscopic techniques were used to characterize the physico- chemical properties of napin purified by preparative chromatography. The molar extinction coefficient was determined (ǫ ) 0.56), and static and dynamic light scattering measurements enabled the average molecular weight (M w ) 13919), the second virial coefficient (A 2 ) 23.95 × 10 -5 mol cm 3 g -2 ), and the hydrodynamic radius (R H ) 1.98 nm) to be determined. No conformational changes were observed by fluorescence and circular dichroism measurements in different buffers at pH 3, 4.6, 7, and 12, confirming the high pH stability of this protein. From MALDI-TOF analysis and after enzymatic digestion, it was found that this purified sample, extracted from the rapeseed variety Express, contained mainly isoform 2SS3_BRANA. KEYWORDS: Rapeseed; napin; isolation; isoform identification; light scattering; mass spectrometry INTRODUCTION Rapeseed (Brassica napus L.) is mainly produced for its high oil content (45-50%). After oil extraction, a meal is obtained containing most of the proteins (30-40%) (1). Originally, its use was limited to animal feeding because of the presence of undesirable substances (glucosinolates, erucic acid). To extend the use of rape oil to human food consumption and to exploit the functional properties of rape proteins in food applications, varieties called “double zero”, without these undesirable mol- ecules, have been developed. In B. napus, there are two major families of storage proteins termed cruciferins and napins, which provide nitrogen and sulfur during germination (2, 3). Cruciferins belong to the 12S globulin class of proteins. These neutral proteins, with a high molecular weight, are composed of several subunits and constitute 26- 65% of the total rape seed protein content, depending on the variety (4). Napins belong to the 2S albumin class of proteins (and are hence water soluble) and represent 15-45% of the total rape seed protein content depending on the variety (4). Napins are low molecular weight proteins (12500-14500). They belong to a multigene family encoded by 10-16 genes (5) and are expressed during seed development as precursors of 21 kDa (6, 7). After their maturation, two peptide chains of 4.5 and 10 kDa result, linked by two disulfide bonds (8, 9). The large chain includes two additional intrachain disulfide bonds, which reinforce the stability of the proteins (10-12). Napins are characterized by their strong basicity (isoelectric point, pI ∼ 11) mainly due to a high amidation of amino acids (13). Many isoforms of napin exist because of the large number of napin genes and differences in proteolytical cleavages. Five isoforms have been first identified according to their molecular weights (10, 14). One of them (isoform BnIb, called 2SSI- _BRANA in the Swiss-prot databank nomenclature) has been totally sequenced and its three-dimensional structure determined by NMR (15). More recently, their sequences and structures were compared from data available from databanks (16). The study clearly identified nine isoforms, but many others exist, which can influence properties such as the hydrophobicity (17). Their separation therefore appears to be complex. The first method of purification developed for these proteins used precipitation by ammonium sulfate (at 40-80% saturation). This method allowed the production of large quantities of proteins, but pigments or contaminants were not totally removed from the protein fractions (18, 19). Chromatographic methods were then developed using either size exclusion chromatography (SEC) (20) or ion exchange chromatography (IEC) (10), which led to highly purified proteins but only on an analytical scale. To study the functional properties of different varieties of napin, a larger scale purification procedure was developed to produce larger amounts of purified napins with most of the contaminating pigments removed (21). Very few studies deal with the physicochemical characteriza- tion of napin because of these difficulties to purify and isolate * Author to whom correspondence should be addressed (fax +33 240 67 5043). ² Unite ´ de Physico-Chimie des Macromole ´cules, INRA. # Universite ´ d’Angers. § Unite ´ de Recherches sur les Prote ´ines Ve ´ge ´tales et leurs Interactions, INRA. J. Agric. Food Chem. 2004, 52, 5995-6001 5995 10.1021/jf0307954 CCC: $27.50 © 2004 American Chemical Society Published on Web 08/20/2004