SummaryDifferent methods of legumin isolation from faba beans were compared with regard to the removal of vicilin taking into consideration the additional influence of variety. SDS-PAGE, analytical ultracentrifugation and SE-HPLC were used for the analysis of protein composition. SDS-PAGE analysis of the isolated legumin samples revealed, that neither combined salt fraction/isoelectric precipitation nor heating to 73 "C resulted in a complete removal of vicilin. Time-consuming isoelectric zonal precipitation and heating at 83 "C as described by SCHLESIER et al. (Biochem. Physiol. Pflanzen 180 (1985) 225) provides legumin preparations free of vicilin in low yields. Acceptable yields in shorter time were obtained by combining isoelectric precipitation/salt fractionation with SCHLESIER'S procedure. SDS-PAGE analysis showed a certain dependence on variety of the patterns of legumin u-polypeptide chains. Zusarnmenfassung lsolierung von Ackerbohnen-Legumin -eine vergleichende Untersuchung verschiedener MethodenVerschiedene, in der Literatur beschriebene Verfahren fur die Isolierung von Legumin aus Akkerbohnen wurden hinsichtlich ihrer Effizienz zur Vicilin-Entfernung an Hand unterschiedlicher Varietiiten mittels SDS-PAGE, analytischer Ultrazentrifugation und SE-HPLC verglichen. Die Analyse der isolierten Legumin-Praparate mittels SDS-PAGE ergab, daI3 weder das Verfahren der kombinierten Salzfraktionierung/isoelektrischen Fiillung noch das Erhitzen auf 73 "C zur vollstiindigen Entfernung des Vicilins fuhrte. Das zeitaufwendige, von SCHLESIER et al. (Biochem. Physiol. Pflanzen 180 (1985) 225) beschriebene Verfahren der kombinierten isoelektrischen Zonenprazipitation/Hitzebehandlung bei 83 "C ergab vicilinfreie Leguminpraparate, jedoch in geringer Ausbeute. Kombination des Verfahrens der Salzfraktionierung/isoelektrischen Fillung mit dem Verfahren von SCHLESIER lieferte in kurzerer Zeit akzeptable Ausbeuten an vicilinfreiem Legumin. Die SDS-PAGE-Muster der a-Polypeptidketten des Legumins zeigten eine gewisse Sortenabhangigkeit.

The effect of progressive acetylation upon the conformation of the 11S globulin legumin from faba bean has been studied using chemical analysis, UV, fluorescence and CD spectroscopy, viscometry and analytical ultracentrifugation. The modification did not induce complete dissociation of the oligomeric protein. Only 30% of the protein was found to be a dissociated 3S subunit after excessive acetylation, whereas 70% was a dimeric legumin aggregate with a molecular mass of about 700 kDa. The aggregation of the highly modified legumin in high‐ionic‐strength buffer solution leads to soluble higher legumin oligomers. The acetylation resulted in a moderate molecular expansion of legumin due to a changed tertiary structure, whereas the far‐UV circular dichroism spectra did not provide definitive evidence of a decrease in domain‐stabilizing β‐sheet conformations in their secondary structure. © 2000 Society of Chemical Industry

SummarySelected physico-chemical properties of a faba bean protein isolate before and after being acetylated have been investigated using analytical ultracentrifugation, SE-and RP-HPLC, viscometry and hydrophobicity measurements using fluorescence probe techniques. The time course of relative chymotryptic hydrolysis rates was followed by means of the TNBS method and RP-HPLC analysis.The extensive decrease of the 11 S and 7 S protein components of the isolate in favour of a low molecular weight 2 S component after exhaustive acetylation is the proof of the dissociated state of the highly modified derivatives. Both the viscometric measurement and the study of proteolysis breakdown give arguments for a rather unfolded state both of the unmodified and the modified protein isolates. The course of relative chymotryptic hydrolysis rate points to a successive transition from a globular state into a more unfolded one depending on the degree of acetylation. Both the viscometric data and the hydrophobicity measurements revealed a conformational transition at about 60% N-acetylation. This level of modification corresponds to that where a sharp increase of OH-acetylation takes place.

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In OjW emulsions, stabilized by acetylated faba bean protein isolate (AFBPI) which are stable against separation of the disperse phase (coalescence) a mechanical barrier for the separation of the continuous phase (creaming) under centrifugal forces has been detected. For any given constant centrifugal acceleration a constant amount of continuous phase is separated. This type of behaviour resembles that ofcoagulation structures in dispersions of solids.One possibility to determine the stability of emulsions against separation of the continuous phase (creaming) for systems stable against coalescence (separation of the disperse phase) is the use of centrifugation in many variations. ONDRACEK et al. [I], for example, used the speed of rotation which caused separation after a given time. In this kinetic method "stability" means that during centrifugation for 10 min at a constant speed of rotation no separation of the continuous phase takes place. The reason for the stability of emulsions against creaming, may be, a mechanical barrier, which breaks down only at higher centrifugal forces, or the rate of separation of the continuous phase is extremely slow. Therefore 10 min are not sufficient to detect separation.