Wright, D. J. (1973) Structural studies on the major reserve proteins of vicia faba (L). Doctoral thesis, Durham University.
The storage globulins of Vicia faba were extracted and the component proteins separated using various procedures. The sub-unit structures of the resulting globulin fractions were investigated and, in the case of legumin, the isolation of the constituent sub-units examined. These sub-units were characterised using gel electrophoresis, isoelectric focussing, amino acid composition, N-terminal amino acid and N-terminal sequence data. Possible causes of artifactual heterogeneity and anomalous behaviour in gel electrophoresis and gel isoelectric focussing were discussed. The onset of synthesis and composition of the globulins during seed development were investigated. Legumin was prepared in a very pure form by extraction in a neutral saline buffer followed by zonal isoelectric precipitation; in contrast, vicilin, prepared by this method, was contaminated with legumin. Structural studies on legumin indicated that it was composed of equimolar proportions of two types of sub-unit, acidic (a) and basic (P), with molecular weights of 36,200 and 22,000 respectively. Isoelectric focussing, sequence data and electrophoresis in a variety of gel systems showed these sub-units to be heterogeneous, and, on the basis of electrophoretic information five sub-units were positively identified viz, ɑ(_1), ɑ(_2),β(_1),β(_2) and β(_3). It was proposed that legumin consisted of twelve sub-units, six acidic and six basic, but because of the heterogeneous nature of the a and g sub-units, the exact composition of the native legumin molecule could not be ascertained. The possibility of naturally-occurring variants of legumin was suggested. The proposed sub-unit model was compared with those published for other legumin-like seed proteins. Vicilin was formed prior to legumin during seed development, although the rate of synthesis of the latter was faster, so that in the mature seed the ratio of legumin to vicilin was between 3:1 and 4:1 by weight. While the sub-unit structure of legumin, as examined by SDS gel electrophoresis, remained reasonably constant during seed development, that of vicilin changed, suggesting the existence of more than one protein in the vicilin fraction. Together with this evidence, results from electrophoretic studies of various preparations of the 4.7 soluble globulin (vicilin) indicated that the latter probably consisted of at least three distinct proteins.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||18 Sep 2013 16:00|