Use of electrophoretic techniques in determining the composition of seed storage proteins in alfalfa

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Abstract
Holoprotein molecular weights and polypeptide composition can be determined for complex mixtures of oligomeric proteins using two-dimensional electrophoretic techniques. The variety of two-dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa. These techniques can be applied to studies of storage proteins in other seeds as well as non-seed storage proteins. The major seed storage proteins in alfalfa are medicagin (a legumin-like globulin), alfin (a vicilin-like globulin) and a family of lower molecular weight albumins (LMW1–3). These comprise 30 %, 10 %, and 20 %, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin is a heterogeneous oligomeric protein (Mr ∼ 150 000) composed of polypeptides ranging in size from Mr 14 000 to 50 000 (α1-α6; 50 000, 38 000, 32 000, 20 000, 16 000 and 14 000, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires high concentrations of salt for solubilisation. It is comprised of a family of individually distinct subunits, each composed of an acidic polypeptide (A1–A9; Mr 49 000 to 39 000) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24 000, 23 000 and 20 000, respectively). This pairing is highly specific and two families are recognizable on the basis of the B polypeptide (B3 or B1 /B2). Subunits (Mr ∼ 50 000–65 000) are assembled as trimers (8S) or larger oligomers (12S–15S) in mature seeds. The lower molecular weight albumins (LMW1–3) are acidic (pI < 6), and consist of sets of disulphide-bonded polypeptides (Mr 15 000 and 11 000).