Subunit Structure of Soybean Agglutinin

Soybean agglutinin purified by affinity chromatography onSepharose-~-aminocaproyl-~-D-galactopyranosylamine was shown to be homogeneous on disc gel electrophoresis, ultracentrifugation, and gel filtration.Isoelectric focusing in a sucrose gradient gave a p1 of 5.81.The extinction coefficient (A:$) was found to be 12.8 cm-'.Measurement of the partial specific volume gave a value of 0.745 ml per g.The intrinsic sedimentation coefficient (s&,~) was estimated as 6.0 =I= 0.12 S, between pH 2.2 and 10.8, and as 2.15 Z!C 0.15 S in 0.1% sodium dodecyl sulfate, 8 M urea, and 6 M guanidine hydrochloride.The molecular weight of the agglutinin, determined by sedimentation equilibrium and by gel filtration, was found to be 122,000 + 1,300 and 120,000 =t 10,000, respectively.Disc gel electrophoresis and gel filtration, both in the presence of sodium dodecyl sulfate, and sedimentation equilibrium in 6 M guanidine hydrochloride gave a subunit molecular weight of 30,000 =t 1,500 and 30,300 & 400, respectively.Four alanine residues per 120,000 g were found by amino-terminal analysis.It is concluded that the agglutinin is a tetramer composed of identical subunits.Two binding sites for N-acetyl-Dgalactosamine were found per 120,000 daltons by equilibrium dialysis and gel filtration, with an association constant K = 3.0 X lo4 liter mole-'.