Characterization of lymphocyte-stimulating blood cell-agglutinating glycoproteins from red kidney beans (Phaseolus vulgaris)

Abstract 1. 1. Commercially available phytohaemagglutinin from red kidney beans (Difco, Phytohemagglutinin P) contains glycoproteins with lymphocyte-stimulating and red and white blood cell-agglutinating activity. By a sequence of steps involving SE-Sephadex C-50, QAE-Sephadex A-50 and Sephadex G-200 chromatography, two different lymphocyte-stimulating agglutinins were isolated in homogeneous form. One was purely leucoagglutinating, while the other was erythroagglutinating and also showed leuco- and mixed agglutinating activity. 2. 2. The physicochemical properties of the agglutinins were studied by analytical ultracentrifugation, isoelectric focusing and polyacrylamide disc electrophoresis. The leucoagglutinin was shown to have the following properties: M r = 140 000, s 0 20, w = 6.8 S, p I = 5.0; the erythroagglutinin respectively: M r = 150 000, s 0 20, w = 7.1 S, p I = 6.5. The partial specific volume of both agglutinins was 0.75 ml/g. 3. 3. The molecular subunit structure of the agglutinins was studied by analytical ultracentrifugation in 5.8 M guanidine hydrochloride, by polyacrylamide electrophoresis in buffers containing sodium dodecyl sulphate and by isoelectric focusing in 8 M urea. Both agglutinins were found to be composed of 4 subunits of similar size held together by noncovalent bonds. Isoelectric focusing of the subunits revealed that the leucoagglutinin contained subunits of only one type, while the erythroagglutinin contained subunits of several types, one type being identical to the subunits of the leucoagglutinin, the other being specific for the erythroagglutinin. A molecular weight of 36 000 was calculated for the subunits. 4. 4. The lymphocyte-stimulating activity of both agglutinins could be abolished by oxidation with NalO 4 , which treatment only slightly affected the agglutinating capacities.