Partial purification of thrombopoietin using lectin chromatography.
Thrombopoietin (TPO) was partially purified from the plasma of thrombocytopenic rabbits by ammonium sulfate fractionation and lectin chromatography. Thrombopoietic activity was measured with an in vivo assay that measures platelet production by the incorporation of 75Se-selenomethionine (75SeM) into newly forming platelets in mice. Thrombopoietin was precipitated from rabbit plasma by an ammonium sulfate saturation of 60%-80%. The ammonium sulfate precipitate was further fractionated by application to a column of wheat germ agglutinin (WGA) bound to agarose. The biological activity was eluted from the WGA column with 0.2 M N-acetylglucosamine. The minimum dose that stimulated thrombopoiesis was decreased 1000-fold to 1.3 microgram/g body weight by this first lectin step. The biological activity eluted from the WGA column was then applied to a column of concanavalin A (ConA) bound to agarose and eluted with 0.1 M alpha-methylmannose. The ConA step produced an additional two-fold increase in specific activity and decreased the minimum dose to 0.65 microgram/g body weight. Stimulation of thrombopoiesis was not associated with an increased peripheral platelet count. Although the purification scheme presented provided a 7000-fold increase in specific activity compared to plasma from thrombocytopenic rabbits, analysis of the WGA and ConA fractions by gel-permeation high-performance liquid chromatography (GP-HPLC) and SDS-PAGE indicated that many proteins were still present at this stage of the purification procedure.
D000818:Animals; D001681:Biological Assay; D001792:Blood Platelets / Q000166:cytology*; D002846:Chromatography, Affinity; D003208:Concanavalin A; D006023:Glycoproteins / Q000302:isolation & purification*; D037102:Lectins; D008970:Molecular Weight; D010976:Platelet Count; D011817:Rabbits; D013926:Thrombopoietin / Q000032:analysis / Q000302:isolation & purification*; D014909:Wheat Germ Agglutinins
D006023:Glycoproteins; D037102:Lectins; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; D013926:Thrombopoietin