Lectin coprecipitative isolation from crudes by Little Rock Orange ligand.
Matrix ligands are intended for upstream use with dilute crudes on a large scale, splitting out sought-for proteins by coprecipitating them as dense, protected aggregates. Matrix ligand coprecipitation is rapidly, quantitatively reversible, by pH shifting and trapping matrix ligands on ion exchange resin, releasing the sought-for protein. Four lectins, wheat germ agglutinin, peanut lectin, concanavalin A, and Phaseolus vulgaris (red kidney bean) lectins, were coprecipitated from crude extracts, 0.05 to 0.4% crude protein, in a single step using Little Rock Orange matrix ligand. All were compared in specific activities (erythrocyte agglutination) and in SDS-PAGE analysis with the four corresponding commercial lectins purified by affinity chromatography. All four matrix-coprecipitated ligands were specifically active within range of the corresponding vendor (Sigma Co.) affinity chromatography-purified lectins. The matrix ligand coprecipitative technique requires optimization of ligand-protein (crude) ratios, denoted y, and determination of suitable pH ranges for coprecipitation relative to lectin isoelectric pH. These parameters control electrostatic ion pair association: ligand head anion binding to cationic target proteins. The coprecipitative and protective powers of new ligands like Little Rock Orange, their ability to scavenge sought-for lectins from dilute crudes, depend on ligand organic tail-tail association. After the strong anion heads of ligands bind to cationic proteins, their organic tails stack and draw the ligand-protein complexes together as aggregated coprecipitates.
Wu
C W
CW
Biochemistry Department, University of Minnesota, St. Paul 55108, USA.
Lovrien
R
R
Matulis
D
D
D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D004912:Erythrocytes / Q000187:drug effects; D006386:Hemagglutination Tests; D006801:Humans; D037102:Lectins / Q000302:isolation & purification* / Q000494:pharmacology; D008024:Ligands
D037102:Lectins; D008024:Ligands