title: Affinity chromatography with collapsibly tethered ligands.
abstract: We introduce a novel affinity chromatography mode in which affinity ligands are secured to the media surface via collapsible tethers. In traditional affinity chromatography, the immobilized ligands act passively, and their local concentration is static. In collapsibly tethered affinity chromatography, the ligand can move dynamically in response to external stimuli, a design that enables marked changes in both the local concentration of the ligand and its surrounding environment without exchange of solvent. Using the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) as a scaffold for ligand and hapten attachment, we were able to achieve controlled mobility and microenvironment alteration of the affinity ligand Ricinus communis agglutinin (RCA120). The glycoprotein target, asialotransferrin, was loaded onto a column in which PIPAAm was partially substituted with both RCA120 and lactose. At 5 degrees C, the column retained the glycoprotein, but released most (95%) of the asialotransferrin upon warming to 30 degrees C. This temperature-induced elution was much greater than can be explained by temperature dependency of sugar recognition by RCA120. The simplest explanation is that upon thermally induced dehydration and collapse of the PIPAAm chains, coimmobilized RCA120 ligand and lactose hapten are brought into closer proximity to each other, enabling immobilized lactose to displace affinity-bound asislotransferrin from the immobilized RCA120 lectin.
authors.affiliation: Department of Research and Development, Amersham Biosciences K.K., 3-25-1 Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan. hidenori.yamanaka@jp.amershambiosciences.com
authors.forename: Hidenori
authors.initials: H
authors.lastname: Yamanaka
authors.forename: Kimihiro
authors.initials: K
authors.lastname: Yoshizako
authors.forename: Yoshikatsu
authors.initials: Y
authors.lastname: Akiyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sota
authors.forename: Yukio
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Yasuro
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
mesh_terms: D001212:Asialoglycoproteins / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D007785:Lactose / Q000378:metabolism; D008024:Ligands; D037121:Plant Lectins / Q000378:metabolism*; D014168:Transferrin / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001212:Asialoglycoproteins; D008024:Ligands; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; D014168:Transferrin; C017031:asialotransferrins; D007785:Lactose