58255-81-5Relevant articles and documents
Two-Step Affinity Chromatography. Model Systems and an Example Using Biotin-Avidin Binding and a Fluoridolyzable Linker
Lin, Wun-Chen,Morton, Thomas Hellman
, p. 6850 - 6856 (2007/10/02)
Two-step affinity chromatography is a general designation for a separation procedure that involves the following pair of sequential steps: specific binding of a biomolecule to a solid support then selective elution using a chemically specific cleaving agent to sever the tether that is attached to the biomolecule.This procedure is exemplified for the case of the enzyme papain covalently modified at its active site.A molecule of biotin is connected to a cysteine residue via a hydrophilic tether incorporating a fluoridolyzable -CH2OSiMe2OCMe2- unit.The biotinylated papain then binds a molecule of streptavidin (a protein that has four identical binding sites for biotin), and this complex in turn binds to a molecule of biotin that is covalently linked to a solid support.After this first step, the covalently modified papain is then selectively eluted by fluoride-catalyzed hydrolysis of an oxygen-silicon bond in the hydrophilic tether.Homogeneous kinetics of model systems in aqueous solution show no evidence for saturation of the rate of fluoride-catalyzed hydrolysis of silicon-oxygen bonds.
