A New Data Analysis Method to Determine Binding Constants of Small Molecules to Proteins Using Equilibrium Analytical Ultracentrifugation with Absorption Optics

@article{citeulike:112860, abstract = {In principle, equilibrium analytical ultracentrifugation (AU) can be used to quantify the binding stoichiometry and affinity between small-molecule ligands and proteins in aqueous solution. We show here that heteromeric binding constants can be determined using a data-fitting procedure which utilizes a postfitting computation of the total amount of each component in the centrifuge cell. The method avoids overconstraining the fitting of the radial concentration profiles, but still permits unique binding constants to be determined using measurements at a single wavelength. The computational program is demonstrated by applying it to data obtained with mixtures of a 500-Da molecule and interleukin-2, a 16-kDa protein. The 1:1 binding stoichiometry and heteromeric dissociation constants (Kab) determined from centrifuge data at two different wavelengths are within the 4-9 [mu]M range independently determined from a functional assay. Values for Kab have been obtained for ligands with affinities as weak as 500 [mu]M. This AU method is applicable to compounds with significant UV absorbance (~0.2) at concentrations within ~5- to 10-fold of their Kab. The method, which has been incorporated into a user procedure for IgorPro (Wavemetrics, Oswego, OR), is included as supplementary material.}, author = {Arkin, Michelle and Lear, J. }, citeulike-article-id = {112860}, doi = {10.1006/abio.2001.5396}, journal = {Analytical Biochemistry}, keywords = {chemical, method}, month = {December}, number = {1}, pages = {98--107}, posted-at = {2006-05-30 15:28:10}, priority = {0}, title = {A New Data Analysis Method to Determine Binding Constants of Small Molecules to Proteins Using Equilibrium Analytical Ultracentrifugation with Absorption Optics}, url = {http://dx.doi.org/10.1006/abio.2001.5396}, volume = {299}, year = {2001} }

TY - JOUR ID - citeulike:112860 L3 - citeulike-article-id:112860 TI - A New Data Analysis Method to Determine Binding Constants of Small Molecules to Proteins Using Equilibrium Analytical Ultracentrifugation with Absorption Optics JF - Analytical Biochemistry VL - 299 IS - 1 SP - 98 EP - 107 N2 - In principle, equilibrium analytical ultracentrifugation (AU) can be used to quantify the binding stoichiometry and affinity between small-molecule ligands and proteins in aqueous solution. We show here that heteromeric binding constants can be determined using a data-fitting procedure which utilizes a postfitting computation of the total amount of each component in the centrifuge cell. The method avoids overconstraining the fitting of the radial concentration profiles, but still permits unique binding constants to be determined using measurements at a single wavelength. The computational program is demonstrated by applying it to data obtained with mixtures of a 500-Da molecule and interleukin-2, a 16-kDa protein. The 1:1 binding stoichiometry and heteromeric dissociation constants (Kab) determined from centrifuge data at two different wavelengths are within the 4-9 [mu]M range independently determined from a functional assay. Values for Kab have been obtained for ligands with affinities as weak as 500 [mu]M. This AU method is applicable to compounds with significant UV absorbance (~0.2) at concentrations within ~5- to 10-fold of their Kab. The method, which has been incorporated into a user procedure for IgorPro (Wavemetrics, Oswego, OR), is included as supplementary material. KW - chemical KW - method AU - Arkin, Michelle AU - Lear, J PY - 2001/12/01/ UR - http://dx.doi.org/10.1006/abio.2001.5396 ER -