title: Use of high-performance size-exclusion, ion-exchange, and hydrophobic interaction chromatography for the measurement of protein conformational change and stability.
abstract: Size-exclusion, ion-exchange, and hydrophobic interaction chromatographic techniques able to detect conformational changes induced by urea were developed for three globular proteins: bovine serum albumin, lysozyme, and trypsin. Alterations in tertiary structure were manifest chromatographically by highly reproducible changes in peak height, retention, and appearance of multiple peaks. Denaturation equilibria and kinetics obtained by classical physical methods, such as fluorescence intensity measurements for bovine serum albumin and enzyme activity for trypsin, could be correlated to particular changes in chromatographic behavior. The chromatographic methods utilized were selective toward specific structural changes and monitored independent denaturation steps via multiphasic kinetics. The correlation of chromatographic behavior to both independent measures of conformational changes and to assays which measure loss in biological activity, in the case of select proteins indicates that non-denaturing high-performance liquid chromatography can be a useful tool to detect and quantitate perturbations of protein three dimensional structure which result in a loss in biological activity.
authors: Pharmaceutics Department, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877.
authors: J
authors: J
authors: Withka
authors: P
authors: P
authors: Moncuse
authors: A
authors: A
authors: Baziotis
authors: R
authors: R
authors: Maskiewicz
mesh_terms: D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D006868:Hydrolysis; D008970:Molecular Weight; D009113:Muramidase / Q000032:analysis; D011487:Protein Conformation; D012710:Serum Albumin, Bovine / Q000032:analysis; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000032:analysis
chemical_list: D012710:Serum Albumin, Bovine; D009113:Muramidase; D014357:Trypsin