title: Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A.
abstract: Aggregation of beta-lactoglobulin A under acidic buffer conditions was studied in hydrophobic interaction chromatography. At high ammonium sulfate concentrations, pH 4.5 and 4 degrees C, UV chromatograms revealed a maximum of three peaks for beta-lactoglobulin A concentrations greater than 5 mg/mL, suggesting three distinct aggregate species. The size of the smallest aggregate (tetramer) and its stoichiometric relationship to the other two aggregates (octamer and dodecamer) were determined from the chromatographic data and a simple mass balance model. These stoichiometries agreed with those determined in a separate study by on-line low-angle laser light scattering. In addition, the association constants describing the formation of octamer from two tetramer molecules and the formation of dodecamer from the octameric and tetrameric species were found to be (2.4 +/- 0.5) X 10(4) M-1 and (3.3 +/- 0.8) X 10(3) M-1, respectively. Analysis of the beta-lactoglobulin A system is based on a model in which aggregates form in solution upon injection before adsorbing to the column matrix. The column retains those species formed in solution and induces little change in the relative amounts of each species. These results illustrate another example by which multiple peaks can arise in high-performance liquid chromatography, beyond the previously described studies of protein conformational changes during chromatography.
authors.forename: N
authors.initials: N
authors.lastname: Grinberg
authors.forename: R
authors.initials: R
authors.lastname: Blanco
authors.forename: D M
authors.initials: DM
authors.lastname: Yarmush
authors.forename: B L
authors.initials: BL
authors.lastname: Karger
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000032:analysis*; D008433:Mathematics; D011506:Proteins / Q000032:analysis*; D013696:Temperature
chemical_list: D007782:Lactoglobulins; D011506:Proteins