The application of size exclusion chromatography and computer simulation to study the thermodynamic and kinetic parameters for short-lived dissociable protein aggregates.
We describe a method to study the monomer-dimer equilibrium of human growth hormone (hGH) making use of a very short size exclusion high-performance liquid chromatographic column and rapid flow rates. By adjusting the flow rate and thus the retention time on the column, the dissociation of the hGH dimer can be observed. Using computer simulation, both the equilibrium constant for dissociation and the dissociation rate constant can be determined directly, followed by the indirect determination of the association rate constant. This method is potentially useful for determining the thermodynamic the kinetic parameters of aggregation of many protein-protein (homodimers and heterodimers) and protein-ligand systems whose rates of interaction are too rapid to be studied by conventional techniques.
Patapoff
T W
TW
Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, California 94080.
Mrsny
R J
RJ
Lee
W A
WA
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003198:Computer Simulation; D005069:Evaluation Studies as Topic; D013006:Growth Hormone / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012985:Software Design; D013816:Thermodynamics
D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D013006:Growth Hormone