Analysis of protein-protein interaction by simulation of small-zone size-exclusion chromatography: application to an antibody-antigen association.
The association of two or more macromolecules results in the formation of a complex characterized by a larger Stokes radius than that of its components. Therefore, analytical procedures such as ultracentrifugation and size-exclusion gel chromatography that resolve molecules on the basis of size have been used to characterize the association. In this paper we describe an iterative computer simulation of small-zone size-exclusion gel filtration. The simulation describes univalent and bivalent interactions of proteins of equal and nonequal molecular weight and appears to have both qualitative and quantitative application to the evaluation of protein-protein interaction as revealed by alteration of chromatographic elution profiles. To test the validity of the simulation, the model was applied to an antibody-antigen interaction by determining the association constant (Ka) for the interaction between the binding fragment derived from a human immunoglobulin A rheumatoid factor and the antigenic fragment obtained from a human myeloma immunoglobulin G. The self-consistency of the estimated Ka values obtained with a valence value of 2 in contrast to the lack of self-consistency if an antigenic valence of 1 was assumed was taken to support the ability of the algorithm to reasonably emulate the chromatographic processes of interacting proteins. In conjunction with the computer simulation, a sensitive microcomputer-interfaced chromatography system was assembled, which is capable of analyzing 300 ng of protein in less than 1 h. This combination of rapid reagent-conservative chromatography and simulation analysis may contribute to the usefulness of small-zone gel filtration in studies of protein-protein interaction.
D000936:Antigen-Antibody Complex / Q000032:analysis*; D002850:Chromatography, Gel / Q000379:methods*; D006801:Humans; D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D012217:Rheumatoid Factor / Q000276:immunology; D012984:Software
D000936:Antigen-Antibody Complex; D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D012217:Rheumatoid Factor