Characterization of HIV-1 p24 self-association using analytical affinity chromatography.
Analytical affinity chromatography (AAC) was used to detect and quantitate the self-association of p24gag, the major structural capsid protein of human immunodeficiency virus (HIV-1). p24gag was immobilized on a hydrophilic polymer (methacrylate) chromatographic support. The resulting affinity column was able to interact with soluble p24, as judged by the chromatographic retardation of the soluble protein upon isocratic elution under nonchaotropic binding conditions. The variation of elution volume with soluble protein concentration fit to a monomer-dimer model for self-association. The soluble p24-immobilized p24 association process was observed using both frontal and zonal elution AAC at varying pH values; the dissociation constant was 3-4 x 10(-5) M at pH 7. That p24 monomer associates to dimers was determined in solution using analytical ultracentrifugation. The solution Kd was 1.3 x 10(-5) M at pH 7. AAC in the zonal elution mode provides a simple and rapid means to screen for other HIV-1 macromolecules that may interact with p24 as well as for modulators, including antagonists, of HIV p24 protein assembly.
Rosé
S
S
Department of Macromolecular Sciences, Biopharmaceuticals R&D, SmithKline Beecham, King of Prussia, Pennsylvania 19406-0939.
Hensley
P
P
O'Shannessy
D J
DJ
Culp
J
J
Debouck
C
C
Chaiken
I
I
D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D015483:HIV Antibodies / Q000276:immunology; D016655:HIV Core Protein p24 / Q000276:immunology / Q000378:metabolism*; D015497:HIV-1 / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D011108:Polymers; D014461:Ultracentrifugation
D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D016655:HIV Core Protein p24; D011108:Polymers