Kinetics of antigenic peptide binding to the class II major histocompatibility molecule I-Ad.
Using high-performance size-exclusion chromatography and fluorescence spectroscopy, we investigated the kinetics of fluorescent peptide reactions with detergent-solubilized I-Ad, a class II molecule of the mouse major histocompatibility complex. At pH 7.0 and 37 degrees C the half-time for the binding of a fluorescein-labeled synthetic peptide representing ovalbumin amino acids 323-339 [FOva-(323-339)Y] to I-Ad was 32 hr, independent of added fluorescent peptide concentration in the range 5-200 microM. Peptide exchange experiments were also carried out, where it was found that the half-time of FOva-(323-339)Y binding was equal to the half-time of dissociation of the Texas Red-labeled peptide TROva-(323-339)Y. These experiments show that slow peptide binding to class II major histocompatibility molecules may be limited by the slow dissociation of prebound peptides. Paradoxically, however, this kinetic behavior--a peptide concentration-insensitive on-reaction with a half-time for peptide binding approximately equal to the half-time for dissociation--can be modeled in more than one way. Models involving a kinetic intermediate are particularly attractive. The kinetics were significantly different at pH 5.0. The half-times for peptide binding and dissociation were approximately 7 times shorter than at pH 7.0. In addition the complex of the I-Ad alpha/beta heterodimer with FOva-(323-339)Y was unstable and dissociated into separate alpha and beta chains with a half-time of approximately 7 hr.
Tampé
R
R
Stauffer Laboratory for Physical Chemistry, Stanford University, CA 94305.
McConnell
H M
HM
D000818:Animals; D000941:Antigens / Q000378:metabolism*; D002460:Cell Line; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008285:Major Histocompatibility Complex; D008433:Mathematics; D051379:Mice; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding
D000941:Antigens; D000949:Histocompatibility Antigens Class II; D010455:Peptides