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-A(d), 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-A(d) was 32 hr, independent of added fluorescent peptide concentration in the range 5-200-mu-M. 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 almost-equal-to 7 times shorter than at pH 7.0. In addition the complex of the I-A(d) alpha/beta heterodimer with FOva-(323-339)Y was unstable and dissociated into separate alpha and beta-chains with a half-time of almost-equal-to 7 hr.