title: Kinetics of nonproteolytic incorporation of a protein ligand into thermally activated alpha 2-macroglobulin: evidence for a novel nascent state.
abstract: We have previously shown that antigens complexed to the receptor-recognized form of alpha(2)-macroglobulin (alpha(2)M*) demonstrate enhanced immune responsiveness mediated by the low density lipoprotein receptor-related protein LRP/CD91. Recently, we developed a proteinase-independent method to covalently bind antigens to alpha(2)M*. Given the potential applications of this chemistry, we analyzed the kinetics, thermodynamics, and pH dependence of this reaction. The incorporation of lysozyme into alpha(2)M* was a mixed bimolecular second-order reaction with a specific rate constant of 91.0 +/- 6.9 m(-1) s(-1), 50.0 degrees C, pH 7.4. The activation energy, activation entropy, and Gibbs' free energy at 50.0 degrees C were 156 kJ mol(-1), 266 J mol(-1) K(-1), and 70 kJ mol(-1), respectively. The rate of incorporation increased as a function of pH from pH 5.0 to 7.0 and was unchanged thereafter. Furthermore, the reaction between alpha(2)M* and lysozyme was irreversible. The data are consistent with a two-step mechanism. In the first step, alpha(2)M* reforms its thiol ester bond, entering a reactive state that mimics the proteolytically induced "nascent state." In the rate-limiting second step, the reformed bond quickly undergoes nucleophilic attack by lysozyme. The kinetic equations derived in this study are the basis for optimizing the formation of stable alpha(2)M*.antigen complexes.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Adlakha
authors.forename: J P
authors.initials: JP
authors.lastname: Hart
authors.forename: S V
authors.initials: SV
authors.lastname: Pizzo
mesh_terms: D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009113:Muramidase / Q000378:metabolism; D013696:Temperature; D000511:alpha-Macroglobulins / Q000378:metabolism*
chemical_list: D008024:Ligands; D000511:alpha-Macroglobulins; C421864:hen egg lysozyme; D009113:Muramidase