Binding of biotin labeled horseradish peroxidase using immobilized palladium ions

Palladium ions can be immobilized onto paramagnetic particles using glutaraldehyde activation followed by covalent addition of 2,3-dimercaptosuccinic acid. Loadings as high as 30 wt.% palladium can be achieved using this method. Particles with Pd(II) surface ions can immobilize active biotin labeled horseradish peroxidase from solution. Using equilibrium data at pH 4, 5, 7 and 8, a Langmuir isotherm fit of the data yields values of qm = 66 nm²/g and K_D = 4.9 × 10^-8 M^-1. Horseradish peroxidase without biotin molecules shows low affinity for immobilized Pd(II) ions, with appreciable binding detected only at higher concentrations and at pH values below 8. At pH 8 no active horseradish peroxidase is detected on the Pd(II) ion modified particle surface. Sodium chloride concentration greater than 0.001 M can lower the level bound of both horseradish peroxidase and its biotinylated counterpart to below detection limits. Unmodified particles containing surface amine groups and the modified particles containing only surface 2,3-dimercaptosuccinic acid groups show much weaker affinity for the enzyme and its biotinylated counterpart than particles with immobilized Pd(II) ions.