The binding of inorganic and organic cations and H+ to cartilage in vitro

The binding of H⁺, Na⁺, Ca²⁺, Ni²⁺, Ce³⁺, chlorpromazine, chloroquine, hexamethonium, paraquat and gentamicin to decalcified cartilage from bovine nasal septum has been studied in vitro. The results indicated that the chondroitin sulphate is the binding material in the cartilage and that a stoichiometric binding occurs to the carboxyl and ester sulphate groups of the chondroitin sulphate. An analysis of the binding by the method of Scatchard was performed. The H⁺ ions were bound to two groups of sites, one representing the carboxyl, the other representing the ester sulphate groups. It was shown that the Scatchard association constants for H⁺ may be converted to pK_a values. Such transformation showed that the carboxyl groups of the chondroitin sulphate had a pK_a value of 4.57 and the ester sulphate groups a pK_a value of 2.60. The data for Na⁺ indicated a binding to three groups of sites. Two of these may represent the carboxyl and ester sulphate groups. The third, which was a small group with a very strong affinity, may represent a specific localised link with a few groups on the chondroitin sulphate. The experiments with Ca²⁺, Ni²⁺, Ce³⁺ and the organic substances indicated that, in contrast to H⁺ and Na⁺, only one binding class was present, which implies that the binding to the carboxyl and ester sulphate groups for these ligands occurs with a similar strength. The affinity of these cations to the cartilage was related to their positive charge, which is the general characteristic for an electrostatic interaction.