The amounts of both Ca
2+ and Na
+, x and y, respectively, bound to C-type chondroitin sulfate (Chs) from an aqueous solution of CaCl
2 mixed with NaCl were obtained by the membrane equilibrium method. To estimate x and y under the effect of the Donnan equilibrium, [Ca
2+] and [Cl
-] for both sides of the dialysing tube and [Chs] for the inner side were analysed. The binding isotherm for Ca
2+ was of the Langmuir type, and both x and k
Ca2+, the binding constant for Ca
2+, decreased with ionic strength, J. On the other hand, the binding isotherm for Na
+ was not of the Langmuir type but its shape was explained as the competitive adsorption of the two components, Na
+ and Ca
2+, because Ca
2+ binds more strongly to Chs than Na
+ and, therefore, the effect of coexistence of Ca
2+ with Na
+ cannot be neglected when the Na
+ binding is examined. It was also suggested that y and k
Na+ decreases with J when the amount of the occupied site by the Ca
2+ is kept constant at x. The degree of the ionization, z, of Chs was found to be 0.40-0.41 for Na
2Chs and 0.20-0.21 for CaChs by this membrane equilibrium method assuming x+y+z=1. These values coincide with those obtained from the vapour pressure osmometry by the authors and with the literature values calculated with considering the Manning theory.
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