The effect of pH and ionic strength on the electrophoretic separation of acidic glycosaminoglycans

Abstract

Using the electrophoretical methods applied to this study it is possible to determinate the dissociation constants (p K) of acid glycosaminoglycans containing a carboxylic group. The p K-values of the six acid glycosaminoglycans separated from animal connective tissues determined in this work were: hyaluronic acid (HA), p K = 3.0; chondroitin sulfate A (CS-A), p K = 2.8; chondroitin sulfate C (CS-C), p K = 3.3; dermatan sulfate (CS-B), p K = 3.3; heparatin sulfate (HeS), p K = 3.1 and heparin (HeP), p K = 2.4 and were measured at a constant ionic strength of I = 0.164 (NaCl) and at 10 ± 2°C. Variation of ionic strength showed that physiological conditions seem to be most suitable for the electrophoretic separation of the glycosaminoglycans studied. A decrease of ionic strength causes increasing mobility but less accurate spots. In the case of increasing ionic strength the results are vice versa. The second spot for HA very often appeared when pH values higher than 2 were used for electrophoresis. The spot had the same form as the original, high intensity, but an undecided migration in the pH range near the p K value of HA (3.0).