| A new approach has been developed to investigate the microenvironment of glycosaminoglycans, and in particular, heparin. It is based on the use of the cationic dye Auramine O as an extrinsic probe; but rather than deriving information from changes in spectral or chiroptical properties, the catalytic influence of heparin on the dye's hydrolysis is exploited. In this regard, heparin is unique among the glycosaminoglycans of animal connective tissue, and therefore the reaction can serve as the basis for a specific heparin assay. The variation of reaction rate with polymer, dye and hydronium ion concentration, ionic strength, and various ratios of these parameters are all consistent with an interpretation of the catalysis in terms of the well known catalytic properties of polyelectrolytes. It is concluded that the catalysis is a consequence of counterion condensation, as strictly defined in Manning's limiting laws of polyelectrolyte solutions. Most significantly, the data demonstrate the participation of hydronium ion in this process, and in the differentiation of heparin from other glycosaminoglycans. Though the rate enhancements cannot be dealt with quantitatively by a Bronsted-Bjerrum-Manning type approach, the data can be straightforwardly explained based on the concept of a critical charge density, as described in Manning's limiting laws. The explanation is offered in terms of Manning's linear charge density parameter, (xi), and the all or none aspect of counterion condensation. Based on the relative catalytic activity of different heparin preparations and subfractions, the implications of this work with regard to the unique biochemical properties of heparin are examined. |
