Berberine sulphate binding to mast cell polyanions: a cytofluorometric method for the quantitation of heparin.

Abstract

A cytofluorometric study on the binding of the fluorescent cationic dye Berberine sulphate to some tissue anions is reported. Measurements were performed on peritoneal cells and on models containing heparin or DNA. The dye was found to be suitable for cytofluorometric quantitation of heparin. At pH 4 a staining equilibrium was established when the dye apparently binds selectively to phosphate and sulphate containing polyanions. Binding to DNA was more resistant to salt extraction than the binding to heparin. Blocking experiments demonstrated that most of the heparin sulphate of mast cells was available for dye binding while the phosphate groups of nuclear DNA were largely blocked by basic protein groups. The fluorescence resulting from binding to nucleic acids was estimated to be 2% or less of the fluorescence intensity of an average mast cell. Model experiments demonstrated a linear relationship between the amount of heparin and the intensity of fluorescence. Fluorescence spectroscopy showed that the dye had a slightly higher emission maximum when bound to heparin than to DNA indicating that the dye binds to heparin in an associated molecular form. The study also revealed some interesting, although yet unexplained differences in the properties of the resulting fluorescence when the dye was bound to different polyanions. Heparin-bound Berberine sulphate showed a rapid exponential fading upon continuous illumination, both in models and in mast cells, while the DNA bound dye showed a slow increase in fluorescence intensity during continuous illumination.