A chelating agent such as EDTA or EGTA used with a dilute TRIS buffer at pH 7.2-7.5 was used in order to effect a good separation of brush borders from the epithelium of the small intestine. A good separation was not obtained in low concentrations of TRIS buffer or saline alone. Brush borders were not obtained when the calcium-chelate complex of EDTA or EGTA was used, and only a partial fractionation was obtained when the magnesium complex of EDTA was tried. The involvement of calcium was further illustrated by adding calcium salts directly to the fractionation medium; separation was prevented when sufficient calcium had been added to saturate the chelating agent. It was found that there was no precise optimum concentration for EDTA but a separation could not be obtained below 2.4 mM/1. The effect of changing the pH of the buffer was also investigated and it was demonstrated that the onset of the ability to release brush borders coincided approximately with the ionization of the third acid radical of the chelating agent. This is in keeping with the suggested hypothesis that EDTA acts by chelating calcium ions.
From these and electron-microscope studies it is suggested that the binding of calcium ions is an important factor in the maintenance of the stability of the epithelial cell membrane.