APPARENT CA2-CONSTANT OF CA2+ CHELATORS INCORPORATED NON-DISRUPTIVELYINTO INTACT HUMAN RED-CELLS( DISSOCIATION)

1. A recently developed method of measuring cytoplasmic Ca2+ buffering in intact red cells was applied to re-evaluate the intracellular Ca2 binding properties of the Ca2+ chelators benz2 and BAPTA. Incorporati on of the free chelators was accomplished by incubating the cells with the acetoxymethyl ester forms (benz2 AM or BAPTA AM). The divalent ca tion ionophore A23187 was used to induce equilibrium distribution of C a2+ between cells and medium. Ca-45(2+) was added stepwise to cell sus pensions in the presence and absence of external BAPTA. To induce full Ca2+ equilibration, the plasma membrane Ca2+ pump was inhibited eithe r by depleting the cells of ATP or by adding vanadate to the cell susp ension. 2. The properties of the incorporated chelators were assessed from the difference in cytoplasmic Ca2+ buffering between chelator-fre e and chelator-loaded cells, over a wide range of intracellular ionize d calcium concentrations ([Ca2+](i)), from nanomolar to millimolar. 3. Under the experimental conditions applied, incorporation of benz2 and BAPTA into the red cells increased their Ca2+ buffering capacity by 3 00-600 mu mol (340 g Hb)(-1). The intracellular apparent Ca2+ dissocia tion constants (K-Di) were about 500 nM for benz2 and 800 nM for BAPTA , values much higher than those reported for standard salt solutions ( K-D) of about 40 and 130 nhl, respectively. These results suggest that , contrary to earlier observations, the intracellular red cell environ ment may cause large shifts in the apparent Ca2+ binding behaviour of incorporated chelators. 4. The possibility that the observed K-D shift s are due to reversible binding of the chelators to haemoglobin is con sidered, and the implications of the present results for early estimat es of physiological [Ca2+](i) levels is discussed.