INTRACELLULAR FREE ZINC AND ZINC BUFFERING IN HUMAN RED-BLOOD-CELLS

Zn2+ has been allowed to equilibrate across the red cell membrane using two agents that increase membrane permeability to this ion: the ionophore A23187 and the specific carrier ethylmaltol. Extracellular free Zn2+ was controlled with EGTA (1,2-di(2-aminoethoxy)ethane-NNN'N'tetra-acetic acid)) buffers, except in the case of ethylmaltol, which itself acts as a buffer. Measurement of cellular zinc content at different levels of free Zn2+ facilitated the study of intracellular Zn2+ binding. It was also possible to estimate intracellular free Zn2+ concentration in untreated cells using a "null-point" technique. Intracellular zinc was found to consist of an inexchangeable component of about 129-mu-mol/10(13) cells and an exchangeable component of 6.7 +/- 1.5-mu-mol/10(13) cells, with a free concentration of about 2.4 x 10(11) M. The main component of Zn2+ buffering is hemoglobin, with a dissociation constant of about 2 x 10(-8) M.