Several amino-reactive chemical probes which differ in hydrophobicity and charge and in their ability to penetrate the red cell membrane were tested for their ability to modify K รท leak and inorganic phosphate (Pi) leak in intact human red cells, Methyl picolinimidate (MP), ethyl acetimidate (EA), methyl acetimidate (MA) are hydrophilic penetrating probes whereas isethionylacetimidate (IA) is a hydrophilic non-penetrating probe. The order of their effectiveness in inhibiting Pi leak was found to be MP>EA>MA>IA. This order is in decreasing hydrophobicity and suggests that some penetration into the bilayer or into hydrophobic domains of the anion transport protein is required to modify an amino group required for Pi permeability through the membrane. These imidoesters have little or no effect on K รท leak in the red ceil.
Trinitrobenzenesulfonate (TNBS) a relatively non-penetrating hydxophohic anionic probe and fluorodinitrobenzene (FDNB) a penetrating hydrophobic neutral probe have markedly different effects on K + and Pi leak. TNBS has little effect on K รท leak but markedly inh~its Pi leak. The effect of TNBS on Pi leak is not blocked by prior treatment with IA suggesting that these probes sense different populations of amino groups in the membrane. FDNB nearly completely blocks Pi leak and markedly increases K รท leak. The results with TNBS and FDNB indicate an asymmetric arrangement of amino groups on the red cell membrane. Certain amino groups on the outer surface of the membrane regulate Pi permeability whereas certain amino groups on the inner surface of the membrane regulate K * permeabilty. The data also suggest that these amino groups are in a hydrophobic domain.