Interaction of phenylisothiocyanate with human erythrocyte band 3 protein. I. Covalent modification and inhibition of phosphate transport.

The hydrophobic probe phenylisothiocyanate is utilized for chemical modification of human erythrocyte band 3 protein. The binding of phenylisothiocyanate to this protein is characterized in whole erythrocytes, erythrocyte ghost membranes and in isolated band 3 protein. The label, reactive with nucleophiles in their deprotonated form is found in all three preparations to be covalently bound to band 3 protein. Under saturation conditions, 4--5 mol phenylisothiocyanate are covalently bound per mol protein (molecular weight 95 000). The described modification effects inhibition of phosphate entry into erythrocytes. 50% inhibition of phosphate transport is obtained following a preincubation of erythrocytes with 0.45 mM phenylisothiocyanate. Both phenylisothiocyanate binding and transport inhibition are saturating processes. The relationship of the two parameters is non-linear.