pThe kinetics of [3H]bilirubin binding to human erythrocyte ghost membranes was investigated. The binding occurred rapidly and was saturable with respect to [3H]bilirubin and membrane concentration. The apparent dissociation constant (Kd) and maximum binding (Bmax.) for bilirubin of the membranes were 2.3 microM and 0.93 nmol/mg of protein respectively. Low-affinity binding, non-saturable at 400 microM, was observed. Thermal dependency of the saturable binding showed a U-shaped curve with the lowest value around 37 degrees C. Affinity labelling of the membrane proteins using [3H]bilirubin-Woodward9s reagent K complex did not define individual proteins. The Kd (12 microM) and Bmax. (4.4 nmol/mg of protein) for bilirubin of the tryptic membranes increased 5.0 and 5.2 times the respective control values (2.4 microM and 0.85 nmol/mg of protein). Heat-treatment of the membranes for 3 min at 100 degrees C increased the saturable binding as much as by 222%. These results indicate that there exist saturable bilirubin-binding sites on the erythrocyte membranes and also suggest that they are not composed of proteins./p
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