Bilirubin binding to normal and modified human erythrocyte membranes: effect of phospholipases, neuraminidase, trypsin and CaCl2

Binding of bilirubin to human erythrocyte membranes was studied after various enzymatic treatments as well as calcium loading. Whereas phospholipase D treatment of erythrocyte membranes resulted in 23% increase in bilirubin binding, phospholipase C-treated membranes showed remarkable enhancement in bilirubin binding. Polar head groups in general and negatively charged phosphate moieties, in particular, of phospholipids of the membrane appear to inhibit a large amount of bilirubin from binding to the membranes. Neuraminidase treatment of the membranes also led to a slight increase in bilirubin binding as compared to untreated membranes. Membrane proteins and carbohydrates seem to play significant regulatory role in bilirubin binding. However, no direct correlation was found between the increase in bilirubin binding and the amount of carbohydrate released upon tryptic digestion of membranes. Increase in bilirubin binding to trypsin-treated membranes can be ascribed to the increase in free bilirubin concentration in the incubation mixture as a result of tryptic hydrolysis of albumin by the membrane-bound tryptic activity. Calcium-loaded erythrocyte membranes also showed remarkable increase in bilirubin binding as a result of negative charge shielding and calcium-induced hydrophobic aggregation. Taken together, these results suggest the inhibitory role of polar head groups of phospholipids (phosphate in particular), carbohydrate and sialic acid in the binding of bilirubin to erythrocyte membranes.