Urokinase Immobilized on Erythrocytes

Dear Sir, Work on insolubilized enzymes has been mainly confined to the coupling of enzymes to nonliving supports. We would like to report on the coupling of the enzyme urokinase to the human red cell membrane of intact erythrocytes by 4,4'-diisothiocyano-2,2'stilbene disulfonate (DIDS). It appears that significant amounts of enzyme activity can be immobilized to the membrane without causing hemolysis of red blood cells. DIDS binds covalently and irreversibly to a small number of ligands on the outer surface of the cell membrane and does not penetrate into the cells (1). Fresh human erythrocytes were extensively washed with PBS (phosphate buffer-NaCl, 300 milliosmolar, pH 7.4) at 4° C. 3 ml of albumin free urokinase (6,000 units) in PBS plus 0.6 ml of a 50% red blood cell suspension were incubated for 10 min at 4° C with 0.5 mg of DIDS in the dark. The cells were then centrifuged, washed 5 times and resuspended in PBS. Control study was performed with same procedure without DIDS. The enzyme