Bilirubin‐sensitized Photoinactivation of Enzymes in the Isolated Membrane of the Human Erythrocyte*

Bilirubin has been found to sensitize the photodynamic inactivation of several enzymes in the isolated membrane (ghost) of the human red cell. When ghosts (pH 8.0, 10°C) + bilirubin (0.1 mM) were irradiated with blue light (350 Wm‐2), the activity of glyceraldehyde 3‐phosphate dehydrogenase decayed with t1/2≃ 15 min. No effect was observed in the absence of pigment or with incident yellow light. Diazabicyclo‐octane (DABCO) sharply reduced the inactivation rate, suggesting that 1O2 is involved. Sodium dodecyl sulfate‐gel electrophoresis of ghosts containing fully inactivated glyceraldehyde 3‐phosphate dehydrogenase revealed no change in the polypeptide band corresponding to the subunit of the enzyme. Solubilized enzyme, which was similarly photosensitive, could be partially protected by nicotinamide adenine dinucleotide or glyceraldehyde 3‐phosphate. The integral enzymes Mg2+‐ATPase, Na+, K+‐ATPase, and acetylcholinesterase were also affected. Under the above conditions and bilirubin = 0.37 mM, these enzymes were photoinactivated in first‐order fashion, k≃ 2, 1.2 and 0.2 h‐1, respectively. The rate of decay of total ATPase was found to vary as the square root of the bilirubin concentration over the range 7–370 μM. At a fixed bilirubin concentration (0.37 mM), this rate was also shown to be directly proportional to light intensity. Inasmuch as the —SH content of bilirubin‐containing ghosts diminished during irradiation, oxidation of essential cysteine residues could be responsible for the inactivation of some of the enzymes studied.