Abstract—D‐α‐tocopherol was found to be an effective quencher of 1O2 molecules (k= 2.5 times 108→mol‐1 s‐1 in pyridine) by measuring its effect on the autosensitized photooxidation of rubrene. The quenching process was shown to be almost entirely ‘physical’, that is, α‐tocopherol deactivated about 120 1O2 molecules before being destroyed. The results suggest that this process may be a mechanism for the protective effect of α ‐ tocopherol in photodynamic action.

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Abstract— Irradiation of protoporphyrin‐sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross‐linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976). Facile oxidative hemolysis can be achieved without observable cross‐linking of membrane proteins upon incubation (37°C) of red cells containing membrane‐bound 3ß‐hydroxy‐5α‐hydroperoxy‐△6‐cholcstene. Thus, protein cross‐linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light‐induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin‐sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross‐linking of membrane proteins. These results suggest that membrane protein cross‐linking and photohemolysis are not coupled processes.

A B S T R A C T Excess erythrocyte protoporphyrins of human congenital erythropoietic protoporphyria and of griseofulvin-induced murine hepatic protoporphyria were found to be associated with hemoglobin and stroma fractions in similar relationships. More than 99.5% of total erythrocyte protoporphyrin was bound to hemoglobin in each case. However, profound differences were found when protoporphyrin concentration was measured in erythrocytes that had been segregated into populations of progressive age on discontinuous density gradients. In erythropoietic protoporphyria, porphyrin content diminished rapidly with age; in murine protoporphyria, the aging erythrocyte populations became progressively more porphyrin rich. In vitro diffusion of protoporphyrin from plasma across the intact erythrocyte membrane was demonstrated. The equimolar binding affinity of protoporphyrin to hemoglobin was shown to be 40 times that of protoporphyrin to serum albumin. This strong affinity provides the driving force for the observed transmembrane diffusion, and explains the high erythrocyte/plasma porphyrin ratio in murine hepatic protoporphyria. The opposite rapid efflux of intraerythrocytic protoporphyrin into plasma previously shown in uncomplicated erythropoietic protoporphyria occurs despite this strong hemoglobin affinity, implying continuous efficient clearance of protoporphyrin from plasma by the liver. Furthermore, these and other This work was presented in part at the 7th International Congress on Photobiology, Rome, Italy, September, 1976. Received for publication 22 November 1976 and in revised form 29 March 1977. data suggest that a hepatic synthetic source for any significant fraction of the blood protoporphyrin in erythropoietic protoporphyria is highly improbable. INTRODUCTION Congenital protoporphyria in humans was first named erythropoietic protoporphyria (EPP)' (1-3) based upon the large amounts of protoporphyrin IX (PP) found in the blood of patients with the disease which was thought to be synthesized in the bone marrow. However, the observation was made that the daily excretion of PP in the feces of these patients approached the total excess PP in the circulating erythrocyte mass. It was clear that this amount could not be derived from senescent erythrocytes only (4). Some investigators proposed the liver as an important additional source of PP in this disease. Acquired protoporphyria is extremely rare in man, but protoporphyria can be easily induced in mice with certain drugs, among which is griseofulvin (GF) (9-11). Murine protoporphyria induced by oral administration of GF has been used as an experimental model for human EPP (12,13). The site of excess PP synthesis in GF-induced murine protoporphyria (GFPP) is thought to be primarily, if not solely, hepatic (14). However, the concentration of PP associated with the circulating erythrocytes in GFPP can become comparable to that observed in EPP (9, 13, 14).We have developed spectrofluorometric techniques which give information about the binding sites of PP ...

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