ELSEVIER Journal of Photochemistry and Photobiology B: Biology 37 ( ! 997 ) 13 !- 140 JOnl'aalof AND PttOt~ B:BIOLOGY Singlet oxygen generation by photodynamic agents Jim M. Fernandez, Mehmet D. Bilgin, Leonard I. Grossweiner * Wenske Laser Center. Ravenswood Hospital Medical Center, 4550 N. Winchester Ave., Chicago, IL 60640 USA Received 5 February 1996; accepted 24 April 1996 Abstract The singlet oxygen quantum yield of photodynamic agents was measured at 546 nm, 630 nm, and on the far-red absorption peak. The technique employed is available in most laboratories, in which the photosensitization of lysozyme is used as an internal actinometer. Measurements in a pH 7.4 phosphate buffer plus 1% Triton X-! 00 (PB/X 100) are scaled to 0.52 for methyleneblue in the phosphate buffer. The average quantum yields are: hematoporphyrin IX (0.73), protoporphyrin IX (0.56) zinc protoporphyrin IX (0.91), mesotetra-(4- sulfonato-phenyl) porphine (0.61), Photofrin s (0.89), benzoporphyrin derivative monoacid ring-A (0.84), chlorin e6 in PB (0.64), pheo- phorbide a (0.69), and aluminum phthalocyanine tetrasulfonate (0.38). Protection factors were measured for added azide ion, 1,4-diazabicyclo [ 2.2.2]-octane, and superoxide dismutase. Spectral evidenceis presented for chlorin e6 interactions with PB/TX 100 and for binding to lysozyme. Keywords: Singlet molecular oxygen; Photodynamic action; Photosensitization 1. Introduction The energy efficiency of a Type II photosensitized process (photodynamic action) is limited by the singlet oxygen quan- tum yield (¢PA). Many different techniques have been employed tbr measurements of q~A, including the "mon- drool"luminescence of singlet oxygen (lAg) at 1270 nm, photothermal methods, EPR measurements of nitroxide rad- icals, oxygen uptake for a sensitized photc~-oxidation, and quantum yields of the photochemical products. To be used in aqueous solution, hydrophobic sensitizers require solubiliz- ing media that may affect the reactivity of tA gwith an external probe. Measurements on self-aggregating sensitizers may depend on the concentration and wavelength owing to dif- ferent cp,, and extinction coefficients of the monomer and higher aggregates. The object of this study was to measure @~ for some photodynamic agents and photodynamic ther- apy (PDT) sensitizers at visible wavelengths. The photosen- sitized inactivation of hen lysozyme (LYS) was used as the probe for tag. Previous measurements on Photofrin a (PF) and hypericin (HY) [ 1-3] show that this technique is sen- sitive and reproducii:1e, requiring only a light source, a wave- length selection :~vice, and a spectrophotometer. The enzyme inactivation quantum yields are scaled to ~ of meth- ylene blue (MB) in pH 7.4 phosphate buffer (PB) and corn- * Corresponding author. pared with the available literature. The involvement of tAg and superoxide (02) were tested by adding azide, 1,4 -dia- zabicyclo [ 2.2.2 ]-octane (DABCO), and superoxide dis- mutase (SOD). 2. Materials and methods 2.1. Chemicals Hematoporphyrin IX (HF), methylene blue chloride (MB), protoporphyrin IX (PPIX), chicken egg white lyso- zyme, lyophilized Micrococcus luteus, bovine erythrocyte superoxide dismutase, sodium azide, 1,4-diazabicy- clo[2.2.2]octane, Triton X-100, sodium laurel sulfate (SDS), and hexadecyltrimethylammonium bromide (CTAB) were obtained from Sigma Chemical Company; zinc protoporphyrin IX (ZnPP), chlorin e6 (Chl-e6), pheo- phorbide a (pheo-a), aluminum phthalocyanine tetrasulfon- ate (AIPcS4), and mesotetra- (4-sulfonato-phenyl) porphine (TPPS) were obtained from Porphyrin Products, Inc. (Logan, UT); Photoffin R (PF) and benzoporphyrin deriva- tive monoacid ring-A (BPD-MA) were obtained from QLT Phototherapeuties Inc. (Vancouver, BC). The chemicals were used as received. Optical absorption spectra were meas- ured with a Perldn-Elmer Lambda 5 spectrophotometer. Flu- orescence spectra were measured with a Fartand manual 1011-1344/97/$17.00 Copyright © 1997 Elsevier Science S.A. All rights reserved PliSiOI !-!344(96)07349-6