Protoporphyrin 9

Zinc phthalocyanine, protoporphyrin IX, methylene Blue and 2,4-bis [4-(N,N-dibenzylamino)-2,6-dihydroxyphenyl] squaraine in powder form were purchased from Sigma-Aldrich. Chlorin e6 was purchased from Frontier Scientific (Logan, UT). Stock solutions of all compounds were prepared in DMSO. Formulations for absorbance measurements and animal studies were made in 10% DMSO.

Under standard reaction conditions, 10 µl of acetone dissolved chlorophyll was added to 3 ml of 0.02% (w/v) n-dodecyl-β-D-maltoside (Sigma) or 0.02% (v/v/) Triton X-100 in 50 mM Tris buffer, pH 8.0 plus 10 µM hemin chloride (MP Biochemicals) and 50 mM β-mercaptoethanol (Sigma). Where stated, hemin chloride was omitted or replaced with 10 µM protoporphyrin IX (Sigma), and β-mercaptoethanol was replaced with 50 mM benzyl mercaptan (Sigma) or 50 mM dithiothreitol (Sigma). For the pH experiments, pH was maintained using a 50 mM Tris buffer for pH 7–9 or 50 mM MES buffer for pH 5.6. After 18 h shaking at room temperature, unless stated otherwise, the chlorophylls and their derivatives were extracted from the reaction mix with saturated NaCl solution and 100% butanol, which was transferred to a new tube and evaporated in a vacuum concentrator. Pigments were resuspended in 100% methanol and immediately subjected to HPLC. The separated pigments were collected from the HPLC, dried using a vacuum concentrator or nitrogen gas stream, and stored at −80°C prior to mass spectrometry or NMR. For β-mercaptoethanol reactions in methanol, a small volume of acetone-solubilised chlorophyll (20 µM) was added to 50 mM β-mercaptoethanol dissolved in 100% methanol and incubated with shaking for 2–4 h. Reactions were concentrated using a nitrogen gas stream before performing HPLC.