Pheophorbide a

Zinc pheophorbide a (Zn-Pheide) was obtained from pheophorbide a (Cayman Chemical, Ann Arbor, MI, USA) via direct metalation with zinc acetate in methanol at 50 °C and purified, as described earlier [10 (link)]. Purity of the compound according to HPLC measurement was at least 96%. The concentration of Zn-Pheide was determined spectrophotometrically in ethanol, using the extinction coefficients at the Q_y band equal to 71,500 M⁻¹ cm⁻¹. Aliquots of the PS were stored dry at −20 °C under nitrogen atmosphere. For each experiment, fresh solutions of the PS were prepared by dissolving them in appropriate volumes of DMSO. All experiments were performed under dim light to avoid degradation of the PS and uncontrolled photodamage of the cells.
Complex of the PS with HSA was prepared by addition of the PS stock solution in DMSO to solution of albumin in PBS, pH 7.4, and incubation at room temperature for 10 min to reach the binding equilibrium.

The NMR spectra using standard pulse programs were recorded at room temperature on a Bruker Avance DPX-400 spectrometer (Bruker, Billerica, MA, USA) operating at 400 (¹H) and 100 (¹³C) MHz. The chemical shift (δ, ppm) values were calibrated using the residual NMR solvent and coupling constant (J) was reported in Hertz (Hz). Column chromatography was done on normal-phase silica gel (230 × 400 mesh, J. T. Baker, Center Valley, PA, USA) or reversed-phase silica gel (C₁₈, 40 μm, J. T. Baker). Silica gel 60 F₂₅₄ TLC plates (Merck, Darmstadt, Germany) and reversed-phase TLC plates (C₁₈, Merck, Darmstadt, Germany) were used for analytical TLC. The plates were visualized by spraying 10% H₂SO₄ followed by heating. The authentic samples pheophorbide a (>95%) and lutein (>90%) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA) and Acros Organics (Pittsburgh, PA, USA), respectively. Their structure and purity were further confirmed by NMR and HPLC in our laboratory. The 37 standards of fatty acid methyl esters (Supelco® 37 components FAME mix) used for GC-MS analysis were purchased from Sigma-Aldrich (St. Louis, MO, USA).