Phylloerythrin. mechanisms for cellular uptake and location, photosensitisation and spectroscopic evaluation

To elucidate the photobiological behaviour of phylloerythrin by studying the cellular uptake and intracellular localisation pattern of phylloerythrin and its spectral properties in Chinese hamster lung fibroblast cells (V79).

Phylloerythrin was diluted in dimethylsulfoxide (DMSO). Fluorescence emission and excitation spectra were measured using a luminescence spectrometer equipped with a red-sensitive photomultiplier. V79 cells were cultured in monolayers and labelled with 0.25 microg/ml phylloerythrin for uptake, cell survival and intracellular localisation studies. For cell survival and intracellular localisation studies, cells were subsequently exposed to blue light at a fluence rate of 9.0 mW/cm2.

The fluorescence excitation spectrum of phylloerythrin in DMSO was characterised by a Soret band exhibiting a maximum peak at 418 nm. The fluorescence emission spectrum had peaks at 643 and 706 nm. The corresponding spectra in cells were red-shifted to 422, 650 and 712 nm, respectively. The cellular uptake of phylloerythrin was complete after about 10 h of incubation. The uptake together with the activation energy and analysis of cells incubated with phylloerythrin at 37 degrees C and 0 degrees C using fluorescence microscopy indicated that the dye is taken up into cells via a diffusion mediated pathway. Measurements of subcellular marker enzymes were performed immediately after light exposure of phylloerythrin-treated cells. The mitochondrial marker enzyme, cytochrome-c oxidase, and the marker enzyme for the Golgi apparatus, UDP galactosyl transferase, but not those for lysosomes, -N-acetyl-D-glucosaminidase (-AGA), and endoplasmic reticulum, NADPH cytochrome-c reductase, were inactivated upon photodynamic treatment.

These results indicate that phylloerythrin is located mainly in the Golgi apparatus and mitochondria of V79 fibroblasts cells.