Development of peptide-based tools to measure cellular free heme after UVA irradiation

Heme plays a vital role in cell biology and the dysregulation of heme levels is implicated in a wide range of diseases. Currently however there is a lack of convenient chemical tools that can be used to measure the changes in heme levels in biological media or in live cells. Exposure of skin cells to solar UVA radiation has been shown to result in elevated levels of free heme and upregulation of Heme oxygenase-1 (HO-1), the oxidant-inducible enzyme that catalyses the breakdown of pro-oxidant heme. There is therefore considerable interest in developing new probes to study such UVA-induced changes in heme levels and gain a greater understanding of the detrimental and potentially beneficial effects of UVA on human skin. The aim of this work is to explore the development of probe molecules that are derived from natural heme-binding proteins. We will describe a tryptophan-containing peptide probe based on a peptide sequence from the natural heme-binding protein, Bach-1 a transcription factor that acts as a negative regulator of the HO-1 gene. The fluorescence of the tryptophan residue can be quenched upon binding of heme to the probe peptide providing a direct readout of heme levels. Here, we describe an efficient and scalable synthesis of the novel fluorescent tryptophan analogue, 7-aza-tryptophan that is incorporated into the Bach-I derived peptide by solid phase peptide synthesis. This peptide probe can sensitively measure changes in heme levels in human dermal fibroblasts, FEK4 cells following exposure to increasing levels of UVA irradiation (up to 500 kJ/m²).