Development of a peptide-based fluorescent probe for biological heme monitoring

A prototype peptide-based probe has been developed for the determination of intracellular heme levels.


S1. Materials and methods
Fmoc-protected amino acids were all obtained from NovaBiochem, with the exception of 7azatryptophan (SigmaAldrich). Peptide grade DMF was purchased from Rathburn Chemicals. All other solvents were purchased from Fisher Scientific.
Solid Phase peptide synthesis was carried out on either Rink Amide MBHA resin (0.78 mmol/g) or Wang resin (0.9 mmol/g) using automated peptide synthesiser (Activotec P11). The peptides were synthesised using a standard Fmoc protocol with PyBOP as the coupling agent. Side chain deprotection and cleavage from the resin was with TFA/H2O/TIS/EDT (92.5/2.5/2.5/2.5, v/v/v/v) at room temperature for 3 h. The peptide was then precipitated in diethyl ether, centrifuged and washed with diethyl ethyl three times. Peptides were purified by semi-preparative reverse-phase HPLC using a Dionex HPLC system equipped with a Phenomenex Gemini 5 μm C-18 (250 × 10 41 mm) column with a flow rate of 2.5 mL/min. The gradient elution system was 0.1% TFA in water (mobile phase A) and 0.1% TFA in acetonitrile (mobile phase B). The peaks were detected at 214 nm, 220 nm, 254 nm and 280 nm. The gradient was T=0 min B=5%, T=30 min B=95%, T=45 min B=95%, T=45.1 min B=5%, T=52 B=5%. Purified peptides were lyophilised and stored at -20 °C.
Mass spectrometry: All peptides were characterised by electrospray mass spectrometry on either a Bruker microTOF LCS spectrometer with time of flight quantitation, or a Bruker MaXis HD ESI-QTOF coupled to a Thermo Scientific Dionex Ultra High Performance Liquid Chromatography (UHPLC) unit. Samples were prepared in acetonitrile or methanol.
Stock solutions of peptide were made at 10 mM in potassium phosphate buffer (PB) (10 mM, pH 7.0). For titration experiments, stock solutions of hemin were prepared at 5 mM in 30 mM NaOH in 10 mM PB. All stock solutions were stored at -20 °C and freshly thawed before use. Cell lysate hemin detection: FEK-4 human skin fibroblasts were cultured in Eagle's Minimum Essential Medium with Earle's salts and sodium bicarbonate (Sigma), supplemented with 2 mM Lglutamine (Invitrogen), 50 U/mL penicillin (Invitrogen) and 50 μg/mL streptomycin and 15% FBS (Sigma). Cells were maintained at 37 °C under 5% CO2 in a humidified incubator.

UV-Vis absorption spectroscopy
Cells were treated with hemin (10 μM) in growth media for 18 h, protected from light. Cells were treated with a UVA dose of 250 kJ/m 2 using a broad-spectrum 4 kW UVA lamp (Sellas, Germany). Irradiation was in the dark at 25 °C. Cells were lysed in lysis buffer (KH 2 PO4, 20 mM; ethylenediaminetetraacetic acid (EDTA), 0.5 mM; PMSF, 0.1%; with a complete Mini EDTA-free protease inhibitor cocktail tablet (Roche)), by sonication for 14 s on ice (Rapidis 300, Ultrasonics, UK). Quantification of cellular protein level was with the Pierce BCA Protein Assay (ThermoFisher Scientific) according to manufacturer's instructions. Cell lysate containing 5 μg of protein was incubated with fluorescent peptide (10 μM) in lysis buffer for 5 min on ice. Fluorescence was measured using a Clariostar microplate reader (BMG Labtech).