Versatility of human cytochrome c non-native forms: pH dependence and relevance for apoptosis

Matthieu Simon, Valérie Metzinger-Le Meuth, Soizic Chevance, Olivier Delalande, Arnaud Bondon

Supplementarymaterials

Human cytochrome c mutation, expression and purification

The plasmid pET21-CCHL-HuCytc was generously provided by Jeng and co-workers [1].The plasmid contains the recombinant human cyt c gene and the CCHL gene of haem lyase. Mutations were introduced by using pET21-CCHL-HuCytc as the template for Quickchange site-directed mutagenesis (Stratagene, La Jolla, CA). Primers for mutations were designed with the web-based software PrimerX ( H26Q, sense primer 5’-GTGGAAAAGGGTGGCAAACAGAAGACTGGTCCAAA CTTGC-3’ and antisense primer 5’-GCAAGTTTGGACCAGTCTTCTGTTTGCCACCCTTTTCCAC-3’; H26QH33N, sense primer 5’-GGGTGGCAAACAGAAGACTGGTCCAAACTTGAATG-GTCTCTTTG-3’ and antisense primer 5’-CAAAGAGACCATTCAAGTTTGGACCAGTCT-TCTGTTTGCCACCC-3’; H33N, sense primer 5'-GGGTGGCAAACATAAGACTGGTCC-AAACTTGAATGGTCTCTTTG-3’ and antisense primer 5'-CAAAGAGACCATTCAAGTT-TGGACCAGTCTTATGTTTGCCACCC-3'. Experiments were performed according to the manufacturer protocol. Mutations were confirmed by gene sequencing on an ABI PRISM 310 Genetic Analyzer (S. Pastezeur, UMR CNRS 6290).

Human cyt c was expressed and purified as previously reported [1-2]. Escherichia coli cells containing the pEC86 plasmid with the ccm genes for cyt c maturation were a kind gift of Dr P. Turano of Florence (CERM, Florence, Italy). The E.coli-pEC86 cells were transformed with pET21-CCHL-Hucytc and grown in LB medium supplemented with ampicillin and chloramphenicol. Expression was induced via the addition of 1 mM IPTG at 30°C, 70 rpm for 19 hours. The cells were harvested by liquid shearing with a French press. Human cyt c was purified on a carboxymethyl cellulose CM52 cation-exchange column (Whatman Biosystems Ltd, Kent, England) and eluted with a linear NaCl gradient (0-500 mM) in 50 mM sodium phosphate buffer at pH 6.8. Fractions containing cyt c were concentrated with an Amicon ultrafiltration system with a molecular weight cutoff of 10 kDa (Millipore Amicon 10 K). Salts were eliminated with PD-10 desalting columns (GE Healthcare Bio-Sciences AB, Sweden). The protein was further characterised by SDS-PAGE and Coomassie Brilliant Blue R (Sigma) staining. The reduced form of purified human cytochrome c had an A415/A280 value > 4. The protein concentration was determined spectrophotometrically using either410 = 106.1 mM-1cm-1 for the oxidised form or 415 = 129.1 mM-1cm-1 for the reduced form. Ferricyanide was used in an equimolar ratio to obtain fully oxidised samples. The exact mass of each protein was verified by electrospray mass spectroscopy ±1 Da, H26Q: 12224.89; H33N: 12210.86; H26QH33N: 12201.85.

References

1.Jeng WY, Chen CY, Chang HC, Chuang WJ (2002) J Bioenerg Biomembr 34:423-431

2.Wegerich F, Turano P, Allegrozzi M, Mohwald H, Lisdat F (2009) Anal Chem 81:2976-2984

Figure S1: Identification of the different iron spin state limit forms of the WT human ferricytochrome c at 20 µM in phosphate buffer 50 mM, pH 6.8. Absorbance measurement in the Q strip region (a), Soret region (b) and near-UV CD measurement in the Soret region (c) as a function of SDS concentrations: 0 mM (solid lines), 1 mM ratio SDS:cyt c 50:1 (dotted lines), 4 mM ratio SDS:Cyt c 200:1 (dashed lines).

Figure S2: Schematic representation of cyt c various forms.