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Cu(I) and Fe(II) were quantitated with BC and BP, respectively. These indicator molecules specifically bind the reduced [Cu(I) and Fe(II)] but not the oxidized [Cu(II) and Fe(III)] forms of copper and iron (23). The Cu(I)-BC and Fe(II)-BP complexes thus formed exhibited absorbance maxima at 480 and 535 nm, respectively (see Figs. 1 and 4), permitting specific quantitation of Cu(I) and Fe(II).
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Chemicals were purchased from Sigma (SOD, S-2515; catalase, C-40; BP, B-1375, hypoxanthine, H-9377; low density lipoprotein, L-5402; ovalbumin, A-5503, KLH, H-1757, and GHL, G-1887), Aldrich (BC, 14.662-59). Fluka (feme citrate, 44941), or Serva (N-ethylmaleimide, 11331).
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2] were separated by reversed-phase high-performance liquid chromatography (SMART-system, equipped with a micro-reversed-phase chromatography C2/C18 SC 2 1/10 column; Pharmacia) Solvent A contained 10 mM ammonium acetate (pH 6.5), solvent B contained 10 mM ammonium acetate (pH 6 5) in 50% acetonitnle. Peptides were separated in a linear gradient running from 50% B to 100% B over 22 min. On-line analysis was performed with a tandem quadrupole mass spectrometer (TSQ7000; Finnigan-MAT, Bremen) equipped with an electrospray ion source Each scan was acquired over the m/z range 600 to 1600 in 2 s The peptides were identified by their molecular mass calculated from the m/z peaks of the single- or multiple-charged ions.
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Supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 317 (SFB317, A/11), the Bundesminister fur Forschung and Technologie, and the Fonds der Chemischen Industrie. C L M. is supported by the National Health and Medical Research Council (Australia)
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