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2-terminal methionine; in addition, ES-MS of Hg-Atx1 yielded one peak at 8287.6 daltons. Gel filtration on an HPLC QC-PAK TSK 200GL (TosoHaas) column yielded molecular sizes of 10,200, 9840, and 9960 daltons for apo-, Hg(II)-, and Cu(I)-Atx1, respectively.
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-). Excess metal was removed by ultrafiltration and the protein was exchanged into a solution containing 20 mM MES (pH 6.0) and 20% (v/v) glycerol. The final protein concentration was 7.84 mM with a Cu/protein ratio of 0.75 ± 0.02. Solutions (>2 mM) of the Cu(I)-protein can be handled in air at 4°C for 30 min or stored long term (>4 months) at -70°C with no observable formation of Cu(II).
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20
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15844409181
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XAS data were obtained at Stanford Synchrotron Radiation Laboratory (SSRL) beam line VII-3 as fluorescence excitation spectra with a Ge solid-state detector array. Samples (150 μl) were maintained at 10 K throughout measurements. Data reduction and analysis were performed according to standard procedures [P. J. Riggs-Gelasco, R. Mei, C. F. Yocum, J. E. Penner-Hahn, J. Am. Chem. Soc. 118, 2387 (1996)]. Data were fitted with amplitude and phase functions calculated with FEFF 6.01 [J. J. Rehr et al., ibid. 113, 5135 (1991)], calibrated by fitting model-compounds of known structure.
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Riggs-Gelasco, P.J.1
Mei, R.2
Yocum, C.F.3
Penner-Hahn, J.E.4
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21
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0001661950
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XAS data were obtained at Stanford Synchrotron Radiation Laboratory (SSRL) beam line VII-3 as fluorescence excitation spectra with a Ge solid-state detector array. Samples (150 μl) were maintained at 10 K throughout measurements. Data reduction and analysis were performed according to standard procedures [P. J. Riggs-Gelasco, R. Mei, C. F. Yocum, J. E. Penner-Hahn, J. Am. Chem. Soc. 118, 2387 (1996)]. Data were fitted with amplitude and phase functions calculated with FEFF 6.01 [J. J. Rehr et al., ibid. 113, 5135 (1991)], calibrated by fitting model-compounds of known structure.
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Rehr, J.J.1
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29
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1842326033
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note
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- was not present in any of the buffers. The final protein concentration was 3.36 mM with a Hg/protein ratio of 0.75 ± 0.06 in 20 mM MES (pH 6.0).
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31
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0029061164
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L. M. Utschig et al., Science 268, 380 (1995).
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1842367756
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2. Safety note: The latter compound is permeable to latex gloves and is exceedingly toxic. Discussion of alternative compounds can be found at www.chem.nwu.edu/∼ohallo/HgNMRStandards
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37
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4, and 40 mM β-mercaptoethanol. They were lysed by homogenization with glass beads after adding 45 μl of 0.1% SDS. The resulting extract (800 μl) was mixed with 200 μl of o-nitrophenyl-β-D-galactopyranoside (4 mg/ml) and incubated at 30°C overnight. After centrifugation, β-Gal activity was measured in the supernatant at 420 nm. In some instances, cells were grown in the presence of the Cu chelator BCS (3 mM). Results are representative of two to four independent samples with ranges of ≤15%. β-Gal activity units for the p53/SV40 positive controls (and vectors) were 9.75 (0.21) in the absence of BCS and 9.95 (0.27) in the presence of BCS.
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note
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2, and A. Duncan for the color illustration.
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