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The results clearly
indicate that the metals associated with MT are acid labile and can be
removed by acidification and dialysis. Figures 1 and 4 show the 254
and 280 absorbance ratios for the chromatographed metallothionein and apo-metallothionein
preparations respectively. Both the chromatographs show a complex
series of unresolved peaks indicative of different isoforms of the protein
in various states of polymerization. However, the major difference
observed between the profiles is the large relative reduction in 254 absorbance
in the apo-MT. This wavelength is characteristic, but not specific
necessarily, for the mercaptide-metal bond, and its attenuation in the
apo-metallothionein preparation is clear indication of metal removal.
The loss of metal from
the protein can be confirmed in Figures 2-3 and 5-6 which show the time
resolved ICP-MS profiles for 63Cu, 64Zn, 95Mo, 98Mo, 114Cd, and 200Hg for
metallothionein and apo-metallothionein respectively. Also shown
in the elemental profiles are the responses obtained from the analysis
of the 200 µl of 5ppb of each analyte by FIA. The mean integrated
areas obtained from these responses were used to quantify the content of
each metal in the two proteins. The data obtained using this procedure
are summarized in tables 1 and 2. Comparisons of the values indicate
approximately 100% removal of Mo, Hg, and Zn, 92.3% removal of Cu, and
97% removal of Cd. These data are consistent with the known hierarchical
strength of binding of MT to different metals under reducing conditions
which follows the order
Hg>Ag>Cu (I)> Cd>Zn>Co>Pb>Mo.
Direct FIA of the apo-MT
shows the presence of Cu and Cd in the sample and, although this data was
not quantified, it does support the argument that the metals associated
in the chromatographed apo-protein were not scavenged from the HPLC system
during fractionation. Future experiments include the addition of
either stable isotopic 64Zn or radioactive 65Zn as traceable markers
to permit the donation of zinc to other apo-metalloproteins, such as alkaline
phosphatase to be quantified. SE-IE HPLC/ICP-MS provides a powerful
analytical technique to quantify the metal content of metalloproteins in
cytosolic samples.
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