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Fig. 2. Size exclusion HPLC profiles at 254 nm
showing the relative retention times of indirectly injected samples (100ul) of
alkaline phosphatase (500ug/ml; Blue), metallothionein (1mg/ml; Pink), oxidized
glutathione (1mM; Red), and reduced glutathione (1mM; green). The trace
for alkaline phosphatase shows a complex chromatogram with multiple absorbing
peaks. However alkaline phospahatase activity was observed only with a
shoulder on the peak centered at 12 minutes. Consequently, this peak was
purified to simplify the interpretation of subsequent chromatograms.
Fig. 3. Diode array spectrum of a mixture of alkaline phosphatase,
metallothionein, GSH, and GSSG fractionated by SE-HPLC. The spectrum
is poorly resolved and clearly shows co-elution of numerous peaks.
Fig. 4. Spectrum max plot of Figure 3 of the protein mixture showing
the presence of 6 partially resolved peaks centered at 12, 14.4, 14.9,
16, 17.2, and 19.8 minutes. Overlying the spectrum max plot are histograms
of the enzymatic activities of alkaline phosphatase (Blue), GSH (Green),
and GSSG (Red) obtained from the 500 ml fractions collected from the column
eluant. The activities confirm the identity of alkaline phosphatase
in peak 1 and GSSG and GSH in peaks 4 and 5.
Fig. 5. Elemental profiles for 64Zn, 63Cu, and 114Cd obtained
from the fractionated mixture of proteins shown in Figure 3 and 4.
Five major peaks with differing elemental compositions are evident eluting
at a, b, c, d, and e. Peak a shows specificity for Zn, which is a
fingerprint of alkaline phosphatase. Peaks b and c contain a mixture
of Cu and Cd while peaks d and e contain mostly Cd with only traces of
other metals. The elements associated with peaks b, c, d, and e are
all characteristic of metallothionein. Also shown in the insert is
the elemental analysis of 200 ml of a 200 ppb (40 ng) solution of mix standards.
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