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Figure 1.
Picture of carbonic anhydrase with zinc.
Figure 2.
Elutions of carbonic anhydrase through a G-10
size exclusion column.
Ten drop fractions were collected and protein
content was tested using a standard Coomassie blue
assay. The
majority of the enzyme eluted between fractions 7
and 10 (100-130 drops).
Figure
3.
Serial dilutions of carbonic anhydrase were
conducted to determine the lowest concentration of
enzyme detectable by the assay.
Protein addition ranged from 50 μg to
3.13 μg, all
showing significant amount of activity.
Figure 4.
Michaelis-Menton Plot of 0.08 mg of carbonic
anhydrase and varying substrate concentrations.
Figure
5.
Comparison of control, apo-, and
reconstituted carbonic anhydrase.
The control enzyme showed a specific reaction
rate/mg
protein of 0.091 while the apo produced a rate/mg
protein of 0.004.
This represents a 96% reduction in activity
of the apoenzyme from the control.
Incubation with ZnCl2 produced a
specific reaction rate/mg
protein of 0.040.
Relative to controls this represents a 44%
increase in activity due to reactivation of the
enzyme.
Proposed
Pathways for Metal Transfer From Metallothionein to
Apo-carbonic Anhydrase
Figure 6.
Two proposed pathways for metal transfer from
metallothionein (MT) to apo-carbonic anhydrase.
One pathway involves the transfer of metal to
a carrier, glutathione (GSH), which delivers the
metal to the apoenzyme.
The second pathway is the direct transfer of
metal from the MT to carbonic anhydrase via direct
interaction of protein with the enzyme.
Figure 7.
Lineweaver-Burke plot showing a Vmax (1.33mM
p-NPA/mg CA/min.) and Km (0.093mM
p-NPA) for Carbonic Anhydrase and the substrate p-nitrophenyl
acetate(p-NPA).
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