|
|
|
|
|
Kelly Thrippleton:
Purification, Isolation and Molecular
Analysis of Apo-Hemocyanin from the Garden Snail, Helix
aspersa, Using Potassium Cyanide and Coupled HPLC/ICP-MS
|
|
Metallothionien (MT) is a
ubiquitously distributed low molecular weight metal
binding protein. The protein is believed to provide
an intracellular store of essential trace metals
such as copper and zinc. However, little is known
about the mechanism of metal transfer from MT to apo-metalloproteins.
In order to study the mechanism of metal transfer,
it is necessary to generate an apo-protein that can
act as the metal recipient.
In vivo, the enzyme carbonic anhydrase
catalyzes the formation of carbonic acid from carbon
dioxide and water. Zinc plays an essential role in
the enzyme catalysis. In this study, the apo form of
carbonic anhydrase was prepared by dialysis against
dipicolinic acid and purified on a molecular
exclusion column. The activity of the enzyme was
quantified by measuring the formation of p-nitrophenol
from p-nitrophenyl acetate. Using this procedure,
the apo-carbonic anhydrase with less than 5% of the
original activity has been routinely prepared. Addition of exogenous ZnCl2 to the apo-enzyme
preparation results in a 44% reconstitution of the
original activity of the enzyme.
|
|
|
|
|
|
|
|
|
