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THE SEPARATION, IDENTIFICATION,
AND MOLECULAR ANALYSIS OF COMPLEX MIXTURES OF METALLOPROTEINS AND POLYPEPTIDES
BY TANDEMLY LINKED TWO-DIMENSIONAL HPLC-ICPMS.
D. Lloyd and A. Z. Mason, Ph.D.
Department
of Biological Sciences, California
State University, Long Beach, CA.
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Approximately 300 different proteins have
been identified that require metals for their physiological functioning.
Metals that are known to have a role in this context include, Fe, Cu, Zn,
V, Cr, Se, Mn, Co, Sr, Ba, Ca, Mg, Na, K, and Ni. More recently,
there is evidence to indicate that Cd can be added to the list of essential
metals since it appears to be utilized by certain phytoplankton in substitute
for Zn for the activation of certain enzymes.
Although the requirement
of these metals for life processes has been well established, the cellular
mechanisms that determine the specificity of the metal - protein associations
are unknown. Recent evidence has indicated that one of the key proteins
involved in regulating the availability of weak Lewis acid metals in the
cell is the protein metallothionein (MT). This ubiquitous, 61 amino
acid protein is characterized by its high cysteine content which accounts
for its capacity to sequester a number of essential and non-essential metals.
Although MT shows no enzymatic activity, data provided by other laboratories
indicate an ambivalent role for this protein that is dependent upon the
prevailing redox conditions in the cell. This redox sensitivity results
from the thiolate clusters which confer oxido-reductive activities to the
protein by providing titratable groups showing altering affinity for metals
with changes in cellular redox state. Thus, a change from reducing
to oxidizing conditions in the cell will cause the oxidation of the sulfhydryl
groups of the cysteine to form disulfide bridges.
These changes cause
a concomitant reduction in the affinity of MT for the metal causing its
release into the cellular environment and thereby increasing its availability
for apo-protein activation. Conversely, shifts to more reducing conditions
cause the sequestration of metals from the cell to MT and change the existing
thermodynamic equilibrium to promote metal removal from metalloenzymes.
In order to experimentally test the role of MT in metal homeostasis,
it is necessary to first develop analytical techniques that can quantitatively
trace the movements of metals between different proteins under varying
redox conditions. These techniques require both exquisite sensitivity
and selectivity together with a multi-elemental capability. This
poster describes preliminary results obtained from the development of an EC/HPLC/ICP-MS system. This system involves directly coupling an
electrochemical flow cell (EC) with a high performance liquid chromatography
system (HPLC) to an inductively coupled plasma mass spectroscopy (ICP-MS)
to form a hybrid instrument.
The development of this technique will allow
metalloenzymes and MT mixtures to be sequentially reduced/oxidized under
clearly defined conditions, chromatographically separated and subsequently
quantified for metal content. The presented data describe the utility
of the technique for separating mixtures of alkaline phosphatase, metallothionein
and reduced and oxidized glutathione (GSH and GSSG respectively).
Glutathione has been hypothesized to be a key intermediary in both affecting
redox change and metal transfer from metallothionein.
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