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Instrumental design of SE-IE HPLC/ICP-MS.
Mixtures of the proteins were fractionated and analyzed by the two dimensional
HPLC/ICP-MS. A schematic of the two dimensional
HPLC/ICP-MS system
is shown in Figure 1. Aliquots (100µL) were fractionated isocratically
at a flow rate of 1ml min-1 by size-exclusion (Phenomenex BioSep 2000;
#5) using a mobile phase of 20mM Tris (pH 7.2) degassed with nitrogen (#2).
A Rheodyne 7000 switching valve (#10) is used to re-direct eluting species
of interest from the size exclusion to an ion-exchange column (Phenomenex
BioSep DEAE-P; #11). A linear gradient of 20mM Tris (#7; pH 8.2) increasing
to 250mM Tris in 500mM NH4Cl (#8) over a twenty one minute period was used
to fractionate the proteins from the ion-exchange column. The UV
absorbance of the eluent was monitored by two diode array detectors (System
Gold 168nm; #6 and #12) positioned down stream of the two columns. An elemental
spectrum was acquired simultaneously by scanning the quadrapole of the ICP-MS in the peak-hopping mode using time resolved acquisition software
(#15). A 200µL flow-injection loop inserted downstream of the
columns (#14) was used to quantify the resulting ion chromatograms from
the integrated peak areas of the ion intensity profiles from the MS.
Fig. 1. Schematic of the SE-IE HPLC/ICP-MS system.
Chemicals. Alkaline Phosphatase (AP), Metallothionien
(Mt), Reduced Glutathione (GSH), Oxidized Glutathione (GSSG), p-nitrophenal
phosphate (p-N), DTNB, and Glutathione reductase (GSR) were all ordered
from Sigma Chemicals. AP was further purified by SE-HPLC to remove
contaminating materials and desalt the sample. Fractions containing
both Zn and AP activity were pooled and concentrated to 100ul for reinjection
using an Amicon concentrator unit with a filter with a 32 KDa exclusion
limit.
Procedure for AP assay. The specific conditions
and procedure used to assay for AP activity are described in the poster
presentation by H. Huynh and A. Z. Mason, Ph.D. In brief 900ul of
1mM p-N suspended in 20mM Tris, pH 7.2, was mixed with the AP sample for
a total reaction volume of 1ml.The reaction was allowed to proceed for
three minutes at 370C at which time the absorbance of the sample was recorded
with a dual beam spectrophotometer at 412nm.
Procedure for GSH and GSSG assay. The assays
used for GSH and GSSG quantification are described in full in the poster
presentation by R. Moeller and A. Z. Mason, Ph.D. In brief, total
GSH and GSSG were analyzed using an enzymatic recycling procedure involving
glutathione reductase and NADPH. The quantities of each compound
were then calculated by masking pre-existing GSH with 2-vinyl pyridine
and repeating the reaction. GSSG content was then calculated by difference
from the two reaction rates.
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