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Materials. E. coli alkaline phosphatase
and all chemicals were purchased from Sigma. Deionized water and
metal-free glassware and pipette tips were used throughout the experiment.
Chelating resin (Iminodiacetic Acid – Chelex 100) was used to sequester
metals released from the enzyme.
Preparation of Apo-Alkaline Phosphatase.
Approximately 1ml of alkaline phosphatase suspended in 2.5 M ammonium sulfate
was transferred to a 1.5ml Eppendorf microcentrifuge tube. The sample
was then centrifuged at 10,000g for ten minutes. The enzyme was collected
and the supernatant was discarded. The pellet of alkaline phosphatase
was dissolved and brought up to the total of 2ml in 1M Tris-HCl, pH = 8.0.
About 0.5 ml of the resuspended enzyme was added to 500mg of Chelex resin.
It was then incubated for 5.5 hours on a rotary shaker. After that
the sample was centrifuged and the supernatant was reprocessed as above.
The final supernatant containing the apo-alkaline phosphatase was stored
at –4OC to await analysis.
Protein and Enzyme Assay. The concentration
of apo-alkaline phosphatase was determined using a Coomassie Protein assay
from a protein standardization curve. Alkaline phosphatase
activity was assayed spectroscopically by measuring the rate of conversion
of p-nitro phenyl phosphate at 412nm. For the assay, exactly 2.7ug,
5.4ug, 8.1ug, and 10.8ug of alkaline phosphatase were pipetted into cuvettes
and made up to 100ul using 20mM Tris-HCl. To this solution, 900ul
of p-nitro phenyl phosphate substrate was added to bring the final
volume in each cuvette to 1ml. The enzyme and the substrate were
mixed and incubated at 31oC. The rate formation of the product was
monitored at 412nm for about 30 minutes. The slopes of the linear
portions of the reaction curves were used to generate a standard curve
relating the rate of activity against total enzyme content.
HPLC-ICPMS. Aliquots of alkaline
phosphatase and apo-alkaline phosphatase ranging from 50-100µL in
volume were fractionated isocratically by size-exclusion (SW 2000 TSK Column)
at a flow rate of 1ml/min using a mobile phase of 20mM Tris, (pH 7.2).
The UV absorbance of the eluent was monitored continuously at a resolution
of 1nm from 200-600nm using a Beckman 168 Diode Array Detector with a 25µL
flow cell. The eluent was directed via a transfer interface to either a
Perkin Elmer 6100 DRC or a HP-4500 ICP-MS operated in the time resolved
mode which was used to simultaneously acquire data on 6 or more elements.
A 250µL flow injection loop installed immediately after the diode
array spectrophotometer and prior to the ICPMS was used to quantify the
elemental composition of the resolved peaks and monitor for substitutive
exchanges of metal occurring during chromatography. Elemental quantification
was conducted off-line using the data obtained from the integrated mass
of analyte from the flow injection loop using a data analysis chromatographic
package.
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