Issue 9, 2007

Determination of “free” iron and iron bound in metalloproteinsvia liquid chromatography separation and inductively coupled plasma-optical emission spectroscopy (LC-ICP-OES) and particle beam/hollow cathode-optical emission spectroscopy (LC-PB/HC-OES) techniques

Abstract

The abilities of liquid chromatography-particle beam/hollow cathode-optical emission spectrometry (LC-PB/HC-OES) and liquid chromatography-inductively coupled plasma-optical emission spectroscopy (LC-ICP-OES) techniques are demonstrated for the quantitative determination of free iron and bound iron in metalloproteins. Separations were performed in the size exclusion and reversed-phase chromatographic modes. Myoglobin, holo-transferrin and iron(II) chloride were separated by size exclusion chromatography and the eluent species were detected by both PB/HC-OES and ICP-OES through the Fe I 259.9 nm and S I 180.7 nm optical emission. Sulfur optical emission was monitored as means of protein identification through the Fe/S empirical formula differences, with the absence of S emission used to identify the “free” Fe. Both techniques demonstrated detection limits for triplicate injections on the nanogram level for iron (0.9 ng mL−1 ICP-OES and 41.9 ng mL−1 PB/HC-OES) over the concentration range of 0.1 ng mL−1–100 μg mL−1 for iron and iron metalloproteins. The corresponding values for sulfur in the proteins were more similar for the two techniques, being of the order of 20 ng mL−1. The SEC retention times and peak shapes of the three analyte peaks for both techniques are similar to those determined by UV absorbance at 220 nm (Fe-containing metalloproteins) and conductivity (inorganic iron), demonstrating the ability of both techniques to preserve the integrity of the separation. While the LODs in “bulk” sampling were lower with ICP-OES, the signal-to-noise values were comparable for both techniques when sampling chromatographic eluents in real-time. In addition, a reversed-phase separation of the same analyte mixture was carried out with Fe I 259.9 nm and S I 180.7 nm optical emission detection by the PB/HC-OES, demonstrating the compatibility of the PB/HC-OES with a wide range of solvent polarities, its ability to detect non-metals and act as a protein specific detector for liquid chromatography of proteins.

Graphical abstract: Determination of “free” iron and iron bound in metalloproteins via liquid chromatography separation and inductively coupled plasma-optical emission spectroscopy (LC-ICP-OES) and particle beam/hollow cathode-optical emission spectroscopy (LC-PB/HC-OES) techniques

Article information

Article type
Paper
Submitted
19 Mar 2007
Accepted
09 Jul 2007
First published
03 Aug 2007

J. Anal. At. Spectrom., 2007,22, 1067-1075

Determination of “free” iron and iron bound in metalloproteins via liquid chromatography separation and inductively coupled plasma-optical emission spectroscopy (LC-ICP-OES) and particle beam/hollow cathode-optical emission spectroscopy (LC-PB/HC-OES) techniques

T. M. Brewer and R. K. Marcus, J. Anal. At. Spectrom., 2007, 22, 1067 DOI: 10.1039/B703812G

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