Detection of metalloproteins in human liver cytosol by synchrotron radiation X-ray fluorescence combined with gel filtration chromatography and isoelectric focusing separation

Abstract

Synchrotron radiation X-ray fluorescence (SRXRF) spectroscopy is an advanced method of quantitative multielemental analysis with space resolution of several μm and sensitivities in the μg g⁻¹ range. It can be used for keeping track of trace elements after an electrophoretic separation of biological samples. In this paper, proteins in human liver cytosol were separated with gel filtration chromatography and thin layer isoelectric focusing (IEF). The contents of metal ions in protein bands were determined by SRXRF. The results showed that in the molecular weight (MW) range of 10∼25 kDa, there were at least 2 Zn-containing bands with isoelectric point (pI) of 5∼6 and 6.2∼7, respectively and about 11 Fe-containing proteins with pI of 4.4, 4.6, 4.8, 5.0, 5.2, 5.3, 5.5, 5.6, 6.6, 6.8, and 7.2, respectively, present in human liver cytosol. The Zn-containing band with pI of 5–6 is the dominant species of zinc in this MW range. In addition, the Cu-containing bands with pI of 5.0 and below 4.8 were also detected. It is demonstrated that the procedure could be widely used in further investigations of the chemical species of trace elements in biological samples.