Quantification of trace elements in protein bands by synchrotron radiation x-ray fluorescence after isoelectric focusing separation of human hemoglobin

The role and effects of a trace element in a particular organism strongly depend on its particular chemical forms in which the element is present. Therefore, the bulk content or concentration of an element in the organism of interest is often meaningless in judging its biological significance. To understand bioavailability, transportation, cell uptake, metabolism, toxicity, and other biological behaviors of trace elements in the body, information is needed about speciation of trace element, especially about distribution of metal-containing proteins. Development of appropriate methods for speciation analysis is therefore required. Synchrotron radiation x-ray fluorescence (SRXRF) is a sensitive method for multielemental analysis with detection limit of 10 ng/g. It has been successfully used for imaging and quantifying trace elements in various pathological and healthy tissues, even in a single cell, to help understand the mechanism of diseases and the biochemistry of elements. In our previous work, the technique was combined with electrophoresis to study distribution of metalloproteins in biological samples, but the quantitative analysis of trace elements in protein bands after electrophoresis was still unrealized. In this study, a procedure has been proposed for quantification of Fe, Cu, and Zn in protein bands with SRXRF analysis after isoelectric focusing (IEF) separation. Calibration standards were prepared by adding certain amounts of metal ions and free-metal proteins to electrophoresis gel. Human hemoglobin was separated with IEF, and Fe, Cu, and Zn in protein bands were analyzed by SRXRF. The calibration curves can be obtained in a range of 0-8 mg/kg metals and a linear relationship between dosage of metals and fluorescent intensity can be observed (r2 > 0.99). The method provides the detection limits of 2.43, 1.12, and 0.96 mg/kg for Fe, Cu and Zn, and the recoveries of 90.4 and 115.7 % for Fe and Zn, respectively. The hyphenated technique of SRXRF and IEF has advantages of quantifying trace elements in proteins and of identifying the proteins simultaneously. It can be employed for speciation analysis of trace elements in biological samples.