Detection of metalloproteins in human liver cytosol by synchrotron radiation X-ray fluorescence after sodium dodecyl sulphate polyacrylamide gel electrophoresis

An improved method of analysis of metals in protein bands with synchrotron radiation X-ray fluorescence (SRXRF) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation is introduced and applied to human liver cytosol. Through a step of drying the gel before SRXRF determination, the continuous background resulting mainly from the Compton-scattering of X-rays by the gel matrix was substantially reduced, and the detection of biological trace elements, such as Cu, Fe, and Zn in protein bands was thereby made possible. With the new procedure, six Zn-containing proteins with molecular weights (MWs) of 17.5, 20.5, 27, 35, 55, and 63kDa, respectively were found in human liver cytosol, among which the 63kDa Zn-containing band was shown to be the dominant form of zinc. In addition, at least four Fe containing proteins with MWs of 20, 23, 43, and 83.5kDa, respectively, were present in the samples. The metal contents in some metalloproteins, such as the 63 kDa Zn-containing protein, the 23 and 83.5kDa Fe-containing proteins, and a 22kDa Cu-containing protein were more closely related to the metal level in the sample. It is demonstrated that the procedure could be widely used to further investigate metal-binding proteins in biological samples.