Direct Detection of Mercury-Bound Metalloproteins (metallothionein and Cu, Zn-Superoxide Dismutase) Using a Combination of Gel Electrophoresis and One Dimensional Synchrotron Radiation X-Ray Fluorescence Analysis

Abstract

Metallothionein-II (MT-II) and Cu, Zn-superoxide dismutase (Cu, Zn-SOD) interacted with mercury were detected by a new method utilizing isoelectric focusing-agarose or -polyacrylamide gel electrophoresis (IEF-AGE or IEF-PAGE) and nondestructive one-dimensional synchrotron radiation X-ray fluorescence (SR-XRF) analysis. When MT-II reacted with mercuric chloride, an obvious change of isoelectric point (pI = 3.7 - 4.7) for the intact form to alkaline pI (9.4) was observed. This marked migration of MT-II by the metal was blocked by addition of glutathione, suggesting that sulfhydryl functions participate in the pI variation. In contrast, interaction of Cu, Zn-SOD with mercury did not cause any changes of its pI although the metal bound tightly to Cu, Zn-SOD after electrophoresis; however, the enzyme activity was drastically suppressed. These observations indicate that combination of electrophoresis with SR-XRF analysis is an useful technique for detecting structural or functional alteration of protein attributable to the binding of the mercury.