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

Metallothionein-II (MT-II) and Cu, Zn-superoxide dismutase (Cu,Zn-SOD) interacted with mercury were detected by a new method utilizing isoel ectric focusing-agarose or -polyacrylamide gel electrophoresis (IEF-AG E or IEF-PAGE) and nondestructive one-dimensional synchrotron radiatio n X-ray fluorescence (SR-XRF) analysis. When MT-II reacted with mercur ic chloride, an obvious change of isoelectric point (pI = 3.7 - 4.7) f or the intact form to alkaline pI (9.4) was observed. This marked migr ation of MT-II by the metal was blocked by addition of glutathione, su ggesting that sulfhydryl functions participate in the pi variation In contrast, interaction of Cu,Zn-SOD with mercury did not cause any chan ges of its pi although the metal bound tightly to Cu,Zn-SOD after elec trophoresis; however, the enzyme activity was drastically suppressed. These observations indicate that combination of electrophoresis with S R-XRF analysis is an useful technique for detecting structural or func tional alteration of protein attributable to the binding of the mercur y.