大鼠肾脏汞结合金属硫蛋白的联用技术表征研究

Characterization of Mercurial-bound Metallothioneins in Rat Kidney by Hyphenated Techniques

通过凝胶色谱(SEC)及反相液相色谱(RPC)分别与电感耦合等离子体质谱(ICP-MS)和电喷雾电离质谱(ESI-MS)联用的技术,对灌喂HgCl2大鼠的肾组织中诱导的多金属结合金属硫蛋白(MT)的结构进行表征分析。通过SEC鄄ICP-MS曲线上的金属信号及紫外检测的MT光谱吸收特征,可确认组织提取液中MT在色谱曲线上的位置。与对照组相比,灌喂HgCl2大鼠肾中Hg-MT的诱导量显著增加,与此同时,Cu-MT在染毒组的诱导量也相应增加,说明MT在染汞大鼠肾脏中起着对汞的解毒和对铜的调节作用。MT样品提取物经葡聚糖凝胶(G-75)柱分离纯化并用透析法脱盐后,用细内径反相色谱柱分离,进行梯度洗脱,并与ESI-MS联用,可获得总离子流色谱图及与色谱峰对应的质谱图,通过对质谱信号的解析及参考脱金属MT构型的有关报道,对汞诱导大鼠肾组织MT的不同亚型和次亚型分子结构进行推断。结果证明,在灌喂HgCl2大鼠的肾组织中存在着一系列汞结合的MT形态。

Multimetal-bound metallothioneins (MT) induced in kidney of rat after oral intake of HgCl2 were characterized with hyphenated techniques as size-exclusion chromatography (SEC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) and reverse phase chromatography (RPC) coupled with electrospray ionization mass spectrometry (EST-MS). The identification of MT fractions in the size-exclusion chromatographic profiles was achieved by both of the chromatogram of SEC-ICP-MS and the corresponding UV spectra of MT. The amount of Hg-MT in rat kidney of HgCl2 group significantly increased compared with that of the control group. In addition, the signal of Cu-MT was largely induced in polluted rat tissue. The results validated the important roles of MT played in the homeostasis of Cu for the detoxification of mercury. After being purified with size-exclusion column (Sephadex G-75) and dialysis (M<1000 Da), the extracts of rat kidney was separated with a narrow-bore reverse phase column and eluted by increasing methanol at gradient mode. The MT polymorphism with different binding-metal (including Hg, Cu and Zn) species was observed by ESI-MS. The tentative formulas of Hg-MT isoforms and subisoforms were interpreted by the measured masses at the vicinity of apex in total Ion current (TIC) of ESI-MS and the reference data of apo-MT reported. The results indicated that many Hg-binding MT species existed in the polluted rat kidney.