Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Barcelona, Spain.
Metallomics. 2019 Feb 20;11(2):349-361. doi: 10.1039/c8mt00294k.
The toxicity of lead, one of the most ubiquitous toxic metals, is well known. Some of its pathological effects are related to its preference for the sulfhydryl groups of proteins. Metallothioneins (MT) are a particular family of metalloproteins characterized by their high Cys content that, among other functions, are linked to the detoxification of heavy metals. In mammals, 4 MT isoforms have been found. The MT3 isoform, also called "neuronal growth inhibitory factor", is mainly synthesized in the brain and contains several structural differences that may contribute to important functional differences between it and other MT isoforms. The abilities of recombinant MT3 and its individual αMT3 and βMT3 fragments to bind Pb(ii) have been investigated here, under different pH conditions, by means of spectroscopy, mass spectrometry and isothermal titration calorimetry. The results obtained show that the binding of Pb(ii) to the intact MT3 protein is relatively unaffected by pH, while the individual domains interact with Pb(ii) in a pH-sensitive manner. The mass spectrometry data reveal the evolution with time of the initially formed Pb-MT complexes. In the case of the full length protein, Pb(ii) remains bound for a long period of time. With the isolated fragments, the lead is eventually released. The Pb-species formed depend on the amount of Pb(ii) present in solution. The thermodynamic data recorded, as measured by ITC, for the replacement of Zn(ii) by Pb(ii) in reactions with Zn-MT3, Zn-αMT3 and Zn-βMT3 are all similar, and in all cases, the displacement of Zn(ii) by Pb(ii) is thermodynamically favorable. Zn-Replete and Pb-replete MT3 have distinctive circular dichroism spectra, suggestive of structural differences with different metallation status.
铅是最普遍存在的有毒金属之一，其毒性众所周知。其一些病理效应与其对蛋白质巯基的偏好有关。金属硫蛋白（MT）是一类特殊的金属蛋白，其特点是 Cys 含量高，除其他功能外，还与重金属解毒有关。在哺乳动物中，已经发现了 4 种 MT 同工型。MT3 同工型，也称为“神经元生长抑制因子”，主要在大脑中合成，含有几个结构差异，可能导致其与其他 MT 同工型之间存在重要的功能差异。本文通过光谱法、质谱法和等温滴定量热法，在不同 pH 条件下，研究了重组 MT3 及其单个αMT3 和βMT3 片段与 Pb（ii）的结合能力。结果表明，完整 MT3 蛋白与 Pb（ii）的结合受 pH 影响相对较小，而单个结构域以 pH 敏感的方式与 Pb（ii）相互作用。质谱数据揭示了最初形成的 Pb-MT 复合物随时间的演变。对于全长蛋白，Pb（ii）长时间保持结合状态。对于分离的片段，铅最终被释放。形成的 Pb 物种取决于溶液中存在的 Pb（ii）量。通过 ITC 记录的热力学数据，用于 Zn-MT3、Zn-αMT3 和 Zn-βMT3 与 Zn（ii）反应中取代 Zn（ii）的 Pb（ii），均相似，在所有情况下，Pb（ii）取代 Zn（ii）在热力学上都是有利的。Zn 充足和 Pb 充足的 MT3 具有独特的圆二色性光谱，表明具有不同金属化状态的结构差异。
