Honda Rubens T, Araújo Roziete Mendes, Horta Bruno Brasil, Val Adalberto L, Demasi Marilene
Instituto Nacional de Pesquisas da Amazônia, INPA, Manaus, AM, Brazil.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Jun 25;820(2):205-10. doi: 10.1016/j.jchromb.2005.03.017. Epub 2005 Apr 25.
We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.
我们描述了一种通过金属螯合柱亲和层析，从注射镉的亚马逊鱼类巨脂鲤肝脏中一步纯化金属硫蛋白，以及从酿酒酵母中负载铜的金属硫蛋白中一步纯化金属硫蛋白的方法。酵母金属硫蛋白从负载Cu2+的树脂中纯化出来，并通过连续的EDTA梯度洗脱，而从鱼类中提取的肝脏金属硫蛋白则通过负载Ni2+的树脂纯化，并通过连续的咪唑梯度洗脱。通过SDS-PAGE对纯化的金属硫蛋白进行评估，并通过脱辅基蛋白和负载Cd2+蛋白的紫外光谱对其进行表征。该方法具有高纯度、高产量以及能快速一步提取负载金属的蛋白和脱辅基蛋白的特点。
