兔肝金属硫蛋白β结构域的制备和鉴定

制备一定量的Ｃｄ、Ｚｎ兔肝金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ，ＭＴ），然后脱掉其中的金属，获得不含金属的硫蛋白（ａｐｏＭＴ）．用一价金属ＣｕＣｌ或ＡｇＮＯ３以５．８１的金属蛋白摩尔比与ａｐｏＭＴ结合，将其β结构域用金属饱和，在３７℃，ｐＨ７．０下用枯草杆菌蛋白酶水解掉未结合金属的α结构域，获得Ｃｕ（Ⅰ）、Ａｇ（Ⅰ）结合的兔肝ＭＴ－Ｉ、ＭＴ－Ⅱ的β结构域．通过ＨＰＬＣ、羧甲基化后的ＳＤＳ－ＰＡＧＥ、氨基酸组成分析、金属定量、Ｎ末端分析和紫外扫描等鉴定，证明确实得到了Ｎ末端为Ｍｅｔ，分子量３０００左右，金属蛋白摩尔比为５～５．５１的ＭＴβ结构域，其氨基酸组成与理论值基本相符

The Preparation and Analysis of Rabbit Liver MT β domain

Following the preparation of rabbit liver MT α domain by our group years ago,the rabbit liver β domain is here prepared by the metal/ion semi saturation method.ApoMT combined with 5.8 times molar concentration of CuCl or AgNO 3 makes its β domain resistant to proteolysis while the α domain is still susceptable.Thus the rabbit liver β domain of MT(Ⅰ)and MT(Ⅱ) were obtained by partial proteolysis of this semi saturated MT with Cu +or Ag +.Some analysis such as HPLC,SDS PAGE,amino acid analysis and N terminal analysis on the Cu、Ag β domain of MT were also done. The results showed,that β domain was prepared as expected including the linker part two lysines at site 30 and 31.