摘要
镉诱导蚯蚓（赤子爱胜，Ｅｉｓｅｎｉａｆｏｅｔｉｄａ）匀浆液经热沉淀，乙醇沉淀后，经凝胶过滤ＳｅｐｈａｃｒｙＳ－２００柱层析，得三个镉结合蛋白峰，分子量依次为４０，１７及９ｋＤ。这三个组份再分别经ＤＥＡＥＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ柱层析，分别得三、三、二个镉结合蛋白峰。根据光谱学特征、巯基含量及氨基酸组成等分析，认为凝胶过滤第二峰为金属硫蛋白（ＭＴ），其经ＤＥＡＥ柱层析所得三个峰为ＭＴ三个亚型。对蚯蚓ＭＴ三个亚型在不同ｐＨ条件下的紫外吸收（ＵＶ）、荧光发射（ＦＳ）、圆二色性（ＣＤ）及扫描隧道显微镜（ＳＴＭ）图象的测定研究结果表明：（１）蚯蚓ＭＴ各亚型在２５０ｎｍ左右均具有一个紫外吸收肩，为Ｃｄ—ＳＨ络合结构特征吸收，随ｐＨ下降而趋于消失。（２）ＭＴ各亚型在２８２ｎｍ激发下，荧光发射的最大波长（Ｐｍ）均在３４０ｎｍ左右。随着ｐＨ下降，Ｐｍ红移且量子产率减小。（３）ｐＨ２．０以上ＭＴ各亚型在２４７ｎｍ附近有一ＣＤ正吸收，２１０ｎｍ附近则有一ＣＤ负吸收。随着ｐＨ降低，在整个２００—３００ｎｍ区间上ＭＴ各亚型的［θ］值均趋负方向变化，当ｐＨ从２．０降至１．０时，则呈现负向骤变。（４）扫描隧道显微镜观察结果显示?
With sephacryl S-200 and DEAE sepharose fast flow column we obtained three isoforms of metallothionein (MT) from earthworm (EW- ⅡA, EW-ⅡB, EW-ⅡC) induced by cadmium . In this context, we measured the ultraviolet absorption(UV), fluroscence emission (FS), circular dichroism (CD) and scanning tunneling microscopy (STM) of all the three MTs under different pH value. The results are reported as follows:1. All the three MTs from earthworm had UV absorption shoulder near 250nm. This shoulder was the specific adsorption for Cd (Ⅱ )-SH complex and disappeared when pH decreased to 1.5, which indicated the metal removal of MT molecule under acid condition.2. With excitation at 282nm the FS emission spectra of the three MTs had a maximum (Pm) near 340nm, and with the pH decreasing the red shift and the quantum yield decreasing could be observed. When pH decreased from 1.5 to 1.0, there was an acute decreasing for the quantum yield near 340nm emission, which showed that the acute metal removal and conformation change of MTs occurred.3. CD spectra showed that above pH 2.0 there were a strong positive ellipticity band located near 247nm and a negative band located near 210nm for the three MTs. With pH decreasing the CD band of the three MTs covering 200 to 300nm decreased, and when pH changed from 2.0 to 1.0 the ellipticity band emerged an acute drop.4. The result of scanning tunneling microscopy showed that the EW-ⅡA was composed of two elliptic domains with diameters of 30-40A and 50-60A, and the diameter of whole molecular was 80-100A.In conclusion, the acid condition causes metal removal from MT, and the combination of MT with metal is the key faCtor MT to keep its tight conformation.
出处
《生物物理学报》
CAS
CSCD
北大核心
1994年第4期529-536,共8页
Acta Biophysica Sinica
基金
国家高科技重点项目No.863-103-21-09研究基金
关键词
蚯蚓
金属硫蛋白
构象
分离
提纯
Earthworm Metallothionein Ultraviolet Fluorescence Circular chroism Scanning tunneling microscopy
