Serda R E, Henzl M T
Department of Chemistry, New Mexico State University, Las Cruces 88003.
J Biol Chem. 1991 Apr 15;266(11):7291-9.
Lanthanide ion luminescence studies and 45Ca2(+)-binding measurements were used to study the metal ion-binding properties of avian thymic hormone. The procedure used to isolate the protein--involving heat-treatment at 80 degrees C, trichloroacetic acid precipitation, DEAE-agarose chromatography, and gel filtration--affords material that is deemed homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as the absence of a detectable tryptophan signal in the fluorescence emission spectrum. Avian thymic hormone exhibits a pI = 4.35 when subjected to isoelectric focusing through polyacrylamide gels. The two ion-binding sites are indistinguishable in their interactions with Ca2+ and Mg2+, displaying KCa = 8 nM and KMg = 68 microM. The Eu3+ 7Fo----5Do excitation spectrum at pH 6 displays a peak at 5795.4 A, with a shoulder at 5792.8 A and is replaced at higher pH values by a broader spectrum with a maximum at 5784.8 A and a shoulder at 5777.1 A. The pKa governing this spectral interconversion is 8.21. All of these properties are very similar to those observed with other parvalbumins. However, polyclonal antibodies to avian thymic hormone do not cross-react with the parvalbumin from chicken leg muscle, as judged by Western blot analysis-further evidence that avian thymic hormone and the muscle-associated chicken parvalbumin are indeed distinct proteins.
利用镧系离子发光研究和⁴⁵Ca²⁺结合测量来研究禽胸腺激素的金属离子结合特性。用于分离该蛋白质的程序——包括80℃热处理、三氯乙酸沉淀、DEAE-琼脂糖色谱和凝胶过滤——得到的物质通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳被认为是均一的，并且在荧光发射光谱中没有可检测到的色氨酸信号。禽胸腺激素在通过聚丙烯酰胺凝胶进行等电聚焦时，其等电点为4.35。这两个离子结合位点在与Ca²⁺和Mg²⁺的相互作用方面无法区分，KCa = 8 nM，KMg = 68 μM。Eu³⁺在pH 6时的⁷F₀→⁵D₀激发光谱在5795.4 Å处有一个峰，在5792.8 Å处有一个肩峰，在较高pH值时被一个更宽的光谱所取代，其最大值在5784.8 Å，肩峰在5777.1 Å。控制这种光谱相互转换的pKa为8.21。所有这些特性与在其他小清蛋白中观察到的非常相似。然而，通过蛋白质免疫印迹分析判断，针对禽胸腺激素的多克隆抗体与鸡腿肌肉中的小清蛋白不发生交叉反应——这进一步证明禽胸腺激素和与肌肉相关的鸡小清蛋白确实是不同的蛋白质。
