Miller K I, van Holde K E, Toumadje A, Johnson W C, Lamy J
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Biochemistry. 1988 Sep 20;27(19):7282-8. doi: 10.1021/bi00419a016.
The C-terminal domain, Od-1, of the 7-domain subunit of Octopus dofleini hemocyanin has been prepared by partial trypsinolysis followed by ion-exchange chromatography. It binds oxygen reversibly and is homogeneous in molecular weight. Its physical properties have been compared with those of the subunit. The domain molecular weight is found by sedimentation equilibrium to be 4.7 X 10(4), in excellent agreement with the result recently obtained in our laboratory from cDNA sequencing of this domain [Lang, W. H. (1988) Biochemistry (preceding paper in this issue)]. It has a sedimentation coefficient of 3.8 S. Both the molecular weight and sedimentation coefficient are consistent with the domain constituting approximately one-seventh of the Mr 3.5 X 10(5) subunit. Its amino acid composition and carbohydrate content differ significantly from that of the whole subunit, confirming the heterogeneity in domains previously established on an immunological basis. Circular dichroism predicts similar secondary structure for the domain and subunit. The domain does not self-associate in the presence of Mg2+ but does bind to the whole molecule in a ratio of approximately 1 domain/subunit. The oxygen affinity of this domain is quite low. It shows intrinsic magnesium and Bohr effects similar to those of the whole molecule but of greatly reduced magnitude.
通过部分胰蛋白酶消化，随后进行离子交换色谱法，制备了太平洋大章鱼血蓝蛋白7结构域亚基的C末端结构域Od-1。它能可逆地结合氧气，且分子量均一。已将其物理性质与该亚基的物理性质进行了比较。通过沉降平衡法测得该结构域的分子量为4.7×10⁴，这与我们实验室最近从该结构域的cDNA测序中得到的结果[朗，W. H.（1988年）《生物化学》（本期前一篇论文）]非常吻合。它的沉降系数为3.8 S。分子量和沉降系数都表明该结构域约占分子量为3.5×10⁵的亚基的七分之一。其氨基酸组成和碳水化合物含量与整个亚基有显著差异，这证实了之前在免疫学基础上确定的各结构域的异质性。圆二色性预测该结构域和亚基具有相似的二级结构。在Mg²⁺存在的情况下，该结构域不会自我缔合，但能以大约1个结构域/亚基的比例与整个分子结合。该结构域的氧亲和力相当低。它表现出与整个分子相似的内在镁效应和玻尔效应，但程度大大降低。
