Schafer M A, Wilson G S
J Biol Chem. 1983 Nov 10;258(21):12835-41.
Cytochrome c has been covalently attached to Sepharose 6MB to study the effects of immobilization on the molecule. A detailed study of the resulting product was conducted based on its three characteristic properties: spectra, oxidation-reduction potential, and biological activity. The spectral properties demonstrated that cytochrome c was essentially the same after attachment. No major conformational changes were indicated. The redox potentials for most samples of immobilized cytochrome c loaded with different amounts of protein were generally 20-25 mV lower than native cytochrome c (270 mV). Heavily loaded samples, however, showed no difference in potential. The Km values for immobilized cytochrome c with cytochrome oxidase and reductase from submitochondrial particles were comparable to the soluble protein. Vmax values are more strongly affected by immobilization, especially for the reductase. It has been demonstrated that the submitochondrial particles cannot penetrate the pores of the support material and therefore only the cytochrome c molecules on the surface are available for reaction. As a support material, Sepharose 6MB, which is CNBr activated, hydrolyzes at a significant rate at the pH of the coupling reaction, and this must be considered in establishing coupling conditions for protein immobilization.
细胞色素c已被共价连接到琼脂糖6MB上，以研究固定化对该分子的影响。基于其三个特性：光谱、氧化还原电位和生物活性，对所得产物进行了详细研究。光谱特性表明，连接后细胞色素c基本相同。未显示出主要的构象变化。大多数负载不同量蛋白质的固定化细胞色素c样品的氧化还原电位通常比天然细胞色素c（270 mV）低20 - 25 mV。然而，高负载样品的电位没有差异。固定化细胞色素c与亚线粒体颗粒中的细胞色素氧化酶和还原酶的Km值与可溶性蛋白质相当。Vmax值受固定化的影响更大，尤其是对于还原酶。已经证明亚线粒体颗粒无法穿透载体材料的孔，因此只有表面的细胞色素c分子可用于反应。作为载体材料，经溴化氰活化的琼脂糖6MB在偶联反应的pH值下会以显著速率水解，在建立蛋白质固定化的偶联条件时必须考虑这一点。
