Smales C M, Moore C H, Blackwell L F
Institute of Fundamental Sciences, Chemistry, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
Bioconjug Chem. 1999 Jul-Aug;10(4):693-700. doi: 10.1021/bc9900441.
Estrone glucuronide conjugates of hen egg white lysozyme were prepared by the mixed anhydride and active ester coupling procedures. Both methods gave good yields of conjugates, but the active ester procedure gave a more diverse range of products, making it less suitable for preparing conjugates for homogeneous enzyme immunoassay. Conjugation of lysozyme with estrone glucuronide by the mixed anhydride procedure gave one major derivative exclusively acylated at lysine residue 33 whereas conjugation by the active ester method gave six derivatives which were acylated at one or more of lysine residues 33, 97, and 116. None of the lysine residues 1, 13, and 96, or the N-terminal alpha-amino group, were acylated in any of the conjugates isolated. The correlation of the conjugate structures with the protein environments of the amino groups in the crystal structure of lysozyme suggested that the sites of acylation were determined not only by the chemical nature of the acylating reagent but also by the surface accessibility and nucleophilicity of the individual lysine residues.
通过混合酸酐法和活性酯偶联法制备了鸡蛋清溶菌酶的雌酮葡萄糖醛酸苷缀合物。两种方法都能得到较高产率的缀合物，但活性酯法得到的产物种类更多，因此不太适合制备用于均相酶免疫分析的缀合物。通过混合酸酐法将溶菌酶与雌酮葡萄糖醛酸苷缀合得到一种主要衍生物，其仅在赖氨酸残基33处被酰化，而通过活性酯法得到六种衍生物，它们在赖氨酸残基33、97和116中的一个或多个处被酰化。在分离得到的任何缀合物中，赖氨酸残基1、13和96以及N端α-氨基均未被酰化。缀合物结构与溶菌酶晶体结构中氨基的蛋白质环境之间的相关性表明，酰化位点不仅由酰化试剂的化学性质决定，还由各个赖氨酸残基的表面可及性和亲核性决定。
