Na D H, Woo B H, Lee K C
Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Korea.
Bioconjug Chem. 1999 Mar-Apr;10(2):306-10. doi: 10.1021/bc980029g.
Determination of the introduced moieties into derivatized proteins is an essential step in the preparation and quality control of chemically defined immunoconjugates. For the derivatized proteins using pyridyl disulfide-containing cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), the derivatization degree (ratio of pyridyl disulfide moieties to protein) has been traditionally determined by measuring the absorbance of both the derivatized protein and 2-thiopyridone (2-TP) released from the dithiothreitol (DTT) treatment (spectrophotometric method). This method, however, causes several problems including false high and low determinations of the protein and 2-TP, respectively, low selectivity, poor reproducibility, and relatively large amounts of sample consumption. A quantitative determination method of the derivatization ratios using bovine serum albumin derivatized with SPDP and SMPT as the model system has been developed. The concentration of protein and 2-TP released from the DTT treatment of derivatized proteins was determined directly without consideration of different reagents used and their concentrations. The present HPLC method was proved to be better in terms of accuracy, selectivity, and reproducibility with micro sample consumption. Moreover, this HPLC method can be directly applied to all derivatized proteins prepared with pyridyl disulfide-containing cross-linkers.
确定引入到衍生化蛋白质中的部分是化学定义免疫缀合物制备和质量控制中的关键步骤。对于使用含吡啶二硫键交联剂（如N-琥珀酰亚胺基3-(2-吡啶二硫基)丙酸酯（SPDP）和4-琥珀酰亚胺氧基羰基-α-甲基-α-(2-吡啶二硫基)甲苯（SMPT））衍生化的蛋白质，传统上通过测量衍生化蛋白质和二硫苏糖醇（DTT）处理释放的2-硫代吡啶酮（2-TP）的吸光度来确定衍生化程度（吡啶二硫键部分与蛋白质的比例）（分光光度法）。然而，该方法存在几个问题，包括分别对蛋白质和2-TP的测定出现假高和假低、选择性低、重现性差以及样品消耗量相对较大。已开发出一种以用SPDP和SMPT衍生化的牛血清白蛋白为模型系统的衍生化比例定量测定方法。直接测定衍生化蛋白质经DTT处理后释放的蛋白质和2-TP的浓度，而不考虑所使用的不同试剂及其浓度。事实证明，目前的高效液相色谱法在准确性、选择性和重现性方面表现更好，且微量样品消耗少。此外，这种高效液相色谱法可直接应用于所有用含吡啶二硫键交联剂制备的衍生化蛋白质。
