Four affinity ligands were designed from 6-chloromethyluracil and 2-aminobenzimidazole and simulated for the interaction with bovine hyaluronidase-1. Regarding sequence alignment, bovine hyaluronidase-1 precursor showed circa 83.6% similarity with human hyaluronidase-1. Regarding structural modeling and molecular docking, bovine hyaluronidase-1 interacted with ligands in the active site. Using epichlorohydrin, 1,3-propanediamine and cyanuric chloride as spacers, 6-chloromethyluracil and 2-aminobenzimidazole were composed to Sepharose beads. The modified Sepharose beads were then subjected to adsorption analysis with bovine hyaluronidase. After one step of affinity adsorption, the samples extracted from bovine testes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and activity assay. As calculated, the densities of four ligands on sorbents (entitled as L-1, L-2, L-3 and L-4) were 37.7 ± 2.3, 36.4 ± 3.2, 42.4 ± 4.2 and 33.7 ± 2.3 μmol/g wet gel; the theoretical maximum adsorption (Q_max) of bovine hyaluronidase on the four sorbents were 63.6 ± 1.6, 72.0 ± 0.7, 111.0 ± 4.1 and 121.7 ± 2.3 mg/g wet gel, respectively; the dissociation constants (K_d) of the four sorbents were 18.5 ± 0.8, 48.1 ± 4.3, 35.0 ± 3.0, 40.6 ± 2.7 μg/g wet gel, respectively. After optimization, the proteins captured by sorbents attaching 2-aminobenzimidazole based ligands (L-3 and L-4) revealed the main single band at approximately 50 kDa, and the purities were about 85.2 and 96.4%; the bioactivity recoveries were 83.5 and 89.4%. In addition, the bands on SDS-PAGE gel were also extracted and confirmed with linear trap quadropole mass spectrometry (LTQ-MS) analysis.

从6-氯甲基尿嘧啶和2-氨基苯并咪唑设计了四个亲和配体，并模拟了它们与牛透明质酸酶-1的相互作用。关于序列比对，牛透明质酸酶-1前体与人透明质酸酶-1显示出约83.6％的相似性。关于结构建模和分子对接，牛透明质酸酶-1在活性位点与配体相互作用。以表氯醇，1，3-丙二胺和氰尿酰氯为间隔基，将6-氯甲基尿嘧啶和2-氨基苯并咪唑合成为琼脂糖珠。然后用牛透明质酸酶对修饰的琼脂糖珠进行吸附分析。经过亲和吸附一步后，将从牛睾丸中提取的样品进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析和活性测定。根据计算，吸附剂上四个配体（分别为L-1，L-2，L-3和L-4）的密度分别为37.7±2.3、36.4±3.2、42.4±4.2和33.7±2.3μmol/ g湿凝胶。理论最大吸附量（四种吸附剂上的牛透明质酸酶的Q _max分别为63.6±1.6、72.0±0.7、111.0±4.1和121.7±2.3 mg / g湿凝胶; 四种吸附剂的解离常数（K _d）分别为湿凝胶18.5±0.8、48.1±4.3、35.0±3.0、40.6±2.7μg/ g。优化后，吸附剂结合2-氨基苯并咪唑基配体（L-3和L-4）捕获的蛋白质显示出约50 kDa的主要单条带，纯度分别为约85.2和96.4％。生物活性回收率分别为83.5％和89.4％。此外，还提取了SDS-PAGE凝胶上的条带，并通过线性阱四极杆质谱（LTQ-MS）分析进行了确认。