Affinity adsorption of bovine hyaluronidase with ligands targeting to active site

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.