title: Affinity purification of aprotinin from bovine lung.
abstract: An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg.
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Jiangsu, P. R. China.
authors: Yu
authors: Y
authors: Xin
authors: Nanchang Wanhua Biochem Products Co., Ltd, Nanchang, Jiangxi, P. R. China.
authors: Lanhua
authors: L
authors: Liu
authors: Nanchang Wanhua Biochem Products Co., Ltd, Nanchang, Jiangxi, P. R. China.
authors: Beizhan
authors: B
authors: Chen
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Jiangsu, P. R. China.
authors: Ling
authors: L
authors: Zhang
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Jiangsu, P. R. China.
authors: Yanjun
authors: Y
authors: Tong
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity; D008168:Lung / Q000737:chemistry*
chemical_list: D007611:Aprotinin
keywords: Affinity ligands; Aprotinin; Bovine lung