Optimizing dye-ligand density with molecular analysis for affinity chromatography of rabbit muscle L-lactate dehydrogenase.
Ligand density is an important factor in determining the binding capacity and separation efficiency for affinity chromatography. A molecular analysis method based on the three-dimensional structure of protein and protein-ligand interactions was introduced to optimize the dye-ligand density for target protein separation. Expanded-bed adsorption (EBA) of L-lactate dehydrogenase (LDH) from rabbit muscle crude extract with Procion Red HE-3B as the dye-ligand was used as the model. After the analysis of LDH three-dimensional molecular structure and dye-protein interaction modes, the rational dye-ligand distance was predicted at about 20 A for efficiently binding LDH. A series of dye-ligand adsorbents with different ligand densities were prepared, and the isotherm adsorption equilibria of LDH were measured. High adsorption capacity of LDH was achieved at about 1600 U/mL adsorbent. Packed-bed chromatography was performed, and the elution effects were investigated. Finally, an EBA process was achieved to capture the LDH directly from rabbit muscle crude extract. The method established in the present work could be expanded to guide the screening of ligand density for other affinity chromatographic processes.
Lin
Dong-Qiang
DQ
State Key Laboratory of Chemical Engineering, College of Materials Science and Chemical Engineering, Zhejiang University, 310027 Hangzhou, People's Republic of China.
Yao
Shan-Jing
SJ
D000327:Adsorption; D000818:Animals; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D002845:Chromatography; D002846:Chromatography, Affinity; D004396:Coloring Agents / Q000737:chemistry*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D008024:Ligands; D008956:Models, Chemical; D008968:Molecular Conformation; D009132:Muscles / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013053:Spectrophotometry / Q000379:methods*
D004396:Coloring Agents; D008024:Ligands; D007770:L-Lactate Dehydrogenase