Immobilization and Characterization of Biochemicals

Part of our research involves the optimization or creation of new schemes for immobilizing and modifying biological agents. The immobilization of biological agents can be accomplished using three general approaches.  These methods include noncovalent techniques like nonspecific and biospecific absorption, covalent coupling techniques, entrapment, and molecular imprinting. Students working in this area explore the use of both organic and inorganic methods of synthesis for the attachment and modification of biochemicals, as needed in high-performance affinity chromatography or other bioanalytical methods.

We have used all of these methods in our group, but most of our work involves the use and development of covalent immobilization methods. Covalent immobilization can be initiated through several different functional groups on the ligand.  Certain immobilization techniques, such as the cyanogen bromide method, the Schiff base method (i.e., reductive amination), the N-hydroxysuccinimide technique, and the carbonyldiimidazole method, use free amine groups during protein immobilization.   Other functional groups that may be employed include sulfhydryl groups, carboxyl groups, and carbonyl groups.   For instance, our group has developed a general method that can be used for the site-selective immobilization of antibodies through their carbohydrate chains.  We have also optimized and studied techniques for the immobilization of the proteins human serum albumin (HSA), alpha 1-acid glycoprotein (AGP), and proteolytic enzymes.  Several patents have been awarded to our group for this research and many papers have resulted for this work, as have appeared in journals like Analytical ChemistryAnalytical BiochemistryBioconjugate Chemistry and the Journal of Chromatography (and B).

Text Box: General approach developed for the entrapment of a protein or large ligand in porous silica Fg. 1

Another interesting type of immobilization that has recently been created and utilized in our group is an entrapment method.  This entrapment method is a noncovalent technique for protein and ligand immobilization that is capable of being used with common HPLC supports.  This entrapment method involves the physical containment of a protein or ligand in a polysaccharide-capped dihydrazide support.  When these oxidized polysaccharides are incubated with the hydrazide-activated support, aldehyde groups on the oxidized polysaccharide will form a stable covalent bond with the hydrazide groups, a reaction known to occur even in a neutral aqueous buffer.  This process will entrap the ligand in the support.

Our group also uses a variety of tools to study immobilized proteins as part of this work in the creation and optimization of immobilization methods.  These tools have included protein assays, flow-injection analysis, infrared spectroscopy, solid-state nuclear magnetic resonance spectroscopy, and mass spectrometry.  Both HPAC and capillary electrophoresis are also used as part of this work.

Examples of Recent Work in the Immobilization & Characterization of Biochemicals

  1. Hee Seung Kim, Young Sik Kye and David S. Hage, “Development & Evaluation of N-Hydroxysuccinimide-Activated Silica for Immobilization of Human Serum Albumin in HPLC Columns”, J. Chromatogr. A, 1049 (2004) 51-61.
  2. Hai Xuan and David S. Hage, “Immobilization of α1-Acid Glycoprotein for Chromatographic Studies of Drug-Protein Binding”, Anal. Biochem., 346 (2005) 300-310.
  3.  Hee Seung Kim and David S. Hage, “Immobilization Methods for Affinity Chromatography”, In: Handbook of Affinity Chromatography, (D. S. Hage, Editor), CRC Press/Taylor & Francis, 2006, Chapter 3.
  4. Hee Seung Kim, Rangan Mallik and David S. Hage, “Chromatographic Analysis of Carbamazepine Binding to Human Serum Albumin. II. Comparison of the Schiff Base and N-Hydroxysuccinimide Immobilization Methods”, J. Chromatogr. B, 837 (2006) 138-146.
  5. Rangan Mallik, Chunling Wa and David S. Hage, “Development of Sulfhydryl-Reactive Silica for Protein Immobilization in High-Performance Affinity Chromatography”, Anal. Chem., 79 (2007) 1411-1424.
  6. Mary Anne Nelson, Annette C. Moser and David S. Hage, “Biointeraction Analysis by High-Performance Affinity Chromatography: Kinetic Studies of Immobilized Antibodies”, J. Chromatogr. B, 878 (2010) 165-171.
  7. Hai Xuan, K.S. Joseph, Chunling Wa and David S. Hage, “Biointeraction Analysis of Carbamazepine Binding to Alpha 1-Acid Glycoprotein by High-Performance Affinity Chromatography”, J. Sep. Sci., 33 (2010) 2294-2301
  8. Abby J. Jackson, Hai Xuan and David S. Hage, “Entrapment of Proteins in Glycogen-Capped and Hydrazide-Activated Supports”, Anal. Biochem., 404 (2010) 106-108.