Specific Covalent Immobilization of Proteins through Dityrosine Cross-Links

• Published in: Langmuir Vol. 22; no. 26; pp. 11305 - 11310
• Main Authors: Endrizzi, Betsy J, Huang, Gang, Kiser, Patrick F, Stewart, Russell J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 19-12-2006
• Subjects: Catalysis; Chemistry; Cross-Linking Reagents - chemistry; Exact sciences and technology; General and physical chemistry; Microspheres; Nickel - chemistry; Oxidation-Reduction; Peptides - chemistry; Protein Array Analysis - methods; Theory of reactions, general kinetics. Catalysis. Nomenclature, chemical documentation, computer chemistry; Tyrosine - analogs & derivatives; Tyrosine - chemistry; Catalytic reaction; Peptides; Immobilization; Metal complex; Washing; EDTA; Complexes; Optimization; Protein; Acrylamide; Acids; Residue; Models; Oxidation; Monomer; Structure
• ISSN: 0743-7463; 1520-5827
• DOI: 10.1021/la0618216

Dityrosine cross-links are widely observed in nature in structural proteins such as elastin and silk. Natural oxidative cross-linking between tyrosine residues is catalyzed by a diverse group of metalloenzymes. Dityrosine formation is also catalyzed in vitro by metal−peptide complexes such as Gly-Gly-His-Ni(II). On the basis of these observations, a system was developed to specifically and covalently surface immobilize proteins through dityrosine cross-links. Methacrylate monomers of the catalytic peptide Gly-Gly-His-Tyr-OH (GGHY) and the Ni(II)-chelating group nitrilotriacetic acid (NTA) were copolymerized with acrylamide into microbeads. Green fluorescent protein (GFP), as a model protein, was genetically tagged with a tyrosine-modified His6 peptide on its carboxy terminus. GFP−YGH6, specifically associated with the NTA−Ni(II) groups, was covalently coupled to the bead surface through dityrosine bond formation catalyzed by the colocalized GGHY−Ni(II) complex. After extensive washing with EDTA to disrupt metal coordination bonds, we observed that up to 75% of the initially bound GFP−YGH6 remained covalently bound to the bead while retaining its structure and activity. Dityrosine cross-linking was confirmed by quenching the reaction with free tyrosine. The method may find particular utility in the construction and optimization of protein microarrays.