A method of reversible biomolecular immobilization for the surface plasmon resonance quantitative analysis of interacting biological macromolecules.
This article presents a new procedure for the immobilization of macromolecules on gold surfaces, with the purpose of studying macromolecular interactions by simple optical configurations rendering surface plasmon resonance. Gold surfaces were covered by a three-layer structure composed of poly-L-lysine irreversibly bound to gold, followed by a second layer of heparin and a third layer of polylysine. The three-layer structure of polylysine-heparin-polylysine remains irreversibly bound to gold, it prevents biomolecules from coming into direct contact with the metal surface, and it allows the irreversible binding of different proteins and polynucleotides. After binding of a macromolecule to the three-layer structure, the interaction with a second macromolecule can be studied, and then the complex formed by the two interacting macromolecules, together with the second heparin layer and the third polylysine layer, can be broken down just by treatment with an alkaline solution having a pH value above the pK value of the amino groups of polylysine. The first polylysine layer remains irreversibly bound to gold, ready to form a new three-layer structure and, therefore, to support a new macromolecular interaction on the same regenerated surface. Polynucleotide interactions, the proteolytic action of chymotrypsin, and the interaction between the component subunits of a heterotetrameric enzyme are described as examples of macromolecular interactions studied by using this system. The method may be especially suitable for developing of low-cost systems aimed to look for surface resonance signals, and it offers the advantage of allowing calculation of parameters related to the size and stoichiometry of the interacting macromolecules, in addition to the kinetic and equilibrium properties of the interaction.
Benítez
María J
MJ
Departamento de Química Física Aplicada, Universidad Autónoma de Madrid, Centro de Biología Molecular Severo Ochoa, Cantoblanco, Madrid 28049, Spain.
Jiménez
Juan S
JS
D047390:Casein Kinase II; D002364:Caseins / Q000032:analysis*; D004800:Enzymes, Immobilized / Q000032:analysis; D006046:Gold / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D011107:Polylysine / Q000737:chemistry; D011119:Polynucleotides / Q000032:analysis*; D017346:Protein Serine-Threonine Kinases / Q000032:analysis*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D020349:Surface Plasmon Resonance / Q000379:methods*; D013499:Surface Properties
D002364:Caseins; D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D011119:Polynucleotides; D011107:Polylysine; D006046:Gold; D006493:Heparin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases