A Strategy for the Generation of Surfaces Presenting Ligands for Studies of Binding Based on an Active Ester as a Common Reactive Intermediate: A Surface Plasmon Resonance Study

Abstract

This paper describes the immobilization of ten proteins and two low-molecular-weight ligands on mixed selfassembled monolayers (SAMs) of alkanethiolates on gold generated from the tri(ethylene glycol)-terminated thiol 1 (HS(CH₂)₁₁(OCH₂CH₂)₃OH) (χ(1) = 1.0−0.0) and the longer, carboxylic acid-terminated thiol 2 (HS(CH₂)₁₁(OCH₂CH₂)₆OCH₂CO₂H) (χ(2) = 0.0−1.0). The immobilization was achieved by a two-step procedure: generation of reactive N-hydroxysuccinimidyl esters from the carboxylic acid groups of 2 in the SAM and coupling of these groups with amines on the protein or ligand. Because this method involves a common reactive intermediate that is easily prepared, it provides a convenient method for attaching ligands to SAMs for studies using surface plasmon resonance spectroscopy (and, in principle, other bioanalytical methods that use derivatized SAMs on gold, silver, and other surfaces). These SAMs were resistant to nonspecific adsorption of proteins having a wide range of molecular weights and isoelectric points. The pH of the coupling buffer, the concentration of protein, the ionic strength of the solution of protein, and the molecular weight of the protein all influenced the amount of the protein that was immobilized. For the proteins investigated in detailcarbonic anhydrase and lysozymethe highest quantities of immobilized proteins were obtained when using a low ionic strength solution at a value of pH approximately one unit below the isoelectric point (pI) of the protein, at a concentration of ∼0.5 mg mL^-1. Comparisons of the kinetic and thermodynamic constants describing binding of carbonic anhydrase and vancomycin to immobilized benzenesulfonamide and N-α-Ac-Lys-d-Ala-d-Ala groups, respectively, on mixed SAMs (by methods described in this paper) and in the carboxymethyl dextran matrix of commercially available substrates yielded (for these systems) essentially indistinguishable values of K_d, k_off, and k_on.