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

This paper describes the immobilization of ten proteins and two low-molecul ar-weight ligands on mixed self-assembled monolayers (SAMs) of alkanethiola tes on gold generated from the hi(ethylene glycol)-terminated thiol 1 (HS(C H2)(11)(OCH2CH2)(3)OH) (chi(1) = 1.0-0.0) and the longer, carboxylic acid-t erminated thiol 2 (HS(CH2)(11)(OCH2CH2)(6)OCH2CO2H) (chi(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 S AM and coupling of these groups with amines on the protein or Ligand. Becau se this method involves a common reactive intermediate that is easily prepa red, it provides a convenient method for attaching ligands to SAMs for stud ies 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 non-specific 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 t he solution of protein, and the molecular weight of the protein all influen ced the amount of the protein that was immobilized, For the proteins invest igated in detail-carbonic anhydrase and lysozyme-the 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 (pl) o f the protein, at a concentration of similar to 0.5 mg mL(-1). Comparisons of the kinetic and thermodynamic constants describing binding of carbonic a nhydrase and vancomycin to immobilized benzenesulfonamide and N-alpha-Ac-Ly s-D-Ala-D-Ala groups, respectively, on mixed SAMs (by methods described in this paper) and in the carboxymethyl dextran matrix of commercially availab le substrates yielded (for these systems) essentially indistinguishable val ues of K-d, k(off), and k(on).