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

• DOI: 10.1021/ac980959t
• Publication Date: 2002-07-26T05:30:26Z
• Authors: Joydeep Lahiri; Lyle Isaacs; Joe Tien; George M. Whitesides
• Keywords: Ethylene Glycol; Sulfonamides; Surface Properties; Proteins; Dextrans; 02 engineering and technology; Buffers; Hydrogen-Ion Concentration; Surface Plasmon Resonance; Ligands; Chemistry Techniques, Analytical; Molecular Weight; Kinetics; Benzenesulfonamides; Vancomycin; Alkanes; Adsorption; Sulfhydryl Compounds; 0210 nano-technology; Carbonic Anhydrases; Protein Binding

This paper describes the immobilization of ten proteins and two low-molecular-weight ligands on mixed self-assembled monolayers (SAMs) alkanethiolates gold generated from tri(ethylene glycol)-terminated thiol 1 (HS(CH2)11(OCH2CH2)3OH) (chi(1) = 1.0-0.0) longer, carboxylic acid-terminated thiol2(HS(CH2)11(OCH2-CH2)6OCH2CO2H) (chi(2) 0.0-1.0). The was achieved by a two-step procedure: generation reactive N-hydroxysuccinimidyl esters acid groups 2 in SAM coupling these with amines protein or ligand. Because this method involves common intermediate that is easily prepared, it provides convenient for attaching to SAMs studies using surface plasmon resonance spectroscopy (and, principle, other bioanalytical methods use derivatized gold, silver, surfaces). These were resistant nonspecific adsorption having wide range molecular weights isoelectric points. pH buffer, concentration protein, ionic strength solution weight all influenced amount immobilized. For investigated detail--carbonic anhydrase lysozyme--the highest quantities immobilized obtained when low at value approximately one unit below point (pI) 0.5 mg mL-1. Comparisons kinetic thermodynamic constants describing binding carbonic vancomycin benzenesulfonamide N-alpha-Ac-Lys-D-Ala-D-Ala groups, respectively, (by described paper) carboxymethyl dextran matrix commercially available substrates yielded (for systems) essentially indistinguishable values Kd, koff, kon.