Application of different surface plasmon resonance biosensor chips to monitor the interaction of the CaM-binding site of nitric oxide synthase I and calmodulin.
Surface plasmon resonance biosensors depend on modified gold surfaces to allow immobilization of proteins or peptides for interaction analysis. We investigated sensor chip surfaces that differ in the geometry of the immobilization matrix: two contain a three-dimensional coupling matrix and two have a surface with immobilization sites on a two-dimensional plane. Properties of sensor chips were compared by studying the interaction of calmodulin with a peptide representing the calmodulin-binding site of nitric oxide synthase I. Apparent K(D) values were determined by three different procedures in order to apply tests for self-consistency. At low surface densities (5-8 fmol/mm(2)) on three of the four tested surfaces, estimated K(D) values were within one order of magnitude and similar to the value found in solution (K(D) = 1-3 nM). When immobilization densities were increased by one to two orders of magnitude, apparent association rate constants were less distorted on a flat carboxymethylated surface than on dextran-coated sensor chips.
Fischer
T
T
Institut für Biologische Informationsverarbeitung 1, Research Center Jülich, Jülich D-52425, Germany.
Beyermann
M
M
Koch
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D020349:Surface Plasmon Resonance / Q000379:methods
D002147:Calmodulin; D010455:Peptides; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat