Real-time surface plasmon resonance imaging measurements for the multiplexed determination of protein adsorption/desorption kinetics and surface enzymatic reactions on peptide microarrays

Wegner, G.J. and Wark, A.W. and Lee, H.J. and Codner, E. and Saeki, T. and Fang, S. and Corn, R.M. (2004) Real-time surface plasmon resonance imaging measurements for the multiplexed determination of protein adsorption/desorption kinetics and surface enzymatic reactions on peptide microarrays. Analytical Chemistry, 76 (19). pp. 5677-5684. ISSN 0003-2700

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Abstract

The kinetics of protein adsorption/desorption onto peptide microarrays was studied using real-time surface plasmon resonance (SPR) imaging. S protein binding interactions were examined using an array composed of five different peptides:  N terminal and C terminal immobilized wild-type S peptide (S1 and S2), an alternate binding sequence derived by phage display (LB2), an NVOC-protected S peptide, and a FLAG peptide control sequence (F). Kinetic measurements of the S protein−S1 peptide interaction were analyzed to determine a desorption rate constant (kd) of 1.1 (±0.08) × 10-2 s-1, an adsorption rate constant (ka) of 1.9 (±0.05) × 105 M-1 s-1, and an equilibrium adsorption constant (KAds) of 1.7 (±0.08) × 107 M-1. SPR imaging equilibrium measurements of S protein to S1 peptide were performed to independently confirm the kinetically determined value of KAds. Rate constants for the S2 and LB2 peptides on the array were measured as follows:  1.6 (±0.04) × 105 M-1 s-1 (ka) and 1.1 (±0.07) × 10-2 s-1 (kd) for S2, 1.2 (±0.05) × 105 M-1 s-1 (ka) and 1.1 (±0.03) × 10-2 s-1 (kd) for LB2. In addition to S protein adsorption/desorption, real-time SPR imaging of peptide arrays was applied to study the surface enzymatic activities of the protease factor Xa. Enzymatic cleavage of the substrate peptide (P1) was shown to follow first-order kinetics and proceed at a rate 10 times faster than that of the mutant peptide (P2), with cleavage velocities of 5.6 (±0.3) × 10-4 s-1 for P1 and 5.7 (±0.3) × 10-5 s-1 for P2.