Author/Authors :
Jolie، نويسنده , , Ruben P. and Duvetter، نويسنده , , Thomas and Houben، نويسنده , , Ken and Vandevenne، نويسنده , , Evelien and Van Loey، نويسنده , , Ann M. and Declerck، نويسنده , , Paul J. and Hendrickx، نويسنده , , Marc E. and Gils، نويسنده , , Ann، نويسنده ,
Abstract :
Two surface plasmon resonance (SPR)-based interaction analysis methods were successfully implemented to explore the binding between plant PME and kiwi PMEI. In a first method, plant PMEs were immobilised on a chip surface via amine coupling. This experimental setup allowed studying the effect of pH and ionic strength on the PME–PMEI interaction kinetics. Strong binding was obtained at pH < 7 and at low salt concentrations, whereas both pH ⩾ 8 and [NaCl] of ca. 1.0 M effectively caused dissociation. In a second method, kiwi PMEI was immobilised on a chip surface to which streptavidin had been covalently attached. Hereto, PMEI was biotinylated by means of a NHS-biotin reagent. With this immobilisation strategy, the effect of (partial) thermal or high pressure-induced denaturation of PME on its affinity towards PMEI was investigated. A notable degree of enzyme inactivation was required before interaction characteristics were significantly altered. Any incomplete inactivation of PME resulted in binding to the PMEI surface.
Keywords :
Pectin methylesterase inhibitor (PMEI) , surface plasmon resonance (SPR) , Interaction analysis , Thermal and high pressure processing , Pectin methylesterase (PME)
