Interaction of cryptogein with its binding sites in tobacco plasma membrane studied using the piezoelectric biosensor

Elicitins are low-molecular-weight proteins representing the elicitor family secreted by many species of the oomycete Phytophthora. Elicitins induce a hypersensitive reaction in tobacco, a process that is triggered by binding of elicitin to the high-affinity site on the plasma membrane. Specific interaction of cryptogein with the binding sites on tobacco plasma membranes was studied using the piezoelectric biosensor in real time in a flow-through mode. Cryptogeins (wild-type and mutant forms) were covalently immobilized on the sensing surface, and membrane vesicles containing receptors were in solution. Kinetic characterization of the interaction provided values of kinetic rate association (k_a) = 5.74 · 10⁶ M¹ s⁻¹ and kinetic rate dissociation (k_d) = 6.87 10⁻⁴ s⁻¹ constants, respectively. The kinetic equilibrium dissociation constant was calculated as K_D = 12.0 nM. The piezoelectric biosensor appeared to be a convenient tool for studying interactions of receptors embedded in membrane vesicles.