Kinetic analysis on nitric oxide binding of recombinant Prolixin-S, a nitric oxide transport protein from the bloodsucking bug, Rhodnius prolixus

Abstract

Kinetics of the NO binding and removal reaction of recombinant Prolixin-S ( rProlixin-S) were analyzed using stopped-flow spectrophotometry. The reaction was observed as a biphasic process. The rate constant of the fast phase increased linearly as NO concentration increased. The rate constant at the slow phase increased as NO concentrations increased at low NO concentration, then reached a plateau at high NO concentration. These NO dependencies of the reaction are characteristic of a bimolecular two-step consecutive reaction. The reaction consisted of the fast NO binding reaction of rProlixin-S and the following slow structural change of NO–protein complex. Kinetic studies revealed that the NO binding rate constant was independent of pH, but the rate constant of the NO removal reaction increased as pH increased. The apparent NO dissociation constant ( K d) of rProlixin-S was also calculated from the values of the kinetic parameters obtained in this work. The K d value increased as pH and temperature increased. The K d value of rProlixin-S and NO was 10–300 nM in regular physiological condition, which is 10 3 higher and 10 3 lower than those of the other ferric and ferrous hemoproteins and NO, respectively. These results indicate that Prolixin-S is one of NO transport proteins regulating blood pressure.