Kinetic analysis of protonation of a specific site on a buffered surface of a macromolecular body

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Abstract
The kinetics of protonation of a specific site on a macromolecular structure (micelle) in buffered solution was studied with the purpose of evaluating the effect of buffer on the observed dynamics. The experimental system consisted of the following elements: Brij 58 micelles serving as homogeneous uncharged macromolecular bodies, bromocresol green, a well-adsorbed proton detector, and 2-naphthol-3,6-disulfonate as a proton emitter in the bulk. Imidazole was the mobile buffer while neutral red, which has a high affinity for the micellar surface, served as the immobile buffer. An intensive laser pulse ejects a proton from the proton emitter, and the subsequent proton-transfer reactions are measured by fast spectrophotometeric methods. The dynamics of proton pulse in buffered solution are characterized by a very rapid trapping of the discharged protons by the abundant buffer molecules. This event has a major effect on the kinetic regime of the reaction. During the first 200 ns the proton flux is rate limited by free-proton diffusion. After this period, when the free-proton concentration decayed to the equilibrium level, the relaxation of the system is carried out by the diffusion of buffer. Thus in the buffered biochemical system, at neutral pH, most of proton flux between active sites and bulk is carried out by buffer molecules, not by diffusion of free protons. Surface groups on a high MW body exchange protons among them at a very fast rate. This reaction has a major role on proton transfer from a specific site to the bulk. The proton can reach the bulk either through dissociation-diffusion or through collisional proton transfer between the mobile buffer and protonable surface groups. The rapid proton exchange between the surface groups increases the efficiency of the latter pathway. It is proposed that a combination of free proton diffusion and buffer-mediated proton diffusion can generate an apparent asymmetry in the bulk to surface vs. surface to bulk proton transfer. Low-pK surface groups will mostly enhance the rate of proton transfer from the bulk to a specific site on the surface, while basic groups on the surface will accelerate the dissipation of the proton to the bulk. This qualitative description is corroborated by accurate quantitative analysis based on experimentally determined rate constants.