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Abstract

2-(p-Toluidino)naphthalene-6-sulfonate (TNS) binds in a reversible bimolecular reaction non-covalently to RUBISCO, the water-soluble enzyme for carbon dioxide fixation. TNS does not change the substrate activity at the active site of RUBISCO. Rate constants k₊ and k₋ for the association and dissociation were measured as (1.2 ± 0.2)×10⁷ dm³ mol⁻¹ s⁻¹ and 1020 ± 300 s⁻¹, respectively. The binding of the steryl dye N-(4-sulfobutyl)-4-{4-[p(dipentylamino)phenyl]butadienyl}pyridinium inner salt (RH421) to RUBISCO is a diffusion-controlled reversible bimolecular reaction with an association rate constant k₊ of (7±0.6)×10⁹ dm³mol⁻¹ s⁻¹ and a dissociation rate constant k₋ of (1.8 ± 0.2)×10⁴ s⁻¹. The dissociation constants K_d for the binding of TNS and RH421 to RUBISCO were calculated from the kinetically determined rate constants. These data are in good agreement with the dissociation constants measured by equilibrium techniques. Upon binding to RUBISCO the fluorescence of TNS and RH421 is more intense and blue shifted. The fluorescence lifetimes of TNS and RH421 are longer compared to those of both dyes dissolved in aqueous solution. The fluorescence anisotropy correlation time of 200 ns for TNS bound to RUBISCO corresponds to a rigidly bound dye which rotates with the same rotational correlation time as the whole protein. Both dyes can be used as probes sensitive to their molecular environment. In further experiments they were applied to the detection of ligand binding events at the active site of RUBISCO as will be described in a forthcoming publication.