Stopped-flow studies of guanine binding by calf spleen purine nucleoside phosphorylase

The binding of guanine to calf spleen purine nucleoside phosphorylase at 20 degrees C, in 20 mM Hepes-NaOH buffer, pH 7.0, at several ionic strength between 5 and 150 mM was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a guanine molecule by each of the binding sites is a two-step process and that symmetrical trimeric calf spleen purine nucleoside phosphorylase represents a system of (identical) interacting binding sites. The interaction is visible through relations between the rate constants and non-additivity of changes in "molar" fluorescence for different forms of PNP-guanine complexes. It is also probable that electrostatic effects in guanine binding are weak, which indicates that it is the neutral form of the ligand which is bound and dissociated by PNP molecule. (c) 2005 Elsevier B.V. All rights reserved.