Kinetics of calcium binding and release from S. mutans

Objective: Streptococci of dental plaque have considerable calcium (Ca) binding capacity, and could be a source of Ca to plaque fluid. However the kinetics of binding and release was not explored, which was evaluated in this study. Methods: Pellets obtained from cultures of S. mutans IB1600 were exposed to PIPES buffer, pH 7.0 containing 0 (control), 1 or 10 mM Ca, for 5, 10, 30 or 60 min, at 37 ºC. Subsequently, bacterial pellets initially equilibrated with 1 mM or 10 mM Ca solutions, were treated with 10 mM or 1 mM Ca, respectively, or with Ca-free buffer at 37 ºC, for 10, 30 or 60 min. Bacteria were separated from the treatment solution by centrifugation and bound Ca was determined in the pellet after acid extraction, using Arsenazo III. Results: The amount of Ca bound (µmol Ca/g) was higher for the higher Ca concentration solution and did not change as a function of time (0 mM Ca, 60 min=2.5±0.1; 1 mM Ca, 5 min=8.0±0.4, 60 min=8.4±1.1; 10 mM Ca, 5 min=23.4±1.9, 60 min=25.2±0.6 µmol Ca/g). In the pellets treated with 10 mM Ca following 1 mM-treatment, a fast binding was also observed. On the other hand, Ca release to lower concentration solutions was only complete after 60 min in the bacteria pre-equilibrated with 1 mM Ca treated with Ca-free PIPES. For the bacteria pre-equilibrated with 10 mM Ca, although at least 70% of release occurred within the first 10 min, it was not complete after 60 min (reaching 80% and 63% of the expected after equilibration with 0 or 1 mM Ca buffers, respectively). Conclusions: Although Ca binding to bacteria is rapid and a function of the surrounding Ca concentrations, the kinetics of release seems dependent on the concentration of Ca in the bacteria and the surrounding media.