Biochromatographic framework for analyzing magnesium chloride salt dependence on nor-NOHA binding to arginase enzyme

Our group demonstrated recently that arginase I inhibition reduces endothelial dysfunction and blood pressure rising in spontaneously hypertensive rats [C. Demougeot, A. Prigent-Tessier, C. Marie, A. Berthelot, J. Hypertens. 23 (2005) 971; C. Demougeot, A. Prigent-Tessier, T. Bagnost, C. Andre, Y. Guillaume, M. Bouhaddi, C. Marie, A. Berthelot, Life Sci. 80 (2007) 1128]. This discovery opens interesting perspectives in the development of new drugs against hypertension. As well, in a previous paper [T. Bagnost, Y.C. Guillaume, M. Thomassin, J.F. Robert, A. Berthelot, A. Xicluna, C. Andre, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 856 (2007) 113], a novel biochromatographic column was developed in our laboratory for studying the binding of Nω-hydroxy-nor-l-arginine (nor-NOHA), an arginase inhibitor, with this enzyme. In this manuscript, using this novel biochromatographic concept, the effect of magnesium chloride on the nor-NOHA/arginase binding was analyzed for the first time. This study demonstrated that the salt ions interacted with arginase and played a great role in the nor-NOHA/arginase association. For a salt concentration (x) in the medium less than 3 mM, the nor-NOHA/arginase binding decreased with x due to a decrease of the charge–charge interactions between nor-NOHA and its arginase binding site. Above 3 mM of salt in the medium, the affinity of nor-NOHA to arginase increased slightly with x because the net number of ions (n) (Mg2+ or Cl−) released or bound upon complex formation is low. As well, it was clearly demonstrated, that above 3 mM the n value depend on the salt concentration in the bulk solvent and was approximately nil for x = 12 mM. This dependence was due to a gradual and conformational change of the arginase enzyme which around 12 mM adopted a less flexible structure; its binding site was thus less accessible to nor-NOHA and nor-NOHA–arginase association decreased slightly.