ABSTRACT
Dithioerythritol (DTE) is frequently employed as a reducing agent or a protective reagent for thiol groups in biological assays. Owing to its known inhibiting properties in enzyme catalysis reactions, lead is also commonly used in such experiments, and often simultaneously with DTE. Given the potential affinity of these two reactants, their measured individual effects may well depend on their interactions in the medium used. Any quantitative assessment of these interactions necessitates, however, that the complex equilibria between lead and DTE be investigated beforehand. To test this hypothesis, the formation constants of lead(II) complexes with DTE under physiological conditions (37 degrees, NaCl 0.15M) have been calculated from the results of glass electrode potentiometry, with the help of the MINIQUAD and ESTA computer programs. The pK values for dissociation of DTE have been found equal to 8.926 +/- 0.003 and 9.840 +/- 0.003. The following lead-DTE species have been characterized: ML (12.774 +/- 0.037), MLH(-1) (2.858 +/- 0.037), M(2)LH(-1) (13.349 +/- 0.025) and M(6)L(5) (86.586 +/- 0.099); the log *beta-values are given in the parentheses. Appropriate computer simulations effectively show that the interactions of the two reactants are indeed quite significant within the concentration ranges commonly used in in vitro biological assays. They should thus be taken into account in interpretation of the effects observed.
ABSTRACT
Dithiothreitol (threo-2,3-dihydroxy-1,4-dithiobutane = DTT) has recently been used to activate 5-aminolevulinic acid dehydratase in kinetic studies for the inhibition of this zinc enzyme by lead. Since the DTT molecule contains donor groups capable of forming metal ion complexes, its presence in the experimental medium used for this kind of assay may largely influence the concentration of lead available for the active sites of the enzyme. Before any quantitative investigation of this phenomenon can be contemplated, all possible complexes formed by lead with DTT must first be identified and their stabilities determined. Accordingly, formation equilibria of DTT complexes with lead(II) have been investigated under physiological conditions (37 degrees C, NaCl, 0.15 mol. dm-3 using glass electrode potentiometry. Corresponding stability constants were refined with MINIQUAD and ESTA computer programs. DTT log protonation constants have been found equal to 9.811 +/- 0.002 and 18.672 +/- 0.002. The following lead-dithiothreitol complexes have been characterized: ML (12.243 +/- 0.063), MLH-1 (2.391 +/- 0.061), M2LH-1 (13.285 +/- 0.059), and M4L3 (51.668 +/- 0.157). Appropriate computer simulations show that the interactions of the two reactants are indeed most significant under the pH and concentration conditions used in the above mentioned biological investigations. In particular, the influence of lead(II)-DTT equilibria on the free concentration of lead available for the active sites of the enzyme is described.
