Microscopic Protonation Equilibria of Oxidized Glutathione

Abstract

The first complete microequilibrium analysis of a tetrabasic acid is presented. A total of 18 protonation microconstants of glutathione disulfide (GSSG) were determined, 16 of them quantitate the overlapping equilibria in acidic medium, where 4 carboxylate sites protonate. The related, pH-dependent concentrations of 16 coexisting microspecies were also obtained, including the 10 different ones and their 6 identical twins of symmetry origin. The GSSG acid-base chemistry in basic medium is also characterized. It is shown for tetra-and further multibasic acids that the number of traditional microconstants mandatorily exceeds that of the pieces of independent information available by any state-of-the-art methodology, making the complete microequilibrium resolution not only highly complex but theoretically impossible on the basis of hitherto reported principles. The GSSG microspeciation was made feasible here by introducing cumulative microconstants, a new equilibrium parameter, and the invariance of the interactivity coefficient, a plausible simplifying principle, on the experimental basis of 1 H NMR-pH titrations in H 2 O/D 2 O 9/1 media. The methods and results were verified by corresponding data of GSSG-(glycyl)-diethyl-ester, a model compound of reduced microequilibrium complexity. The obtained log k U ) 2.44 and log k Y ) 3.59 "core" microconstants of the respective glutamyl and glycyl carboxylates of GSSG are close to those of the sulfhydryl (GSH) form of glutathione. The GSSG intermoiety interactions were found to be weak, apparently of Coulombic nature. The pH-dependent distribution of the 18 microspecies is depicted.

Determination of microscopic acid?base parameters from NMR?pH titrations

“…Virtually the same interactivity parameter has been found for CysGly, Cys methyl ester, and reduced oxytocin, which contain both groups in the same distance and intramolecular environment [102]. Indeed, the interactivity parameter of a pair of functional groups proved to be less perturbed by actual molecular environment and thus it is a more transferable parameter between molecules than microconstants [18,112].…”

“…Microconstants are highly detailed parameters to quantitate equilibria at the submolecular level, which are surpassed only by their rotamer-specific counterparts [13,14], which are discussed in a separate paper in this issue of Analytical and Bioanalytical Chemistry. Other fundamental details on microscopic protonation equilibria are discussed in several publications [9,10,11,12,13,15,16,17,18,19,20].…”

“…8) [17]. The theory and practice used to analyze a genuine tetraprotic system has recently been published [18]. However, the complexity of the full analysis of rAVP can be reduced significantly by "decoupling" the independently protonating phenolate (O) and Cys 6 thiolate (S′) sites from protonation equilibria of the strongly interacting Cys 1 amino (N) and thiolate (S) groups in the evaluation procedure.…”

“…In order to alleviate difficulties in the formalism of description, we introduced cumulative microconstants [17, 18], designated by κ, the number of which is 2 n -1. This Hessian-type microconstant is assigned to every microspecies containing at least one proton, and it unifies the various, alternative microscopic routes of microspecies formation in one single parameter [17,18]. Taking the example in Eq.…”

Physico-chemical Profiling of ACE-inhibitor Lisinopril: Acid-base Properties

“…The mutual basicity-modifying effect of these moieties is quantified by the interactivity parameter, defined as the difference in the corresponding microconstants: pE CD = 2.15 -1.68 = 3.10 -2.63 = 0.47. This pE value suggests an interaction exceeding the random, Coulombic value of 0.2-0.3 [27]; presumably, these sites communicate through space with a simultaneous change in their geometric positions. A conformational change at pH < 4.5 is also substantiated by the changing multiplet patterns in the NMR spectra, but a detailed analysis of vicinal 3 J HH values holding conformational information is beyond the scope of this article.…”