Speciation 98: Abstracts

Histidine and histamine are absolutely fundamental compounds of human biology and medicine. Their membrane-penetration, receptor-binding, metal-complexation and all other chemical and biochemical reactions depend on the conformation and protonation stages of these multiconform molecules.

For histidine and histamine, the number of protonation states is 8 and 4, respectively. The related proton-binding microconstants were determined by a previously undescribed C-2 proton-deuterium exchange rate method, which showed a D log k ~ 0.9 interactivity parameter for the imidazole and amino groups.

As every microspecies can be decomposed into three rotamers, the histidine and histamine solutions contain 24 and 12 non-identical rota-microspecies, respectively.

Of these, concentration of the major ones could be determined, by using three-bond ¹H-¹H and ¹H-¹³C NMR coupling constants. NMR-pH titrations were performed in 90% H₂O-10% D₂O solutions, using t-butanol reference.

As histamine protonates in largely separated stages and possess an ABXY spin system for protons, rotamer populations, for its major protonation forms could be determined in a redundant set of equations. Histidine proton-binding takes place in the of amino, imidazole, carboxylate order in a sufficiently separated fashion, rotamer populations of the major microspecies could be calculated directly from the coupling constants. In order to mimic the minor microspecies, histidine-amid and imidazole-lactic acid were used, whose NMR coupling constants provided fairly good estimates for the rotamer concentrations of the minor microspecies.

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