, Volume 22, Issue 5, pp 771–778 | Cite as

Determination of the pKa value of the hydroxyl group in the α-hydroxycarboxylates citrate, malate and lactate by 13C NMR: implications for metal coordination in biological systems

  • Andre M. N. Silva
  • Xiaole Kong
  • Robert C. HiderEmail author


Citric acid is an important metal chelator of biological relevance. Citric acid helps solubilizing metals, increasing their bioavailability for plants and microbes and it is also thought to be a constituent of both the extracellular and cytoplasmic low molecular iron pools occurring in plants and vertebrates. Metal coordination by citric acid involves coordination both by the carboxylate and hydroxyl groups, of particular interest is its α-hydroxycarboxylate function. This structural feature is highly conserved in siderophores produced by evolutionarily distant species and seems to confer specificity toward Fe(III) binding. In order to understand the mechanism of metal coordination by α-hydroxycarboxylates and correctly evaluate the respective complex stability constants, it is essential to improve the knowledge about the ionisation of the alcohol group in these compounds. We have evaluated the hydroxyl pKa value of citric, malic and lactic acids with the objective of understanding the influence of α-carbon substitution. Studies at high pH values, utilizing 13C NMR, permitted estimation of the pKa values for the three acids. The pKa (alcohol) values (14.4 for citric acid, 14.5 for malic acid, and 15.1 for lactic acid) are considerably higher than the previously reported value for citric acid (11.6) but still lower than the value of 15.5 for methanol. A comparative analysis of the three compounds indicates that different substitutions on the α-carbon introduce changes to the inductive effect experienced by the hydroxyl group thereby modulating its ionisation behaviour. Comparison with the siderophore rhizoferrin, which pKa (alcohol) values were confirmed to be 10 and 11.3, suggests that intra-molecular hydrogen bonding may also aid in the hydroxyl ionisation by stabilizing the resulting anion. Studies of metal coordination by α-hydroxycarboxylates should take these factors into account.


Citrate Malate Lactate pKa Siderophores α-Hydroxycarboxylate 



Silva A. M. N. would like to acknowledge “Fundacao para a Ciencia e Tecnologia”, Lisboa Portugal for his PhD grant [SFRH/BD/22633/2005]. The authors would like to thank Jane Hawkes for assistance with NMR spectroscopy.


  1. Bakkeren DL, De Jeu-Jaspers CMH, Van Der Heul C, Van Eijk HG (1985) Analysis of iron-binding components in the low molecular weight fraction of rat reticulocyte cytosol. Int J Biochem 17:925–930. doi: 10.1016/0020-711X(85)90177-6 PubMedCrossRefGoogle Scholar
  2. Barton PA, Pai MP, Depczynski J, McQuade CR, Mercier RC (2006) Non-transferrin-bound iron is associated with enhanced Staphylococcus aureus growth in hemodialysis patients receiving intravenous iron sucrose. Am J Nephrol 26:304–309. doi: 10.1159/000094343 CrossRefGoogle Scholar
  3. Bino A, Shweky I, Cohen S, Bauminger ER, Lippard SJ (1998) A novel nonairon(III) citrate complex: a “ferric triple-decker”. Inorg Chem 37:5168–5172. doi: 10.1021/ic9715658 CrossRefGoogle Scholar
  4. Carrano CJ, Drechsel H, Kaiser D, Jung G, Matzanke B, Winklemann G, Rochel N, Albrecht-Gary AM (1996) Coordination chemistry of the carboxylate type siderophore rhizoferrin: the iron(III) complex and its metal analogs. Inorg Chem 35:6429–6436. doi: 10.1021/ic960526d PubMedCrossRefGoogle Scholar
  5. Cistola DP, Small DM, Hamilton JA (1982) Ionization behavior of aqueous short-chain carboxylic acid: a carbon-13 NMR study. J Lipid Res 23:795–799PubMedGoogle Scholar
  6. Drechsel H, Winklemann G (2000) Iron chelation and siderophores. In: Winklemann G, Carrano C (eds) Transition metals in microbial metabolism. Harwood Academic Publishers, AmsterdamGoogle Scholar
  7. Field TB, McCourt JL, McBryde WAE (1974) Composition and stability of iron and copper citrate complexes in aqueous solution. Can J Chem 52:3119–3124. doi: 10.1139/v74-458 CrossRefGoogle Scholar
  8. Gampp H, Maeder M, Meyer CJ, Zuberbühler AD (1985) Calculation of equilibrium constants from multiwavelength spectroscopic data—I, mathematical considerations. Talanta 32:95–101. doi: 10.1016/0039-9140(85)80035-7 PubMedCrossRefGoogle Scholar
  9. Gans P, Sabatini A, Vacca A (1996) Investigation of equilibria in solution. Determination of equilibrium constants with the HYPERQUAD suite of programs. Talanta 43:1739–1753. doi: 10.1016/0039-9140(96)01958-3 PubMedCrossRefGoogle Scholar
  10. Gautier-Luneau I, Merle C, Phanon D, Lebrun C, Biaso F, Serratrice G, Pierre JL (2005) New trends in the chemistry of iron(III) citrate complexes: correlations between X-ray structures and solution species probed by electrospray mass spectrometry and kinetics of iron uptake from citrate by iron chelators. Chem Eur J 11:2207–2219. doi: 10.1002/chem.200401087 CrossRefGoogle Scholar
  11. Grootveld M, Bell JD, Halliwell B, Aruoma OI, Bomford A, Sadler PJ (1989) Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy. J Biol Chem 15:4417–4422Google Scholar
  12. Guerinot ML, Meidl E, Plessner L (1990) Citrate as a siderophore in Bradyrhizobium japonicum. J Bacteriol 172:3298–3303PubMedGoogle Scholar
  13. Harle C, Kim IS, Angerer AM, Braun W (1995) Signal transfer through 3 compartments–transcription initiation of the Escherichia coli ferric citrate transport-system from the cell surface. EMBO J 14:1430–1438PubMedGoogle Scholar
  14. Kourgiantakis M, Matzapetakis M, Raptopoulou CP, Terzis A, Salifoglou A (2000) Lead-citrate chemistry. Synthesis, spectroscopic and structural studies of a novel lead(II)-citrate aqueous complex. Inorg Chim Acta 297:134–138. doi: 10.1016/S0020-1693(99)00339-4 CrossRefGoogle Scholar
  15. Martell AE, Smith RM (1977) Critical stability constants V.3. Plenum Press, New YorkGoogle Scholar
  16. Martinez JS, Butler A (2007) Marine amphiphilic siderophores: marinobactin structure, uptake, and microbial partitioning. J Inorg Biochem 101:1692–1698. doi: 10.1016/j.jinorgbio.2007.07.007 PubMedCrossRefGoogle Scholar
  17. Matzapetakis M, Raptopoulou CP, Tsohos A, Papaefthymiou V, Moon N, Salifoglou A (1998) Synthesis, spectroscopic and structural characterization of the first mononuclear, water soluble iron–citrate complex, (NH4)5Fe(C6H4O7)2·2H2O. J Am Chem Soc 120:13266–13267. doi: 10.1021/ja9807035 CrossRefGoogle Scholar
  18. Matzapetakis M, Raptopoulou CP, Terzis A, Lakatos A, Kiss T, Salifoglou A (1999) Synthesis, structural characterization, and solution behavior of the first mononuclear, aqueous aluminum citrate complex. Inorg Chem 38:618–619. doi: 10.1021/ic9806131 CrossRefGoogle Scholar
  19. Matzapetakis M, Dakanali M, Raptopoulou CP, Tangoulis V, Terzis A, Moon N, Giapintzakis J, Salifoglou A (2000a) Synthesis, spectroscopic, and structural characterization of the first aqueous cobalt(II)-citrate complex: toward a potentially bioavailable form of cobalt in biologically relevant fluids. J Biol Inorg Chem 5:469–474. doi: 10.1007/s007750050007 PubMedCrossRefGoogle Scholar
  20. Matzapetakis M, Karligiano N, Bino A, Dakanali M, Raptopoulou CP, Tangoulis V, Terzis A, Giapintzakis J, Salifoglou A (2000b) Manganese citrate chemistry: syntheses spectroscopic studies, and structural characterizations of novel mononuclear, water-soluble manganese citrate complexes. Inorg Chem 39:4044–4051. doi: 10.1021/ic9912631 PubMedCrossRefGoogle Scholar
  21. Matzapetakis M, Kourgiantakis M, Dakanali M, Raptopoulou CP, Terzis A, Lakatos A, Kiss T, Banyai I, Iordanidis L, Mavromoustakos T, Salifoglou A (2001) Synthesis, pH-dependent structural characterization, and solution behavior of aqueous aluminum and gallium citrate complexes. Inorg Chem 40:1734–1744. doi: 10.1021/ic000461l PubMedCrossRefGoogle Scholar
  22. Migal PK, Sychen AY (1958) Ustoichivost limonnokislykh kompleksov nekotorykh metallov. Zh Neorg Khim 3:314–324Google Scholar
  23. Pitch A, Scholz G, Seifert K (1991) Effect of nicotianamine on iron uptake and citrate accumulation in two genotypes of tomato, Lycopersicon esculentum Mill. J Plant Physiol 137:323–326Google Scholar
  24. Quirt AR, Lyerla JR, Peat IR, Cohen JS, Reynolds WF, Freedman MH (1974) Carbon-13 nuclear magnetic resonance titration shifts in amino acids. J Am Chem Soc 96:570–574. doi: 10.1021/ja00809a038 PubMedCrossRefGoogle Scholar
  25. Shweky I, Bino A, Goldberg DP, Lippard SJ (1994) Synthesis, structures and magnectic-properties of 2 dinuclear iron(III) citrate complexes. Inorg Chem 30:5161–5162. doi: 10.1021/ic00101a001 CrossRefGoogle Scholar
  26. Strouse J, Layten SW, Strouse CE (1977) Structural studies of transition-metal complexes of triionized and tetraionized citrate—models for coordination of citrate ion to transition-metal ions in solution and at active-site of aconitase. J Am Chem Soc 99:562–572. doi: 10.1021/ja00444a041 PubMedCrossRefGoogle Scholar
  27. Sykes P (1986) A guide book to mechanism in organic chemistry, 6th edn. Pearson Education Ltd, EssexGoogle Scholar
  28. Villafranca JJ, Mildvan AS (1972) The mechanism of aconitase action. J Biol Chem 247:3454–3463PubMedGoogle Scholar
  29. Volhardt KPC, Schore NE (2003) Organic chemistry, structure and function, 4th edn. WH Freeman and Company, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • Andre M. N. Silva
    • 1
  • Xiaole Kong
    • 1
  • Robert C. Hider
    • 1
    Email author
  1. 1.Pharmaceutical Sciences Research DivisionKing’s College LondonLondonUK

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