Polyamines pp 493-503 | Cite as

Measurement of Polyamine pKa Values

  • Ian S. Blagbrough
  • Abdelkader A. Metwally
  • Andrew J. Geall
Part of the Methods in Molecular Biology book series (MIMB, volume 720)


The extent of ionization of the polyamines is an important factor in their interactions with cellular components. The pKa is the pH at which a functional group is 50% ionized. For compounds such as polyamines with more than one ionizable center (atom or functional group), there is a pKa value for each center of ionization. This chapter describes the pKa values for each amine group in many important polyamines, the factors influencing these values and methods for their determination using potentiometric titration and nuclear magnetic resonance spectroscopy.

Key words

Ionization Potentiometry Nuclear magnetic resonance spectroscopy DNA condensation Henderson–Hasselbalch equation 



We thank the BBSRC (A.J.G.) and the Egyptian Government (A.A.M.) for PhD studentships and Celltech Therapeutics, Slough, UK for a CASE award (to A.J.G.).


  1. 1.
    Henderson LJ (1908) Concerning the relationship between the strength of acids and their capacity to preserve neutrality. Am J Physiol 21:173–179Google Scholar
  2. 2.
    Henderson LJ (1908) The theory of neutrality regulation in the animal organism. Am J Physiol 21:427–448Google Scholar
  3. 3.
    Hasselbalch KA (1916) Die Berechnung der Wasserstoffzahl des Blutes aus der freien und gebundenen Kohlensäure desselben, und die Sauerstoffbindung des Blutes als Funktion der Wasserstoffzahl. Biochem Z 78:112–144Google Scholar
  4. 4.
    Po N, Senozan M (2001) Henderson–Hasselbalch equation: its history and limitations. J Chem Educ 78:1499–1503CrossRefGoogle Scholar
  5. 5.
    Frassineti C, Ghelli S, Gans P, Sabatini A, Moruzzi MS, Vacca A (1995) Nuclear ­magnetic resonance as a tool for determining protonation constants of natural polyprotic bases in solution. Anal Biochem 231:374–382PubMedCrossRefGoogle Scholar
  6. 6.
    Meloun M, Bordovská S (2007) Benchmarking and validating algorithms that estimate pKa values of drugs based on their molecular structures. Anal Bioanal Chem 389:1267–1281PubMedCrossRefGoogle Scholar
  7. 7.
    Basu HS, Schwietert HCA, Feuerstein BG, Marton LJ (1990) Effects of variation in the structure of spermine on the association with DNA and the induction of DNA conformational changes. Biochem J 269:329–334PubMedGoogle Scholar
  8. 8.
    Usherwood PNR, Blagbrough IS (1989) Amino acid synapses and receptors. In: McFarlane NR (ed) Progress and prospects in insect control, Monograph No 43. Farnham, London, pp 45–58Google Scholar
  9. 9.
    Usherwood PNR, Blagbrough IS (1989) Antagonism of insect muscle glutamate receptors - with particular reference to arthropod toxins. In: Narahashi T, Chambers JE (eds) Insecticide action from molecule to organism, ACS Symposium Series. Plenum, New York, pp 13–31Google Scholar
  10. 10.
    Usherwood PNR, Sudan H, Standley C, Blagbrough IS, Bycroft BW, Mather AJ (1990) The mechanisms of neurotoxicity of low molecular weight spider toxins. In: Volans GN, Sims J, Sullivan FM, Turner P (eds) Basic science in toxicology. Taylor & Francis, London, pp 569–579Google Scholar
  11. 11.
    Usherwood PNR, Blagbrough IS (1991) Spider toxins affecting glutamate receptors: polyamines in therapeutic neurochemistry. Pharmacol Ther 52:245–268PubMedCrossRefGoogle Scholar
  12. 12.
    Usherwood PNR, Blagbrough IS, Brackley PTH, Kerry CJ, Sudan HL, Nakanishi K (1992) Polyamines and polyamine-containing toxins - modulators and antagonists of excitatory amino acid receptors. In: Kawai N, Nakajima T, Barnard E (eds) Neuroreceptors, ion channels and the brain. Elsevier, Amsterdam, pp 11–20Google Scholar
  13. 13.
    Usherwood PNR, Blagbrough IS (1994) Electrophysiology of polyamines and polyamine amides. In: Carter C (ed) The neuropharmacology of polyamines. Academic, London, pp 185–204Google Scholar
  14. 14.
    Bergeron RJ, McManis JS, Weimar WR, Schreier KM, Gao FL, Wu QH, Ortiz-Ocasio J, Luchetta GR, Porter C, Vinson JRT (1995) The role of charge in polyamine analog recognition. J Med Chem 38:2278–2285PubMedCrossRefGoogle Scholar
  15. 15.
    Leroy D, Heriche JK, Filhol O, Chambaz EM, Cochet C (1997) Binding of polyamines to an autonomous domain of the regulatory subunit of protein kinase CK2 induces a conformational change in the holoenzyme – a proposed role for the kinase stimulation. J Biol Chem 272:20820–20827PubMedCrossRefGoogle Scholar
  16. 16.
    Ouameur A, Mangier E, Diamantoglou S, Rouillon R, Carpentier R, Tajmir-Riahi H (2004) Effects of organic and inorganic polyamine cations on the structure of human serum albumin. Biopolymers 73:503–509PubMedCrossRefGoogle Scholar
  17. 17.
    Ahmed OAA, Pourzand C, Blagbrough IS (2006) Varying the unsaturation in N4, N9-dioctadecanoyl spermines: nonviral lipopolyamine vectors for more efficient plasmid DNA formulation. Pharm Res 23:31–40PubMedCrossRefGoogle Scholar
  18. 18.
    Rege K, Ladiwala A, Hu SH, Breneman CM, Dordick JS, Cramer SM (2005) Investigation of DNA-binding properties of an aminoglycoside-polyamine library using quantitative structure-activity relationship (QSAR) models. J Chem Inf Model 45:1854–1863PubMedCrossRefGoogle Scholar
  19. 19.
    Perrin DD (1965) Dissociation constants of organic bases in aqueous solution Butterworths, London, p 137Google Scholar
  20. 20.
    Albert A, Serjeant EP (1971) The determination of ionization constants. Chapman & Hall, London, pp 91–96Google Scholar
  21. 21.
    Baillon JG, Mamont PS, Wagner J, Gerhart F, Lux P (1988) Fluorinated analogs of spermidine as substrates of spermine synthase. Eur J Biochem 176:237–242PubMedCrossRefGoogle Scholar
  22. 22.
    Kimberly MM, Goldstein JH (1981) Determination of pKa values and total proton distribution pattern of spermidine by Carbon-13 nuclear magnetic resonance titrations. Anal Chem 53:789–793CrossRefGoogle Scholar
  23. 23.
    Palmer BN, Powell HKJ (1974) Polyamine complexes with seven-membered chelate rings: Complex formation of 3-azaheptane-1,7-diamine, 4-azaoctane-1,8-diamine (spermidine), and 4,9-diazadodecane-1,12-diamine (spermine) with copper(II) and hydrogen ions in aqueous solution. J Chem Soc Dalton Trans 2089–2092Google Scholar
  24. 24.
    Frassineti C, Alderighi L, Gans P, Sabatini A, Vacca A, Ghelli S (2003) Determination of protonation constants of some fluorinated polyamines by means of 13C NMR data ­processed by the new computer program HypNMR2000. Protonation sequence in polyamines. Anal Bioanal Chem 376:1041–1052PubMedCrossRefGoogle Scholar
  25. 25.
    Takeda Y, Samejima K, Nagano K, Watanabe M, Sugeta H, Kyogoku Y (1983) Determination of protonation sites in thermospermine and in some other polyamines by 15N and 13C nuclear magnetic resonance spectroscopy. Eur J Biochem 130:383–389CrossRefGoogle Scholar
  26. 26.
    Palmer BN, Powell HKJ (1974) Complex formation between 4,9-diazadodecane-1,12-diamine (spermine) and copper(II) ions and protons in aqueous solution. J Chem Soc Dalton Trans 2086–2089Google Scholar
  27. 27.
    Geall AJ, Taylor RJ, Earll ME, Eaton MAW, Blagbrough IS (2000) Synthesis of cholesteryl polyamine carbamates: pK a studies and condensation of calf thymus DNA. Bioconjug Chem 11:314–326PubMedCrossRefGoogle Scholar
  28. 28.
    Szczepanik W, Kaczmarek P, Sobczak J, Bal W, Gatner K, Jezowska-Bojczuk M (2002) Copper(II) binding by kanamycin A and hydrogen peroxide activation by resulting complexes. New J Chem 26:1507–1514Google Scholar
  29. 29.
    Clouet-d’Orval B, Stage TK, Uhlenbeck OC (1995) Neomycin inhibition of the hammerhead ribozyme involves ionic interactions. Biochemistry 34:11186–11190Google Scholar
  30. 30.
    Geall AJ, Blagbrough IS (2000) Rapid and sensitive ethidium bromide fluorescence quenching assay of polyamine conjugate-DNA interactions for the analysis of lipoplex formation in gene therapy. J Pharm Biomed Anal 22:849–859PubMedCrossRefGoogle Scholar
  31. 31.
    Glasoe PK, Long FA (1960) Use of glass electrodes to measure acidities in deuterium oxide. J Phys Chem 64:188–190CrossRefGoogle Scholar
  32. 32.
    Remy J-S, Sirlin C, Vierling P, Behr J-P (1994) Gene transfer with a series of lipophilic ­DNA-binding molecules. Bioconjug Chem 5:647–654PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ian S. Blagbrough
    • 1
  • Abdelkader A. Metwally
    • 1
  • Andrew J. Geall
    • 1
  1. 1.Department of Pharmacy and PharmacologyUniversity of BathBathUK

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