Kinetic analysis of the transphosphorylation with creatine kinase by pressure-assisted capillary electrophoresis/dynamic frontal analysis


Kinetic reactions of the transphosphorylation with creatine kinase (CK) were individually investigated between creatine (Cr) and creatine phosphate (CrP) by pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The transphosphorylations are reversible between Cr and CrP, and reverse reactions inevitably accompany in general batch analyses. In pCE/DFA, the kinetic reaction proceeds in a separation capillary and the product is continuously resolved from the substrate zone. Therefore, the formation rate is kept constant at the substrate zone without the reverse reaction, and the product is detected as a plateau signal. This study demonstrates the direct and individual analyses of both the forward and the backward kinetic reactions with CK by pCE/DFA. A plateau signal was detected in the pCE/DFA with ADP or ATP as one of the products on either the forward or the backward reactions. The Michaelis-Menten constants of Km,ATP (from Cr to CrP) and Km,ADP (from CrP to Cr) were successfully determined through the plateau signal. Determined values of Km,ATP and Km,ADP by pCE/DFA were smaller than the ones obtained by the pre-capillary batch analyses. The results agree with the fact that the reverse reaction is excluded in the analysis of the kinetic reactions. The proposed pCE/DFA is useful on individual analyses of both forward and backward kinetic reactions without any interference from the reverse reaction.

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  1. 1.

    Faber K. Biotransformations in organic chemistry. 5th ed. Berlin: Springer; 2004. p. 123–34.

    Google Scholar 

  2. 2.

    Min K-L, Steghens J-P. ADP is produced by firefly luciferase but its synthesis is independent of the light emitting properties. Biochimie. 2001;83:523–8.

    CAS  PubMed  Google Scholar 

  3. 3.

    Rudolph FB, Fromm HJ. Kinetic studies of the adenosine 5′-triphosphatase activity of yeast hexokinase and its relationship to the mechanism of action of the enzyme. J Biol Chem. 1970;245:4047–52.

    CAS  PubMed  Google Scholar 

  4. 4.

    Li Y, Liu D, Bao JJ. Characterization of tyrosine kinase and screening enzyme inhibitor by capillary electrophoresis with laser-induced fluoresce detector. J Chromatogr B. 2011;879:107–12.

    CAS  Google Scholar 

  5. 5.

    Yangyuoru PM, Otieno AC, Mwongela SM. Determination of sphingosine kinase 2 activity using fluorescent sphingosine by capillary electrophoresis. Electrophoresis. 2011;32:1742–9.

    CAS  PubMed  Google Scholar 

  6. 6.

    Santacruz L, Arciniegas AJL, Darrabie M, Mantilla JG, Baron RM, Bowles DE, et al. Hypoxia decreases creatine uptake in cardiomyocytes, while creatine supplementation enhances HIF activation. Physiol Rep. 2017;5:e13382.

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Suresh Babu CV, Cho SG, Yoo YS. Method development and measurements of endogenous serine/threonine Akt phosphorylation using capillary electrophoresis for systems biology. Electrophoresis. 2005;26:3765–72.

    CAS  PubMed  Google Scholar 

  8. 8.

    Zhang M, Liang S, Lu Y-T. Cloning and functional characterization of NtCPK4, a new tobacco calcium-dependent protein kinase. Biochim Biophys Acta. 1729;2005:174–85.

    Google Scholar 

  9. 9.

    Ni F, Kung A, Duan Y, Shah V, Amador CD, Guo M, et al. Remarkably stereospecific utilization of ATP α,β-halomethylene analogues by protein kinases. J Am Chem Soc. 2017;139:7701–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Strätker K, Haidar S, Amesty Á, El-Awaad E, Götz C, Estévez-Braun A, et al. Development of an in vitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors. FEBS J. 2020;287:3042–64.

    PubMed  Google Scholar 

  11. 11.

    Bessman SP, Carpenter CL. The creatine-creatine phosphate energy shuttle. Annu Rev Biochem. 1985;54:831–62.

    CAS  PubMed  Google Scholar 

  12. 12.

    Friedman DL, Perryman MB. Compartmentation of multiple forms of creatine kinase in the distal nephron of the rat kidney. J Biol Chem. 1991;266:22404–10.

    CAS  PubMed  Google Scholar 

  13. 13.

    Wallimann T, Dolder M, Schlattner U, Eder M, Hornemann T, Kraft T, et al. Creatine kinase: an enzyme with a central role in cellular energy metabolism. Magn Reson Mater Phy. 1998;6:116–9.

    CAS  Google Scholar 

  14. 14.

    Cai Y, Lee J, Wang W, Yang J-M, Qian G-Y. Effect of Cd2+ on muscle type of creatine kinase: inhibition kinetics integrating computational simulations. Int J Biol Macromol. 2016;83:233–41.

    CAS  PubMed  Google Scholar 

  15. 15.

    Morandi L, Angelini C, Prelle A, Pini A, Grassi B, Bernardi G, et al. High plasma creatine kinase: review of the literature and proposal for a diagnostic algorithm. Neurol Sci. 2006;27:303–11.

    CAS  PubMed  Google Scholar 

  16. 16.

    Fan Y, Scriba GKE. Advances in-capillary electrophoretic enzyme assays. J Pharm Biomed Anal. 2010;53:1076–90.

    CAS  PubMed  Google Scholar 

  17. 17.

    Bao J, Regnier FE. Ultramicro enzyme assays in capillary electrophoretic system. J Chromatogr A. 1992;608:217–24.

    CAS  Google Scholar 

  18. 18.

    Harmon BJ, Patterson DH, Regnier FE. Mathematical treatment of electrophoretically mediated microanalysis. Anal Chem. 1993;65:2655–62.

    CAS  PubMed  Google Scholar 

  19. 19.

    Harmon BJ, Patterson DH, Regnier FE. Electrophoretically mediated microanalysis of ethanol. J Chromatogr A. 1993;657:429–34.

    CAS  PubMed  Google Scholar 

  20. 20.

    Patterson DH, Harmon BJ, Regnier FE. Electrophoretically mediated microanalysis of calcium. J Chromatogr A. 1994;662:389–95.

    CAS  PubMed  Google Scholar 

  21. 21.

    Iqbal J. An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases. Anal Biochem. 2011;414:226–31.

    CAS  PubMed  Google Scholar 

  22. 22.

    Iqbal J, Iqbal S, Müller CE. Advances in immobilized enzyme microbioreactors in capillary electrophoresis. Analyst. 2013;138:3104–16.

    CAS  PubMed  Google Scholar 

  23. 23.

    Cheng M, Chen Z. Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking. Electrophoresis. 2017;38:486–93.

    CAS  PubMed  Google Scholar 

  24. 24.

    Nehme H, Nehme R, Lafite P, Routier S, Morin P. New development in in-capillary electrophoresis techniques for kinetic and inhibition study of enzymes. Anal Chim Acta. 2012;722:127–35.

    CAS  PubMed  Google Scholar 

  25. 25.

    Krylova SM, Okhonin V, Krylov SN. Transverse diffusion of laminar flow profiles - a generic method for mixing reactants in capillary microreactor. J Sep Sci. 2009;32:742–56.

    CAS  PubMed  Google Scholar 

  26. 26.

    Farcaş E, Pochet L, Fillet M. Transverse diffusion of laminar flow profiles as a generic capillary electrophoresis method for in-line nanoreactor mixing: application to the investigation of antithrombotic activity. Talanta. 2018;188:516–21.

    PubMed  Google Scholar 

  27. 27.

    Whisnant AR, Johnston SE, Gilman SD. Capillary electrophoretic analysis of alkaline phosphatase inhibition by theophylline. Electrophoresis. 2000;21:1341–8.

    CAS  PubMed  Google Scholar 

  28. 28.

    Craig DB, Nichols ER. Continuous flow assay for the simultaneous measurement of the electrophoretic mobility, catalytic activity and its variation over time of individual molecules of Escherichia coli β-galactosidase. Electrophoresis. 2008;29:4298–303.

    CAS  PubMed  Google Scholar 

  29. 29.

    Ma J, Peng X, Cheng K-W, Chen F, Yang D, Chen B, et al. Use of capillary electrophoresis to evaluate protective effects of methylglyoxal scavengers on the activity of creatine kinase. J Sep Sci. 2008;31:2846–51.

    CAS  PubMed  Google Scholar 

  30. 30.

    Fujima JM, Danielson ND. Determination of creatine kinase activity and phosphocreatine in off-line and on-line modes with capillary electrophoresis. Anal Chim Acta. 1998;375:233–41.

    CAS  Google Scholar 

  31. 31.

    Takayanagi T, Mine M, Mizuguchi H. Capillary electrophoresis/dynamic frontal analysis for the enzyme assay of 4-nitrophenyl phosphate with alkaline phosphatase. Anal Sci. 2020;36:829–34.

    CAS  PubMed  Google Scholar 

  32. 32.

    Mine M, Mizuguchi H, Takayanagi T. Inhibition assay of theophylline by capillary electrophoresis/dynamic frontal analysis on the hydrolysis of p-nitrophenyl phosphate with alkaline phosphatase. Chem Lett. 2020;49:681–4.

    CAS  Google Scholar 

  33. 33.

    Mine M, Mizuguchi H, Takayanagi T. Kinetic analysis of substrate competition in enzymatic reactions with β-D-galactosidase by capillary electrophoresis / dynamic frontal analysis. J Pharm Biomed Anal. 2020;188:113390.

    CAS  PubMed  Google Scholar 

  34. 34.

    Mine M, Matsumoto N, Mizuguchi H, Takayanagi T. Kinetic analysis of an enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase by pressure-assisted capillary electrophoresis/dynamic frontal analysis. Anal Methods. 2020;12:5846–51.

    CAS  PubMed  Google Scholar 

  35. 35.

    Nehmé H, Nehmé R, Lafite P, Routier S, Morin P. Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing. J Chromatogr A. 2013;1314:298–305.

    PubMed  Google Scholar 

  36. 36.

    Nehmé R, Nehmé H, Roux G, Destandau E, Claude B, Morin P. Capillary electrophoresis as a novel technique for screening natural flavonoids as kinase inhibitors. J Chromatogr A. 2013;1318:257–64.

    PubMed  Google Scholar 

  37. 37.

    Nehmé R, Nehmé H, Saurat T, de-Tauzia M-L, Buron F, Lafite P, et al. New in-capillary electrophoretic kinase assays to evaluate inhibitors of the PI3k/Akt/mTOR signaling pathway. Anal Bioanal Chem. 2014;406:3743–54.

    PubMed  Google Scholar 

  38. 38.

    Nehmé H, Chantepie S, Defert J, Morin P, Papy-Garcia D, Nehmé R. New methods based on capillary electrophoresis for in vitro evaluation of protein tau phosphorylation by glycogen synthase kinase 3-β. Anal Bioanal Chem. 2015;407:2821–8.

    PubMed  Google Scholar 

  39. 39.

    The R Project for Statistical Computing, available from <>, (accessed 2020-11-16).

  40. 40.

    Haynes WM, editor. CRC handbook of chemistry and physics. 91st ed. Boca Raton: CRC Press; 2010. p. 7–2.

    Google Scholar 

  41. 41.

    Bickerstaff GF, Price NC. Reversible denaturation of rabbit muscle creatine kinase. Biochem Soc T. 1977;5:761–4.

    CAS  Google Scholar 

  42. 42.

    Zhao T-J, Feng S, Wang Y-L, Liu Y, Luo X-C, Zhou H-M, et al. Impact of intra-subunit domain–domain interactions on creatine kinase activity and stability. FEBS Lett. 2006;580:3835–40.

    CAS  PubMed  Google Scholar 

  43. 43.

    Savabi F, Geiger PJ, Bessman SP. Myofibrillar end of the creatine phosphate energy shuttle. Am J Phys. 1984;247:C424–32.

    CAS  Google Scholar 

  44. 44.

    Wu C-L, Li Y-H, Lin H-C, Yeh Y-H, Yan H-Y, Hsiao C-D, et al. Activity and function of rabbit muscle-specific creatine kinase at low temperature by mutation at gly268 to asn268. Comp Biochem Phys B. 2011;158:189–98.

    Google Scholar 

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This work was supported by a Grant-in-Aid for Scientific Research (C) (No. 20K05568) from the Japan Society for the Promotion of Sciences (JSPS).

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M. Mine: methodology, investigation, data curation, formal analysis, visualization, writing—original draft; H. Mizuguchi: investigation, resources, validation; T. Takayanagi: conceptualization, funding acquisition, project administration, resources, supervision, writing—review and editing.

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Correspondence to Toshio Takayanagi.

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Mine, M., Mizuguchi, H. & Takayanagi, T. Kinetic analysis of the transphosphorylation with creatine kinase by pressure-assisted capillary electrophoresis/dynamic frontal analysis. Anal Bioanal Chem 413, 1453–1460 (2021).

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  • Capillary electrophoresis
  • Dynamic frontal analysis
  • Creatine kinase
  • Transphosphorylation
  • Kinetic analysis