Time-Resolved Fluorescence-Based Assay for the Determination of Alkaline Phosphatase Activity and Application to the Screening of Its Inhibitors

Abstract

A single-step end point method is presented for determination of the activity of the enzyme alkaline phosphatase (ALP) using the effect of enhancement of fluorescence of the easily accessible europium(III)-tetracycline 3:1 complex (Eu₃TC). Its luminescence, peaking at 616 nm if excited at 405 nm, is enhanced by a factor of 2.5 in the presence of phosphate. Phenyl phosphate was used as a substrate that is enzymatically hydrolyzed to form phenol and phosphate. The latter coordinates to Eu₃TC and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. The assay is performed in a time-resolved (gated) mode, which is shown to yield larger signal changes than steady-state measurement of fluorescence. The limit of detection for ALP is 4 µmol L^—1. Based on this scheme, a model assay for theophylline as inhibitor for ALP was developed with a linear range from 14 to 68 µmol L^{— 1} of theophylline. (Journal of Biomolecular Screening 2008:9-16)

Keywords

alkaline phosphatase phosphate probe europium enzyme inhibition screening assay

References

Christenson RH : Biochemical markers of bone metabolism: an overview. Clin Biochem 1997;30:573-593.

Low MG : The glycosyl-phosphatidylinositol anchor of membrane proteins. Biochim Biophys Acta 1989;988:427-454.

Fenoll J. , Jourquin G. , Kauffmann JM : Flourimetric determination of alkaline phosphatase in solid and fluid dairy products. Talanta 2002;56: 1021-1026.

Zhao L. , Lin JM , Li Z. : Comparison and development of two different solid phase chemiluminescence ELISAs for the determination of albumin in urine. Anal Chim Acta 2005;541:199-207.

Nören-Müller A. , Reis-Corrêa I. Jr , Prinz H. , Rosenbaum C. , Saxena K. , Schwalbe HJ , et al: Discovery of protein phophatase inhibitor classes by biology-oriented synthesis. Proc Natl Acad Sci U S A 2006;103:10606-10611.

Bialy L. , Waldmann H. : Inhibitors of protein tyrosine phophatases: next-generation drugs? Angew Chem Int Ed Engl 2005;44:3814-3839.

Zheng W. , Brandish PE , Kolodin DG , Scolnick EM , Strulovici B. : High-throughput cell-based screening using scintillation proximity assay for the discovery of inositol phosphatase inhibitors. J Biomol Screen 2004;9: 132-140.

Huang Z. , Wang Q. , Ly HD , Gorvindarajan A. , Scheigetz J. , Zamboni R. , et al: 3,6-Fluorescein diphosphate: a sensitive fluorogenic and chromogenic substrate for protein tyrosine phosphatases. J Biomol Screen 1999;4:327-334.

Doman TN , McGovern SL , Witherbee BJ , Kasten TP , Kurumbail R. , Stallings WC , et al: Molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase-1B. J Med Chem 2002; 45:2213-2221.

10.

Taylor SD : Inhibitors of protein tyrosine phosphatase 1B (PTP1b). Curr Top Med Chem 2003;3:759-782.

11.

Alkaline phosphatase assay. Standard operation protocol. Berlin: Diaglobal GmbH.

12.

Hempen C. , Karst U. : A dual fluorophore system for simultaneous bioassays. Anal Chim Acta 2004;521:117-122.

13.

Wolfbeis OS , Koller E. : Photometric and fluorimetric assay of alkaline phosphatase with new coumarin-derived substrates. Microchim Acta 1985;85:389-395.

14.

Whisnant AR , Johnston SE , Gilman SD : Capillary electrophoretic analysis of alkaline phosphatase inhibition by theophylline . Electrophoresis 2000;21:1341-1348.

15.

Whateley JG , inventor: Methods for measuring protein kinase and phosphatase activity . 2003; US2003228646.

16.

Serra B. , Morales MD , Raviejo AJ , Hall EH , Pingarron JM : Rapid and highly sensitive electrochemical determination of alkaline phosphatase using a composite tyrosinase biosensor. Anal Biochem 2005;336:289-294.

17.

Webb MR : A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 1992;89:4884-4887.

18.

Molecular Probes : Handbook of Fluorescent Probes and Research Products. 9th ed. Retrieved from http://probes.invitrogen.com/handbook/sections/2102.html

19.

Brune M. , Hunter JL , Corrie JT , Webb MR : Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase . Biochemistry 1994;33:8262-8271.

20.

Bisswanger H. : Practical Enzymology. Chichester (UK): Wiley-VCH; 2004.

21.

Richardson FS : Terbium(III) and europium(III) ions as luminescent probes and stains for biomolecular systems. Chem Rev 1982;82:541-552.

22.

Wolfbeis OS , Dürkop A. , Wu M. , Lin Z. : A europium-ion based luminescent sensing probe for hydrogen peroxide. Angew Chem Int Ed Engl 2002;41:4495-4498.

23.

Lei W. , Duerkop A. , Lin Z. , Wu M. , Wolfbeis OS : Detection of hydrogen peroxide in river water via a microplate luminescence assay with time-resolved (“gated”) detection. Microchim Acta 2003;143:269-274.

24.

Chen QX , Zheng WZ , Lin JY , Shi Y. , Xie WZ , Zhou HM : Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase. In J Biochem Cell Biol 2000;32:879-885.

25.

Sigma-Aldrich: Specification sheet. Retrieved from www.sigmaaldrich.com/catalog/search/SpecificationSheetPage/SIGMA/P7640

26.

Wu M. , Lin Z. , Duerkop A. , Wolfbeis OS : Time-resolved enzymatic determination of glucose using a fluorescent europium probe for hydrogen peroxide. Anal Bioanal Chem 2004;380:619-626.

27.

Lin JM , Tsuji A. , Maeda M. : Chemiluminescent flow injection determination of alkaline phosphatase and its application to enzyme immunoassays. Anal Chim Acta 1997;339:139-146.

28.

Ciana LD , Bernacca G. , Bordin F. , Fenu S. , Garetto F. : Highly sensitive amperometric measurement of alkaline phosphatase activity with glucose oxidase amplification. J Electroanal Chem 1995;382:129-135.

29.

Bagel O. , Limoges B. , Schöllhorn B. , Degrand C. : Subfemtomolar determination of alkaline phosphatase at a disposable screen-printed electrode modified with a perfluorosulfonated ionomer film . Anal Chem 1997;69:4688-4694.

30.

Jourquin G. , Kauffmann JM : Fluorimetric determination of theophylline in serum by inhibition of bovine alkaline phosphatase in AOT based water/in oil microemulsion. J Pharm Biomed Anal 1998 ;18:585-596.

31.

Vinet B. , Zizian L. : Enzymatic assay for serum theophylline. Clin Chem 1979;25:1370-1372.

32.

Sànchez-Cabezudo M. , Fernàndez-Romero JM , Luque de Castro MD : Fluorimetric-flow injection determination of theophylline based on its inhibitory effect on immobilized alkaline phosphatase. Anal Chim Acta 1995;308:159-163.

33.

Mazzei F. , Botrè F. , Montilla S. , Pilloton R. , Podesta E. , Botrè C. : Alkaline phosphatase inhibition based electrochemical sensors for the detection of pesticides. J Electroanal Chem 2004;574:95-100.

34.

Cano-Raya C. , Fernádez Ramos MD , Capitán Vallvey LF , Wolfbeis OS , Schäferling M. : Fluorescence quenching of the europium tetracycline hydrogen peroxide complex by copper (II) and other metal ions. Appl Spectrosc 2005;59:1209-1216.

35.

Duerkop A. , Turel M. , Lobnik A. , Wolfbeis OS : Microtiter plate assay for phosphate using europium-tetracycline complex as sensitive luminescent probe. Anal Chim Acta 2006;555:292-295.

36.

Schäferling M. , Wolfbeis OS : Europium tetracycline as a luminescent probe for nucleoside phosphates, and its application to the determination of kinase activity. Chem Eur J 2007;13:4342-4349.

37.

Molecular Devices : IMAP technology . Retrieved from www.moleculardevices.com/pages/reagents/imap.html

38.

Promega : Abstract for ProFluor ® tyrosine phosphatase assay. Retrieved from www.promega.com/tbs/tb334/tb334.html