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Quantitative single-step purification of dinucleoside polyphosphates
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 316 (2003) 135–138 www.elsevier.com/locate/yabio
Notes & Tips
Quantitative single-step purification of dinucleoside polyphosphates Michael Wright, Julian A. Tanner, and Andrew D. Miller* Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, South Kensington, London SW7 2AZ, UK Received 19 September 2002
50 ,5000 -P1 ; P4 -Dinucleoside polyphosphates are an important family of nucleotides with both intracellular and extracellular biological roles [1,2]. Accordingly, there have been a number of synthetic methods reported for the preparation of dinucleoside polyphosphates, allowing these biological roles to be studied in more detail. One of the most successful methods involves the use of the heat-inducible Escherichia coli lysyl-tRNA synthetase (LysU)1 as a flexible enzyme catalyst for the synthesis of a wide range of dinucleoside polyphosphates using purified adenosine 50 -triphosphate (ATP) with a second nucleotide as substrate [3]. LysU biosynthesis of dinucleoside polyphosphates is an efficient alternative to multistep chemical syntheses [4]. Traditionally, thinlayer chromatography (TLC) has been used to monitor reactions and subsequent purifications carried out using reversed-phase high-performance liquid chromatography (HPLC). We have consistently observed that TLC is frequently unreliable and gives poor quantitative analyses. Furthermore, reversed-phase HPLC poorly resolves dinucleoside polyphosphates with similar properties. Accordingly, we have encountered frequent problems in the isolation and purification of dinucleoside polyphosphates and their analogues, limiting our attempts at characterizing their biological activity. Here we report a fast (<5 min), quantitative method of dinucleoside polyphosphate analysis and purification that is reproducible and has solved all our basic problems with TLC and HPLC analysis/purifications. This
* Corresponding author. Fax: +44-20-7594-5803. E-mail address: [email protected] (A.D. Miller). 1 Abbreviations used: LysU, lysyl-tRNA synthetase; Ap2 CH2 p2 ,A, b-c-methylenediadenosine 50 ,500 -P1 ; P4 -tetraphosphate; Ip4 I, diinosine 50 ,5000 -P1 ; P4 -tetraphosphate; Ip2 CH2 p2 I, b-c-methylenediinosine 50 ,5000 P1 ; P4 -tetraphosphate; FPLC, fast protein liquid chromatography; TEAB, triethylammonium hydrogen carbonate buffer; FAB, fast atom bombardment; ES, electrospray.
method is a single step ion-exchange technique that we have found to give high-resolution purification without the need for subsequent desalting of the purified product. By way of illustration, we describe the updated syntheses and subsequent purification of three different dinucleoside polyphosphates: b; c-methylenediadenosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ap2 CH2 p2 A), diinosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ip4 I), and b; c-methylenediinosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ip2 CH2 p2 IÞ: Ap2 CH2 p2 A was prepared by means of the LysU biosynthesis procedure updated from previously [3]. Briefly, LysU (30 lM, dimer) was dialyzed at 4 °C against 50 mM Tris–HCl, pH 8.0 (1 L) and left to reactivate for a minimum of 48 h. Reaction mixture (4 ml) was then prepared consisting of 9 lM LysU, 2 mM L -lysine, 10 mM MgCl2 , and 75 lg yeast inorganic pyrophosphatase (Roche) in 50 mM Tris–HCl buffer, pH 8.0. This mixture was incubated at 30 °C with stirring and 5 mM ATP, 7.5 mM b; c-methylene-ATP (AMP-PCP, Sigma), and 160 lM ZnCl2 was slowly added. Incubation was continued for a further hour or until the formation of Ap2 CH2 p2 A was judged to be complete. Ip2 CH2 p2 I was prepared from Ap2 CH2 p2 A and Ip4 I directly from Ap4 A (Sigma) using the enzyme 50 -adenylic acid deaminase from Aspergillus (Sigma). This unspecific enzyme is able to deaminate adenosine nucleotides to their inosine counterparts [5]. Our updated procedure was to prepare a reaction mixture (2 ml) containing 50 adenylic acid deaminase (0.22 U) and diadenosine polyphosphate (20 mM) in 50 mM Tris–HCl, pH 6.5. This mixture was incubated at 35 °C for 1 h with stirring or until the formation of product was judged to be complete. In all cases reaction progress could be closely monitored using a Resource Q anion-exchange column (6 ml), connected to a fast protein liquid chromatography (FPLC) system equipped with UV lamp (254 nm) and
0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00035-6
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Notes & Tips / Analytical Biochemistry 316 (2003) 135–138
fraction collector (all from Amersham Biosciences). The column was equilibrated in 10 mM Tris–HCl, pH 8.0, and eluted with 0–50% gradient of 1 M NaCl in 50 mM Tris–HCl, pH 8.0, at a flow rate of 10 ml/min over 5 min. Aliquots (5 ll) were taken from the reaction mixture and diluted to 300 ll in deionized water before injection via a 200-ll injection loop. Adenosine 50 -monophosphate (AMP), adenosine 50 -diphosphate (ADP), AMP-PCP, ATP, and Ap4 A solutions of known concentration were used to calibrate the column (Fig. 1a). Using this method, quantitative completion of both types of enzyme-catalyzed reactions could be observed with little difficulty in comparison to the situation encountered with TLC methods. For dinucleoside polyphosphate purification from reaction mixtures, the Resource Q/NaCl system was not found appropriate owing to the small column size and the requirement for a difficult desalting step. Instead, we developed a new protocol adapted from Lenzen et al. [6] in which a HR 16/10 column was packed with 20 ml of
Fig. 1. (a) FPLC calibration trace, using known concentrations of nucleotides and 125 lM Ap4 A (all Sigma, 95%). Inosine analogues elute slightly after their adenosine equivalents, as shown (b) FPLC trace showing the separation of 75 lM Ap4 A and Ip4 I. In both figures the bold line shows the increasing NaCl gradient (0–0.4 M).
Source 15Q ion-exchange media (the same as used in the Resource Q) that was then equilibrated in 5 mM triethylammonium hydrogen carbonate buffer (TEAB), pH 8–9. Upon completion of enzyme-catalyzed reactions, the mixtures were microcentrifuged and loaded onto the column in appropriate aliquots. The column was eluted with 0–50% 2 M TEAB at a flow rate of 6 ml/ min over 25 min in order to elute pure dinucleoside polyphosphate product. Column retention behavior and resolution were found to be comparable to Resource Q/NaCl, with nucleotides eluting in the same order. This gave a useful correlation between analytical and preparative systems and made fraction identification significantly easier. The high concentration TEAB was prepared by bubbling CO2 evolved from dry ice through aqueous triethylamine (279 ml made up to 1 L with deionized water) with stirring for 48 h. Since TEAB is volatile, it was easily removed by freeze drying to leave dinucleoside polyphosphate product in a crystalline state. Yields of pure product were in the range 60–70% with respect to initial ATP for the LysU biosynthesis and greater than 80% for the deaminase conversions. Previously we reported a complex method for obtaining identification of dinucleoside polyphosphates by mass spectrometry in which polyphosphates were precipitated from acetone solution using NaCl and then characterized by negative ion fast atom bombardment (FAB) mass spectrometry [7]. In this case, the high purity of each dinucleoside polyphosphate product and the low levels of TEAB contamination after freeze drying allowed us to obtain facile mass spectral characterization of each product by electrospray (ES) mass spectrometry using a Bruker Esquire 3000 electrospray mass spectrometer set to negative polarity. Typical results are shown for two of the dinucleoside polyphosphates, Ap2 CH2 p2 A (Fig. 2a) and Ip4 I (Fig. 2b). The use of anion-exchange Source/Resource Q media allows for rapid, facile, and reliable reaction monitoring compared to TLC and similarly eases purification of dinucleoside polyphosphates from reaction mixtures. Moreover, these chromatographic methods are sufficiently high resolution to resolve even compounds as chemically similar as Ap4 A and Ip4 I (Fig. 1b). Finally, the use of high concentration TEAB solution allows for the preparation of dinucleoside polyphosphate products in a crystalline state with minimal salt contamination that can be analyzed directly by ES mass spectrometry without the need for the complex preparation procedures that were described previously for mass spectral analysis [7]. We would suggest that the approaches described here could be applied potentially to a wide range of nucleotides and nucleotide analogues including even fluorescent polyphosphate analogues [6,8,9].
Notes & Tips / Analytical Biochemistry 316 (2003) 135–138
137
Fig. 2. Negative ion electrospray mass spectra of Ap2 CH2 p2 A (a) and Ip4 I (b) immediately following purification and freeze drying. The expected mass of singly charged Ap2 CH2 p2 A is 833.04 and Ip4 I is 837.0 but in both cases single and double sodium complexes are also seen as higher m=z satellite peaks.
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Notes & Tips / Analytical Biochemistry 316 (2003) 135–138
References [1] J. Pintor, J. Gualix, M.T. Miras-Portugal, Diinosine polyphosphates, a group of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor, Mol. Pharm. 51 (1997) 277– 284. [2] A.G. McLennan, Dinucleoside polyphosphates-friend or foe?, Pharmacol. Ther. 87 (2000) 73–89. [3] M.-E. Theoclitou, E.P.L. Wittung, A.D. Hindley, T.S.H. ElThaher, A.D. Miller, Characterisation of stress protein LysU. Enzymic synthesis of diadenosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ap4 A) analogues by LysU, J. Chem. Soc., Perkin Trans. 1 (1996) 2009–2019. [4] A.G. McLennan (Ed.), Ap4 A Other Dinucleoside Polyphosphates, CRC Press, Boca Raton, FL, 1992. [5] A. Guranowski, E. Starzynska, M.A. Gunther Sillero, A. Sillero, Conversion of adenosine (50 ) oligophospho(50 ) adenosines into
[6]
[7]
[8]
[9]
inosine(50 ) oligophospho(50 ) inosines by non-specific adenylate deaminase from the snail Helix pomatia, Biochem. Biophys. Acta 1243 (1995) 78–84. C. Lenzen, R.H. Cool, A. Wittinghofer, Analysis of intrinsic and CDC25-stimulated guanine nucleotide exchange of p21ras-nucleotide complexes by fluorescence measurements, Methods Enzymol. 255 (1995) 95–109. M.-E. Theoclitou, A.D. Miller, Enhancement of molecular ion intensity of synthetic analogues of diadenosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ap4 A) in negative-ion mode fast atom bombardment mass spectrometry, Anal. Biochem. 218 (1994) 235–237. Q. Ni, J. Shaffer, J.A. Adams, Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives, Protein Sci. 9 (2000) 1818–1827. T. Hiratsuka, New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes, Biochem. Biophys. Acta 742 (1983) 496–508.
Notes & Tips
Quantitative single-step purification of dinucleoside polyphosphates Michael Wright, Julian A. Tanner, and Andrew D. Miller* Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, South Kensington, London SW7 2AZ, UK Received 19 September 2002
50 ,5000 -P1 ; P4 -Dinucleoside polyphosphates are an important family of nucleotides with both intracellular and extracellular biological roles [1,2]. Accordingly, there have been a number of synthetic methods reported for the preparation of dinucleoside polyphosphates, allowing these biological roles to be studied in more detail. One of the most successful methods involves the use of the heat-inducible Escherichia coli lysyl-tRNA synthetase (LysU)1 as a flexible enzyme catalyst for the synthesis of a wide range of dinucleoside polyphosphates using purified adenosine 50 -triphosphate (ATP) with a second nucleotide as substrate [3]. LysU biosynthesis of dinucleoside polyphosphates is an efficient alternative to multistep chemical syntheses [4]. Traditionally, thinlayer chromatography (TLC) has been used to monitor reactions and subsequent purifications carried out using reversed-phase high-performance liquid chromatography (HPLC). We have consistently observed that TLC is frequently unreliable and gives poor quantitative analyses. Furthermore, reversed-phase HPLC poorly resolves dinucleoside polyphosphates with similar properties. Accordingly, we have encountered frequent problems in the isolation and purification of dinucleoside polyphosphates and their analogues, limiting our attempts at characterizing their biological activity. Here we report a fast (<5 min), quantitative method of dinucleoside polyphosphate analysis and purification that is reproducible and has solved all our basic problems with TLC and HPLC analysis/purifications. This
* Corresponding author. Fax: +44-20-7594-5803. E-mail address: [email protected] (A.D. Miller). 1 Abbreviations used: LysU, lysyl-tRNA synthetase; Ap2 CH2 p2 ,A, b-c-methylenediadenosine 50 ,500 -P1 ; P4 -tetraphosphate; Ip4 I, diinosine 50 ,5000 -P1 ; P4 -tetraphosphate; Ip2 CH2 p2 I, b-c-methylenediinosine 50 ,5000 P1 ; P4 -tetraphosphate; FPLC, fast protein liquid chromatography; TEAB, triethylammonium hydrogen carbonate buffer; FAB, fast atom bombardment; ES, electrospray.
method is a single step ion-exchange technique that we have found to give high-resolution purification without the need for subsequent desalting of the purified product. By way of illustration, we describe the updated syntheses and subsequent purification of three different dinucleoside polyphosphates: b; c-methylenediadenosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ap2 CH2 p2 A), diinosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ip4 I), and b; c-methylenediinosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ip2 CH2 p2 IÞ: Ap2 CH2 p2 A was prepared by means of the LysU biosynthesis procedure updated from previously [3]. Briefly, LysU (30 lM, dimer) was dialyzed at 4 °C against 50 mM Tris–HCl, pH 8.0 (1 L) and left to reactivate for a minimum of 48 h. Reaction mixture (4 ml) was then prepared consisting of 9 lM LysU, 2 mM L -lysine, 10 mM MgCl2 , and 75 lg yeast inorganic pyrophosphatase (Roche) in 50 mM Tris–HCl buffer, pH 8.0. This mixture was incubated at 30 °C with stirring and 5 mM ATP, 7.5 mM b; c-methylene-ATP (AMP-PCP, Sigma), and 160 lM ZnCl2 was slowly added. Incubation was continued for a further hour or until the formation of Ap2 CH2 p2 A was judged to be complete. Ip2 CH2 p2 I was prepared from Ap2 CH2 p2 A and Ip4 I directly from Ap4 A (Sigma) using the enzyme 50 -adenylic acid deaminase from Aspergillus (Sigma). This unspecific enzyme is able to deaminate adenosine nucleotides to their inosine counterparts [5]. Our updated procedure was to prepare a reaction mixture (2 ml) containing 50 adenylic acid deaminase (0.22 U) and diadenosine polyphosphate (20 mM) in 50 mM Tris–HCl, pH 6.5. This mixture was incubated at 35 °C for 1 h with stirring or until the formation of product was judged to be complete. In all cases reaction progress could be closely monitored using a Resource Q anion-exchange column (6 ml), connected to a fast protein liquid chromatography (FPLC) system equipped with UV lamp (254 nm) and
0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00035-6
136
Notes & Tips / Analytical Biochemistry 316 (2003) 135–138
fraction collector (all from Amersham Biosciences). The column was equilibrated in 10 mM Tris–HCl, pH 8.0, and eluted with 0–50% gradient of 1 M NaCl in 50 mM Tris–HCl, pH 8.0, at a flow rate of 10 ml/min over 5 min. Aliquots (5 ll) were taken from the reaction mixture and diluted to 300 ll in deionized water before injection via a 200-ll injection loop. Adenosine 50 -monophosphate (AMP), adenosine 50 -diphosphate (ADP), AMP-PCP, ATP, and Ap4 A solutions of known concentration were used to calibrate the column (Fig. 1a). Using this method, quantitative completion of both types of enzyme-catalyzed reactions could be observed with little difficulty in comparison to the situation encountered with TLC methods. For dinucleoside polyphosphate purification from reaction mixtures, the Resource Q/NaCl system was not found appropriate owing to the small column size and the requirement for a difficult desalting step. Instead, we developed a new protocol adapted from Lenzen et al. [6] in which a HR 16/10 column was packed with 20 ml of
Fig. 1. (a) FPLC calibration trace, using known concentrations of nucleotides and 125 lM Ap4 A (all Sigma, 95%). Inosine analogues elute slightly after their adenosine equivalents, as shown (b) FPLC trace showing the separation of 75 lM Ap4 A and Ip4 I. In both figures the bold line shows the increasing NaCl gradient (0–0.4 M).
Source 15Q ion-exchange media (the same as used in the Resource Q) that was then equilibrated in 5 mM triethylammonium hydrogen carbonate buffer (TEAB), pH 8–9. Upon completion of enzyme-catalyzed reactions, the mixtures were microcentrifuged and loaded onto the column in appropriate aliquots. The column was eluted with 0–50% 2 M TEAB at a flow rate of 6 ml/ min over 25 min in order to elute pure dinucleoside polyphosphate product. Column retention behavior and resolution were found to be comparable to Resource Q/NaCl, with nucleotides eluting in the same order. This gave a useful correlation between analytical and preparative systems and made fraction identification significantly easier. The high concentration TEAB was prepared by bubbling CO2 evolved from dry ice through aqueous triethylamine (279 ml made up to 1 L with deionized water) with stirring for 48 h. Since TEAB is volatile, it was easily removed by freeze drying to leave dinucleoside polyphosphate product in a crystalline state. Yields of pure product were in the range 60–70% with respect to initial ATP for the LysU biosynthesis and greater than 80% for the deaminase conversions. Previously we reported a complex method for obtaining identification of dinucleoside polyphosphates by mass spectrometry in which polyphosphates were precipitated from acetone solution using NaCl and then characterized by negative ion fast atom bombardment (FAB) mass spectrometry [7]. In this case, the high purity of each dinucleoside polyphosphate product and the low levels of TEAB contamination after freeze drying allowed us to obtain facile mass spectral characterization of each product by electrospray (ES) mass spectrometry using a Bruker Esquire 3000 electrospray mass spectrometer set to negative polarity. Typical results are shown for two of the dinucleoside polyphosphates, Ap2 CH2 p2 A (Fig. 2a) and Ip4 I (Fig. 2b). The use of anion-exchange Source/Resource Q media allows for rapid, facile, and reliable reaction monitoring compared to TLC and similarly eases purification of dinucleoside polyphosphates from reaction mixtures. Moreover, these chromatographic methods are sufficiently high resolution to resolve even compounds as chemically similar as Ap4 A and Ip4 I (Fig. 1b). Finally, the use of high concentration TEAB solution allows for the preparation of dinucleoside polyphosphate products in a crystalline state with minimal salt contamination that can be analyzed directly by ES mass spectrometry without the need for the complex preparation procedures that were described previously for mass spectral analysis [7]. We would suggest that the approaches described here could be applied potentially to a wide range of nucleotides and nucleotide analogues including even fluorescent polyphosphate analogues [6,8,9].
Notes & Tips / Analytical Biochemistry 316 (2003) 135–138
137
Fig. 2. Negative ion electrospray mass spectra of Ap2 CH2 p2 A (a) and Ip4 I (b) immediately following purification and freeze drying. The expected mass of singly charged Ap2 CH2 p2 A is 833.04 and Ip4 I is 837.0 but in both cases single and double sodium complexes are also seen as higher m=z satellite peaks.
138
Notes & Tips / Analytical Biochemistry 316 (2003) 135–138
References [1] J. Pintor, J. Gualix, M.T. Miras-Portugal, Diinosine polyphosphates, a group of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor, Mol. Pharm. 51 (1997) 277– 284. [2] A.G. McLennan, Dinucleoside polyphosphates-friend or foe?, Pharmacol. Ther. 87 (2000) 73–89. [3] M.-E. Theoclitou, E.P.L. Wittung, A.D. Hindley, T.S.H. ElThaher, A.D. Miller, Characterisation of stress protein LysU. Enzymic synthesis of diadenosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ap4 A) analogues by LysU, J. Chem. Soc., Perkin Trans. 1 (1996) 2009–2019. [4] A.G. McLennan (Ed.), Ap4 A Other Dinucleoside Polyphosphates, CRC Press, Boca Raton, FL, 1992. [5] A. Guranowski, E. Starzynska, M.A. Gunther Sillero, A. Sillero, Conversion of adenosine (50 ) oligophospho(50 ) adenosines into
[6]
[7]
[8]
[9]
inosine(50 ) oligophospho(50 ) inosines by non-specific adenylate deaminase from the snail Helix pomatia, Biochem. Biophys. Acta 1243 (1995) 78–84. C. Lenzen, R.H. Cool, A. Wittinghofer, Analysis of intrinsic and CDC25-stimulated guanine nucleotide exchange of p21ras-nucleotide complexes by fluorescence measurements, Methods Enzymol. 255 (1995) 95–109. M.-E. Theoclitou, A.D. Miller, Enhancement of molecular ion intensity of synthetic analogues of diadenosine 50 ,5000 -P1 ; P4 -tetraphosphate (Ap4 A) in negative-ion mode fast atom bombardment mass spectrometry, Anal. Biochem. 218 (1994) 235–237. Q. Ni, J. Shaffer, J.A. Adams, Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives, Protein Sci. 9 (2000) 1818–1827. T. Hiratsuka, New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes, Biochem. Biophys. Acta 742 (1983) 496–508.