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Lanthanide ions as luminescent chromophores for the liquid chromatographic detection of polynucleotides and nucleic acids
Journal of Chromatography,
436 (1988) 299-307
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. 20 147
LANTHANIDE IONS AS LUMINESCENT CHROMOPHORES FOR THE LIQUID CHROMATOGRAPHIC DETECTION OF POLYNUCLEOTIDES AND NUCLEIC ACIDS
THOMAS J. WENZEL* and LISA M. COLLETTE Department
of Chemistry,
Bates College, Lewiston,
ME 04240 (U.S.A.)
(First received June 22nd, 1987; revised manuscript received October 16th, 1987)
SUMMARY
Europium(II1) and terbium(II1) can be used as luminescent chromophores for the liquid chromatographic detection of certain nucleotides and nucleic acids. The method is dependent upon an energy transfer from the nucleic acid to the lanthanide ion. Of the base moieties, only xanthine, guanine, and thiouridine have appropriate excited state energy levels for efficient energy transfer. The lanthanide ion can be added in a pre- or post-column mode. The applicability of the method was demonstrated for the detection of homologous polynucleotides such as poly X and poly G. The method was also used to detect transfer RNA from Escherichia coli.
INTRODUCTION
We have reported the use of lanthanide ions as luminescent chromophores for the liquid chromatographic (LC) detection of aromatic aldehydes and ketones’. The lanthanide detection scheme is based on the principle of sensitized luminescenceZ. An organic compound is first excited by light. If an appropriate match between the energy of the triplet state of the organic and an excited state of a lanthanide ion exists, a transfer of energy to the lanthanide ion can occur. The lanthanide ion can then emit visible luminescence that is detected3-6. The two best lanthanide ions for this method, based on energy level considerations and luminescence quantum yields, are terbium(II1) and europium(II1). Tb(II1) emits a green luminescence at 545 nm, whereas Eu(II1) emits a red luminescence at 613 nm. We will now describe the use of Tb(II1) and Eu(II1) as luminescent chromophores for the LC detection of certain nucleotides and nucleic acids. A number of previous reports have discussed aspects of the energy transfer from nucleotides7-9 and nucleic acids10-23 to lanthanide ions. All of the common bases of nucleic acids can transfer energy to Tb(III)8,11,16J3. Th e efficiency of this energy transfer is largest, however, with guanine*,’ 1,16,23,xanthine9*16, and thiouridine17-2 l. Modification of guanine residues significantly descreased Tb(II1) luminescence9~16. Incorporation of the nucleotides into polymer chains increased the efficiency of the energy transfer9v1 2. Nucleic acids must be single-stranded to observe energy transfer9v1 1,12,22,24. The 0021-9673/88/%03.50
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absence of lanthanide luminescence with double-stranded nucleic acids is attributed to an alteration of the energy of the triplet levels of the bases9. Unlike aromatic aldehydes and ketones, which transfer energy to the lanthanide ions by an intermolecular process 24-2*, the nucleotides and nucleic acids transfer energy by an intramolecular process 8. Most previous work has concluded that bonding of the lanthanide ion occurs at the phosphate group, however, some evidence for a degree of bonding at the basesa or sugar moiety13 has been reported. As our work will demonstrate, the lanthanide ions can be added to the LC system in either a preor post-column mode. The residence time in a post-column reaction coil is suflicient for bonding to occur. Detection based on lanthanide luminescence is suitable for polynucleotides or single-stranded nucleic acids containing xanthine, guanine, or thiouridine residues. Hydrolysis of the nucleotides and nucleic acids in the presence of lanthanide ions is slow enough so that it does not cause problems in LC detection. If a fluorescence instrument with a pulsed source is employed, improvements in the sensitivity and detection limits can be achieved by incorporating a delay time between the lamp pulse and the acquisition of data. Such an improvement in sensitivity is possible because of the long excited state lifetimes of Tb(II1) and Eu(II1). EXPERIMENTAL
Reagents
All reagents were used as received without further purification. The nucleotides, polynucleotides, and nucleic acids were purchased from Sigma (St. Louis, MO, U.S.A.). HPLC-grade water was obtained from Fisher (Fairlawn, NJ, U.S.A.). The chloride salts of Tb(II1) and Eu(II1) were prepared as previously described’ using lanthanide oxides (Alfa Products, Danvers, MA, U.S.A.) and Ultrex hydrochloric acid(J. T. Baker, Phillipsburg, NJ, U.S.A.). Sodium chloride and sodium acetate were obtained from J. T. Baker. Apparatus
A diagram of the apparatus employed for obtaining liquid chromatograms is shown in Fig. 1. Beckman Model 110B pumps were used for introducing the mobile and post-column phases. The mixing tee was stainless-steel and a 25 ft. x 0.01 in. I.D. stainless-steel reaction coil was placed between the mixing tee and the spectrofluorometer. The flow-rate of the LC phase and post-column phase were set at 0.5 ml/min. A steric exclusion column (Synchropak GPC-500; Alltech, Deerfield, IL, U.S.A.) was used for separating the homopolymers and transfer RNA (tRNA). Samples were introduced from a 20-4 injection loop.
r--@J Q-l PUMP
PUMP
REACTION COIL
&I
MOBILE
PHASE
FLUORESCENCE DETECTOR
LANTHANIDE PHASE
Fig. 1. System set-up for post-column addition of the lanthanide ions.
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A Perkin-Elmer LS-5 spectrofluorometer was used for all fluorescence measurements. The LS-5 can be used in the cuvette mode or an optional flow-cell for LC detection can be employed. Excitation and emission slit widths were set to 5 nm for cuvette work and 10 nm for LC detection. The response time was typically adjusted to setting 1 on the instrument. The apparatus for flow-injection analyses (FIA) was identical to that shown in Fig. 1 except the analytical column was removed. For phosphorescence measurements, which utilize a delay time between the lamp pulse and the start of data acquisition, the instrument was set to the phosphorescence mode and appropriate delay and gate times were set. Unless otherwise noted, all measurements were performed in the fluorescence mode. Procedures
All solutions of the nucleotides, nucleic acids, and lanthanide salts were prepared in an acetate buffer that had been adjusted to pH 6 using Ultrex hydrochloric acid. The acetate buffer consisted of a solution of sodium chloride (0.1 M) and sodium acetate (0.01 M) in HPLC-grade doubly distilled water. Unless stated otherwise, all solutions of the nucleotides and nucleic acids were prepared the day of use. Solutions containing nucleotides or nucleic acids were prepared by weighing the compound into a volumetric flask and dissolving it in acetate buffer or acetate buffer containing the lanthanide ion. The concentration of lanthanide ion was lop4 A4 in cuvette studies. In LC applications, the concentration of the lanthanide ion in the post-column phase was 10e4 M and therefore 5 . lO+ M at the detector. The concentration of lanthanide ion was selected after consideration of previous findingsl’j. No steps were taken to remove dissolved oxygen from mobile and post-column phases. It has been reported that the intensity of lanthanide luminescence based on intramolecular transfer processes is not significantly influenced by dissolved oxygen29. RESULTS AND DISCUSSION
The range of nucleotides and nucleic acids for which lanthanide luminescence represented a viable LC detection method were evaluated by cuvette and FIA studies. A typical emission spectrum is shown in Fig. 2a for a mixture of poly G (2.5 . 1O-4 M in Guo-5’-P moieties) and terbium(II1) chloride (low4 M). The excitation wavelength was 290 nm. The bands in the emission spectrum at 489 and 544 nm are characteristic of emission from Tb(II1). The band at 544 nm is usually the most intense for Tb(II1). Fig. 2b is the emission spectrum of a solution of terbium(II1) chloride without poly G. All conditions were identical to those of the spectrum shown in Fig. 2a. The Tb(II1) bands seen in the spectrum in Fig. 2a are therefore the result of energy transfer from guanine. The emission spectra of mixtures of Tb(II1) with other nucleotides and polynucleotides were essentially identical to that shown in Fig. 2. Our work has been carried out using an instrument equipped with an emission monochromator. The use of a filter instrument with an emission cut-off of 470 nm should be sufficient in LC detection. The emission spectrum obtained for a mixture of europium(II1) chloride ( 1W4 M) and tRNAPh’ (100 pg/ml) is shown in Fig. 3a. The excitation wavelength was 345 nm. Fig. 3b shows the emission spectrum of europium(II1) chloride (10e4 A4) run under identical conditions. The Eu(II1) bands at 590 and 614 nm with tRNAPh”
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_(d
/
530
500
(b)
(bl
_
560
_j
(a)
470nm
670
640
610
560nm
Fig. 2. Emission spectrum from 470 to 560 nm for (a) a mixture of poly G (2.5 . 10m4M in Guo-S-P moiety) and terbium(III) chloride (10e4 M), and (b) terbium(II1) chloride (lO+ M). Both solutions were prepared in acetate buffer. Excitation wavelength: 290 nm. Fig. 3. Emission spectrum from 570 to 670 nm for (a) a mixture of tRNAPb” (100 pg/ml) and europium(II1) chloride (lo-“ M) and (b) europium(II1) chloride ( 10m4M). Both solutions were prepared in acetate buffer. Excitation wavelength: 345 nm.
results because of an energy transfer from a thiouridine residue. Only Eu(II1) emission is observed at wavelengths higher than 580 nm, permitting the use of a filter instrument for LC detection. Table I reports relative luminescent intensities of terbium(II1) chloride with several nucleotides and polynucleotides. These responses were measured by cuvette studies. Our results are generally consistent with previous literature reports9J2,16,23. The nucleotides transfer energy more efficiently when incorporated into polymers9,12,16. This enhancement in the polymer form is independent of whether or not TABLE I RELATIVE LUMINESCENT INTENSITY AT 545 nm FOR A SOLUTION OF TERBIUM(II1) CHLORIDE (lo-“ A4) AND NUCLEOTIDE (2.5 . lo-“ M) The homopolymers were 2.5
lo-“ M in nucleotide moieties.
Compound
Luminescent intensity
Compound
Luminescent intensity
Poly x Poly G 5’-GMP 5’-XMP 5’-dGMP
10000 510 67 67 55
Poly Poly Poly Poly
32 30 14 11
u I c A
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the series is homologous 16. Tb(II1) luminescence is greater with ribose nucleotides than with deoxyribose nucleotides i6. The enhancement of Tb(II1) luminescence by guanosine S-diphosphate (GDP) and guanosine 5’-triphosphate (GTP) is greater than that of guanosine 5’-monophosphate (GMP) at concentrations less than 100 pM16. At concentrations of GDP and GTP higher than 100 PM, the Tb(II1) luminescence is suppressed16. Only xanthine and guanine nucleotides exhibit sufficient energy transfer to permit useful LC detection. One concern in LC detection is the method of addition of the lanthanide chromophore. Both pre- and post-column modes have decided advantages. In post-column addition the chromophore will not influence retention properties. Postcolumn addition will dilute the column eluent, however, and cause a reduction in sensitivity. If a small-volume reciprocating pump is used for the post-column phase, the pulsing of the pump will increase the noise and further raise the detection limits. The viability of post-column addition of the lanthanide ions was tested by FIA using poly X and poly G. The same apparatus as shown in Fig. 1 was employed except the analytical column was removed. In one case, samples of the polynucleotide were prepared with Tb(II1). The polynucleotide-Tb(II1) solution was injected into a mobile phase containing Tb(II1) and acetate buffer. This was mixed with a “postcolumn” phase consisting only of the acetate buffer. In the second case, samples of the polynucleotide were prepared without Tb(II1) and injected into a mobile phase that contained no Tb(II1). The Tb(II1) was introduced in the “post-column” phase, and the only contact between the Tb(II1) and polynucleotide prior to detection occurred in the reaction coil. If the reaction coil provides enough time for bonding to occur, the response measured in the two experiments should be identical. The response we obtained in the two experiments were essentially the same for both poly X and poly G. Lanthanide ions can therefore be added in a post-column mode with nucleotides and nucleic acids. Pre-column introduction of the lanthanide ions is also suitable. Mixtures of the lanthanide ions and the homopolymers were found to be stable with no observable decomposition for at least 24 h. Mixtures of the lanthanide ions with nucleotides and RNA were stable with no observable decomposition for at least 2 h. The emission from Tb(II1) or Eu(II1) in these samples remained the same over these periods of time. The range of mobile phases and buffers with which our detection system is compatible are also of concern. In our previous work with aromatic aldehydes and ketones, which transfer energy to the lanthanide ions by an intermolecular process, we reported that water caused a significant quenching of the lanthanide luminescence’. This quenching could be reduced by adding a species that displaced water from the first coordination sphere of the lanthanide ion’. Nucleotides and nucleic acids transfer energy by an intramolecular process, and in bonding presumably displace some or all of the water in the first coordination sphere of the lanthanide ion. As a result, sufficient sensitivity of the lanthanide luminescence is observed when detecting nucleotides and nucleic acids in aqueous solvents. The intensity of lanthanide luminescence from a sample of poly X or poly G (2.5 . low4 A4) and terbium(II1) chloride (low4 A4) was essentially the same whether dissolved in aqueous buffer (see experimental section for description), aqueous buffer-acetonitrile (80:20),
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or aqueousbuffer-methanol (80:20). Mobile phases employing acetonitrile or methanol as organic modifiers are therefore suitable for use with lanthanide luminescence detection of nucleotides and nucleic acids. Not all buffers are compatible with our detection method. Complexes with the lanthanide ion that are insoluble may form and block the flow through the column or post-column reaction system. It is also possible that a buffer may disrupt the energy transfer process. Acetate*J4J5J7,1g, cacodylategJ-13J6J8~20-22, and Tris” buffers have been used with mixtures of nucleotides and lanthanide ions. Phosphate buffers, which are frequently employed with nucleic acids and nucleotides, form highly insoluble complexes with lanthanide ions. One means of circumventing this problem is to add a ligand that complexes with the lanthanide ion and prevents precipitation with the phosphate ion. This added ligand must bond in a way that permits the energy transfer to occur. Ethylenediaminetetraacetic acid (EDTA) was evaluated as one such ligand. Lanthanide complexes with EDTA are soluble in phosphate buffers, and it is known that the EDTA ligand does not fully encapsulate the metal ion30. As a result the nucleotide should be able to bond to the lanthanide ion. Lanthanide chelates of EDTA have been used as NMR shift reagents to assign the conformation of AMP3’. No transfer was observed, however, in solutions of S-GMP (2.5 . 10e4 M) and Tb(EDTA) (10e4 A4) at pH 6. The use of Tb(II1) as a detection chromophore was demonstrated on mixtures of the homopolymers of xanthine, guanine, adenine, cytosine, and uracil. An example is shown in Fig. 4 for the separation of a mixture of poly I and poly X by steric exclusion chromatography. The chromatogram obtained using UV detection at 254 nm is shown in Fig. 4a. Peaks are observed for both poly X and poly I. The response obtained using fluorescence detection with post-column addition of Tb(II1) is shown in Fig. 4b. The fluorescence instrument was set to an excitation wavelength of 290 nm and an emission wavelength of 545 nm. The poly I is not detected because it does not effectively transfer energy to Tb(II1). POLY
POLY
I
x
(a)
__/\
0
(b)
2
4
6
6
IO
12 min
Fig. 4. Chromatogram of a mixture of poly X and poly 1 with (a) UV detection at 254 mn, and (b) fluorescence detection at 545 nm with Tb(II1). Mobile phase: sodium chloride (0.1 A4)and sodium acetate (0.01 M) adjusted to pH 6,0.5 ml/min. Post-column phase: sodium chloride (0.1 M), sodium acetate (0.01 M), and terbium(II1) chloride (10m4M) adjusted to pH 6, 0.5 ml/min. Column: Synchropak GPC-500. Concentration of poly X and poly I based on nucleotide moieties: 2.5 . lo-4 hf.
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The Tb(II1) could not be added directly to the mobile phase when chromatographing the homopolymers. No peak was observed for poly X when Tb(II1) was added to the mobile phase. Apparently the Tb(II1) complexes with poly X in such a way as to cause irreversible adsorption on the column or an excessively long retention time. The selectivity of our detection method allows one to simplify liquid chromatograms. Using Tb(III), poly X or poly G may be detected in the presence of the other homopolymers. Detection limits of approximately 7 ng for poly X and 81 ng for poly G were obtained by our method using FIA and a 20-,nl injection volume. The ability of ribonucleic acids to transfer energy to the lanthanide ions has been demonstrateda*’ 6-Zl. Energy transfer will occur if the single-stranded portions of RNA contain bases capable of transferring energy. tRNA from Escherichia coli have been particularly well studied. The compounds tRNAPh”, tRNAWe’, tRNAG’“, and tRNA”” from E. coli are capable of transferring energy to Eu(III)‘~-~~ or Tb(III)17,1g through a thiouridine residue (excitation = 345 nm). This is usually a 4-thiouridine residue; however, in the case of tRNAG’“, the transfer occurs through a modified 2-thiouridine residue”. Yeast tRNAPh’, which does not have a thiouridine residue, will not transfer energy to Eu(II1) or Tb(II1) if an excitation wavelength of 345 nm is used17J8. An alternative is to excite the tRNA at 290 nm, which corresponds to absorption by guanine residues. Emission spectra of tRNAfM” (180 pg/ml) with either Eu(II1) ( lO-4 i%f)or Tb(II1) (IO” M) are shown in Fig. 5. In the spectra shown in Fig. 5a and b, the excitation wavelength was set at 345 nm. Emission from Eu(II1) and Tb(II1) are both observed and either ion can be used as an LC detection chromophore. The emission from Tb(II1) at 543 nm is more intense than that from Eu(III) at 590 or 615 nm. In the spectra shown in Fig. 5c and d, the excitation wavelength was 290 nm. Both Tb(II1) and Eu(II1) emission is observed, however, the emission from Tb(II1) is significantly larger than that from Eu(II1). The instrumental conditions are identical for all four spectra. A comparison of the emission intensities indicates that, in this example, detection based on transfer from 4-thiouridine is preferable to that from guanine. Transfer from guanine residues can be used to detect yeast tRNAPh” (ref. 17), yeast RNA18, and ribosomal RNA16s21. The applicability of our detection method for RNA was demonstrated using steric exclusion chromatography. Detection of tRNA from E. coli was successfully performed using either Tb(II1) or Eu(II1). As expected from cuvette studies, the response was larger with Tb(II1). The lanthanide ion was added to the mobile phase at a concentration of lop4 M. The retention times of tRNAG’“, tRNAr”“, and tRNAPh” were not altered by the presence of the lanthanide ion in the mobile phase. Detection limits were estimated to be 1.4 pg, 1.0 pg, and 1.6 pg for tRNAG’“, tRNAfM”‘, and tRNAPh’ respectively based on a 20-~1 injection volume. Mixtures of the tRNA compounds were not studied because of similar retention times. Since the LS-5 spectrofluorometer employed in our work uses a pulsed source., it is possible to incorporate a delay time between the lamp pulse and the start of data acquisition. In a previous study of biacetyl, which has a long excited state lifetime, it was found that the reduction in background scatter and fluorescence caused by the delay time, more than offset the reduction in signal intensity for biacety132. As a result, the signal-to-noise ratio was better in the phosphorescence mode. Since lanthanide ions have relatively long excited state lifetimes, the influence of a delay time
306
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T. J. WENZEL, L. M. COLLETTE
630
580
550
510
470
nm
Fig. 5. Emission spectrum from 470 to 670 run for tRNAfMc’ (180 pg/ml) in acetate buffer with (a) terbium(II1) chloride (10m4M), excitation wavelength, 345 nm; (b) europium(II1) chloride (lo-4 M), excitation wavelength, 345 nm; (c) terbium(II1) chloride (lo* M), excitation wavelength, 290 nm; (d) europium(II1) chloride (lo* M), excitation wavelength, 290 nm. The large peak at 580 nm in (c) and (d) is an artifact due to the second order band of the excitation grating.
on the sensitivity and detection limits for polynucleotides and nucleic acids was investigated. For samples of poly X and poly G with Tb(III), and tRNA with Tb(II1) and Eu(III), better sensitivity and detection limits were achieved in the phosphorescence mode. A range of delay and gate times were evaluated in an attempt to optimize the sensitivity. At a gate time of 1 ms, the signal-to-noise ratio remained almost constant for delay times from 10 to 140 ps. For delay times longer than 140 ,US,the signalto-noise ratio decreased. At a delay time of 30 ,US,the signal-to-noise ratio was comparable at gate times of 0.5 and 1 ms, and decreased at gates times greater than 1 ms. A delay time of 30 ps and gate time of 1 ms were selected for further measurements. At these settings the sensitivity and detection limits for poly X with Tb(II1) were improved by a factor of three, and poly G with Tb(II1) by a factor of two. A four-fold improvement in the sensitivity and detection limit was observed for tRNAfMe’ from E. coli with Tb(II1). A two-fold improvement was observed with Eu(II1). With the tRNA samples the excitation wavelength was set to that of the 4thiouridine residue. These improvements are considerably less than the 50-fold enhancement observed for biacetyl 32. It is possible that lower concentrations of fluorescent impurities, or less emission at the wavelengths of Tb(II1) and Eu(III), were present with our mobile phases. In the previous work, the sensitizing donor did exhibit a slight fluorescence at the emission wavelength of biacetyl (516 nm)j2.
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CONCLUSIONS
Sensitized lanthanide luminescence is a viable detection method for any single-stranded nucleic acid or polynucleotide containing guanine, thiouridine, or xanthine residues. It has been applied to homopolymers such as poly X and poly G, and transfer RNA from E. coli. It should be applicable as an LC detection method to single-stranded DNA and almost all types of RNA. The lanthanide ion can be added in a post-column mode. Improvements in sensitivity and detection limits are achieved if the fluorescence spectrofluorometer has a pulsed source and is used in the phosphorescence mode. ACKNOWLEDGEMENTS
We wish to thank Research Corporation, through a Leo H. Baekeland Grant, and the National Science Foundation (Biological Instrumentation Program, spectrofluorometer; College Science Instrumentation Program, liquid chromatograph) for supporting this work. REFERENCES 1 E. E. DiBella, J. B. Weissman, M. J. Joseph, J R. Schultz and T. J. Wenzel, J. Chromatogr., 328 (1985) 101. 2 J. J. Donkerbroek, C. Gooijer, N. H. Velthorst and R. W. Frei, Anal. Chem., 54 (1982) 891. 3 S. I. Weissman, J. Chem. Phys., 10 (1942) 214. 4 R. E. Whan and G. A. Crosby, J. Mol. Spectrosc., 8 (1962) 315. 5 G. A. Crosby, R. E. Whan and J. J. Freeman, J. Phys. Chem., 66 (1962) 2493. 6 A. P. B. Sinha, in C. N. R. Rao and J. R. Ferraro (Editors), Spectroscopy in Inorganic Chemistry,Vol. II, Academic Press, New York, 1971, p. 255. 7 A. A. Lamola and J. Eisinger, Biochim. Biophw. Acta, 240 (1971) 313. 8 C. Formoso, Biochem. Biophys. Res. Commun.. 53 (1973) 1084. 9 D. S. Gross and H. Simpkins, J. Biol. Chem., 256 (1981) 9593. 10 G. Yonuschot, G. Robey, G. W. Mushrush, D. Helman and G. van de Woude, Bioinorg. Chem., 8 (1978) 397. 11 D. P. Ringer, B. A. Howell and D. E. Kizer, Anal. Biochem., 103 (1980) 337. 12 M. D. Topal and J. R. Fresco, Biochemistry, 19 (1980) 5531. 13 T. Haertle, J. Augustyniak and W. Guschlbauer, Nucleic. Acids Res., 9 (1981) 6191. 14 G. Yonuschot and G. W. Mushrush, Biochemistry, 14 (1975) 1677. 15 C. Formoso, Photochem. Photobiol., 26 (1977) 159. 16 D. P. Ringer, S. Burchett and D E. Kizer, Biochemistry, 17 (1978) 4818. 17 M. S. Kayne and M. Cohn, Biochemistry, 13 (1974) 4159. 18 J. M. Wolfson and D. R. Keams, Biochemistry, 14 (1975) 1436. 19 D. Pavlick and C. Formoso, Biochemistry, 17 (1978) 1537. 20 J. M. Wolfson and D. R. Keams, J. Am. Chem. Sot., 96 (1974) 3653. 21 T. D. Barela, S. Burchett and D. E. Kizer, Biochemistry, 14 (1975) 4887. 22 D. P. Ringer, J. L. Etheredge and D. E. Kizer, J. Inorg. Biochem., 24 (1985) 137. 23 G. Yonuschot, D. Helman, G. Mushrush, G. van de Woude and G. Robey, Bioinorg. Chem., 8 (1978) 405. 24 A. Heller and E. Wasserman, J. Chem. Phys., 42 (1965) 949. 25 M. L. Bhaumik and M. A. El-Sayed, J. Phys. Chem., 69 (1965) 275. 26 W. J. McCarthy and J. D. Winefordner, Anal. Chem., 38 (1966) 848. 27 N. Filipescu and G. W. Mushrush, J. Phys. Chem., 72 (1968) 3516. 28 J. Eisinger and A. A. Lamola, Biochim. Biophys. Acta, 240 (1971) 299. 29 L. M. Hirschy, E. V. Dose and J. D. Winefordner, Anal. Chim. Acta, 147 (1983) 311. 30 M. D. Lind, B. Lee and J. L. Hoard, J. Am. Chem. Sot., 87 (1965) 1611. 31 P. Tanswell, E. W. Westhead and R. J. P. Williams, Biochem. Sot. Trans., 2 (1974) 79. 32 R. A. Baumann, C. Gooijer, N. H. Velthorst and R. W. Frei, Anal. Chem., 57 (1985) 1815.
436 (1988) 299-307
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. 20 147
LANTHANIDE IONS AS LUMINESCENT CHROMOPHORES FOR THE LIQUID CHROMATOGRAPHIC DETECTION OF POLYNUCLEOTIDES AND NUCLEIC ACIDS
THOMAS J. WENZEL* and LISA M. COLLETTE Department
of Chemistry,
Bates College, Lewiston,
ME 04240 (U.S.A.)
(First received June 22nd, 1987; revised manuscript received October 16th, 1987)
SUMMARY
Europium(II1) and terbium(II1) can be used as luminescent chromophores for the liquid chromatographic detection of certain nucleotides and nucleic acids. The method is dependent upon an energy transfer from the nucleic acid to the lanthanide ion. Of the base moieties, only xanthine, guanine, and thiouridine have appropriate excited state energy levels for efficient energy transfer. The lanthanide ion can be added in a pre- or post-column mode. The applicability of the method was demonstrated for the detection of homologous polynucleotides such as poly X and poly G. The method was also used to detect transfer RNA from Escherichia coli.
INTRODUCTION
We have reported the use of lanthanide ions as luminescent chromophores for the liquid chromatographic (LC) detection of aromatic aldehydes and ketones’. The lanthanide detection scheme is based on the principle of sensitized luminescenceZ. An organic compound is first excited by light. If an appropriate match between the energy of the triplet state of the organic and an excited state of a lanthanide ion exists, a transfer of energy to the lanthanide ion can occur. The lanthanide ion can then emit visible luminescence that is detected3-6. The two best lanthanide ions for this method, based on energy level considerations and luminescence quantum yields, are terbium(II1) and europium(II1). Tb(II1) emits a green luminescence at 545 nm, whereas Eu(II1) emits a red luminescence at 613 nm. We will now describe the use of Tb(II1) and Eu(II1) as luminescent chromophores for the LC detection of certain nucleotides and nucleic acids. A number of previous reports have discussed aspects of the energy transfer from nucleotides7-9 and nucleic acids10-23 to lanthanide ions. All of the common bases of nucleic acids can transfer energy to Tb(III)8,11,16J3. Th e efficiency of this energy transfer is largest, however, with guanine*,’ 1,16,23,xanthine9*16, and thiouridine17-2 l. Modification of guanine residues significantly descreased Tb(II1) luminescence9~16. Incorporation of the nucleotides into polymer chains increased the efficiency of the energy transfer9v1 2. Nucleic acids must be single-stranded to observe energy transfer9v1 1,12,22,24. The 0021-9673/88/%03.50
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absence of lanthanide luminescence with double-stranded nucleic acids is attributed to an alteration of the energy of the triplet levels of the bases9. Unlike aromatic aldehydes and ketones, which transfer energy to the lanthanide ions by an intermolecular process 24-2*, the nucleotides and nucleic acids transfer energy by an intramolecular process 8. Most previous work has concluded that bonding of the lanthanide ion occurs at the phosphate group, however, some evidence for a degree of bonding at the basesa or sugar moiety13 has been reported. As our work will demonstrate, the lanthanide ions can be added to the LC system in either a preor post-column mode. The residence time in a post-column reaction coil is suflicient for bonding to occur. Detection based on lanthanide luminescence is suitable for polynucleotides or single-stranded nucleic acids containing xanthine, guanine, or thiouridine residues. Hydrolysis of the nucleotides and nucleic acids in the presence of lanthanide ions is slow enough so that it does not cause problems in LC detection. If a fluorescence instrument with a pulsed source is employed, improvements in the sensitivity and detection limits can be achieved by incorporating a delay time between the lamp pulse and the acquisition of data. Such an improvement in sensitivity is possible because of the long excited state lifetimes of Tb(II1) and Eu(II1). EXPERIMENTAL
Reagents
All reagents were used as received without further purification. The nucleotides, polynucleotides, and nucleic acids were purchased from Sigma (St. Louis, MO, U.S.A.). HPLC-grade water was obtained from Fisher (Fairlawn, NJ, U.S.A.). The chloride salts of Tb(II1) and Eu(II1) were prepared as previously described’ using lanthanide oxides (Alfa Products, Danvers, MA, U.S.A.) and Ultrex hydrochloric acid(J. T. Baker, Phillipsburg, NJ, U.S.A.). Sodium chloride and sodium acetate were obtained from J. T. Baker. Apparatus
A diagram of the apparatus employed for obtaining liquid chromatograms is shown in Fig. 1. Beckman Model 110B pumps were used for introducing the mobile and post-column phases. The mixing tee was stainless-steel and a 25 ft. x 0.01 in. I.D. stainless-steel reaction coil was placed between the mixing tee and the spectrofluorometer. The flow-rate of the LC phase and post-column phase were set at 0.5 ml/min. A steric exclusion column (Synchropak GPC-500; Alltech, Deerfield, IL, U.S.A.) was used for separating the homopolymers and transfer RNA (tRNA). Samples were introduced from a 20-4 injection loop.
r--@J Q-l PUMP
PUMP
REACTION COIL
&I
MOBILE
PHASE
FLUORESCENCE DETECTOR
LANTHANIDE PHASE
Fig. 1. System set-up for post-column addition of the lanthanide ions.
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A Perkin-Elmer LS-5 spectrofluorometer was used for all fluorescence measurements. The LS-5 can be used in the cuvette mode or an optional flow-cell for LC detection can be employed. Excitation and emission slit widths were set to 5 nm for cuvette work and 10 nm for LC detection. The response time was typically adjusted to setting 1 on the instrument. The apparatus for flow-injection analyses (FIA) was identical to that shown in Fig. 1 except the analytical column was removed. For phosphorescence measurements, which utilize a delay time between the lamp pulse and the start of data acquisition, the instrument was set to the phosphorescence mode and appropriate delay and gate times were set. Unless otherwise noted, all measurements were performed in the fluorescence mode. Procedures
All solutions of the nucleotides, nucleic acids, and lanthanide salts were prepared in an acetate buffer that had been adjusted to pH 6 using Ultrex hydrochloric acid. The acetate buffer consisted of a solution of sodium chloride (0.1 M) and sodium acetate (0.01 M) in HPLC-grade doubly distilled water. Unless stated otherwise, all solutions of the nucleotides and nucleic acids were prepared the day of use. Solutions containing nucleotides or nucleic acids were prepared by weighing the compound into a volumetric flask and dissolving it in acetate buffer or acetate buffer containing the lanthanide ion. The concentration of lanthanide ion was lop4 A4 in cuvette studies. In LC applications, the concentration of the lanthanide ion in the post-column phase was 10e4 M and therefore 5 . lO+ M at the detector. The concentration of lanthanide ion was selected after consideration of previous findingsl’j. No steps were taken to remove dissolved oxygen from mobile and post-column phases. It has been reported that the intensity of lanthanide luminescence based on intramolecular transfer processes is not significantly influenced by dissolved oxygen29. RESULTS AND DISCUSSION
The range of nucleotides and nucleic acids for which lanthanide luminescence represented a viable LC detection method were evaluated by cuvette and FIA studies. A typical emission spectrum is shown in Fig. 2a for a mixture of poly G (2.5 . 1O-4 M in Guo-5’-P moieties) and terbium(II1) chloride (low4 M). The excitation wavelength was 290 nm. The bands in the emission spectrum at 489 and 544 nm are characteristic of emission from Tb(II1). The band at 544 nm is usually the most intense for Tb(II1). Fig. 2b is the emission spectrum of a solution of terbium(II1) chloride without poly G. All conditions were identical to those of the spectrum shown in Fig. 2a. The Tb(II1) bands seen in the spectrum in Fig. 2a are therefore the result of energy transfer from guanine. The emission spectra of mixtures of Tb(II1) with other nucleotides and polynucleotides were essentially identical to that shown in Fig. 2. Our work has been carried out using an instrument equipped with an emission monochromator. The use of a filter instrument with an emission cut-off of 470 nm should be sufficient in LC detection. The emission spectrum obtained for a mixture of europium(II1) chloride ( 1W4 M) and tRNAPh’ (100 pg/ml) is shown in Fig. 3a. The excitation wavelength was 345 nm. Fig. 3b shows the emission spectrum of europium(II1) chloride (10e4 A4) run under identical conditions. The Eu(II1) bands at 590 and 614 nm with tRNAPh”
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_(d
/
530
500
(b)
(bl
_
560
_j
(a)
470nm
670
640
610
560nm
Fig. 2. Emission spectrum from 470 to 560 nm for (a) a mixture of poly G (2.5 . 10m4M in Guo-S-P moiety) and terbium(III) chloride (10e4 M), and (b) terbium(II1) chloride (lO+ M). Both solutions were prepared in acetate buffer. Excitation wavelength: 290 nm. Fig. 3. Emission spectrum from 570 to 670 nm for (a) a mixture of tRNAPb” (100 pg/ml) and europium(II1) chloride (lo-“ M) and (b) europium(II1) chloride ( 10m4M). Both solutions were prepared in acetate buffer. Excitation wavelength: 345 nm.
results because of an energy transfer from a thiouridine residue. Only Eu(II1) emission is observed at wavelengths higher than 580 nm, permitting the use of a filter instrument for LC detection. Table I reports relative luminescent intensities of terbium(II1) chloride with several nucleotides and polynucleotides. These responses were measured by cuvette studies. Our results are generally consistent with previous literature reports9J2,16,23. The nucleotides transfer energy more efficiently when incorporated into polymers9,12,16. This enhancement in the polymer form is independent of whether or not TABLE I RELATIVE LUMINESCENT INTENSITY AT 545 nm FOR A SOLUTION OF TERBIUM(II1) CHLORIDE (lo-“ A4) AND NUCLEOTIDE (2.5 . lo-“ M) The homopolymers were 2.5
lo-“ M in nucleotide moieties.
Compound
Luminescent intensity
Compound
Luminescent intensity
Poly x Poly G 5’-GMP 5’-XMP 5’-dGMP
10000 510 67 67 55
Poly Poly Poly Poly
32 30 14 11
u I c A
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the series is homologous 16. Tb(II1) luminescence is greater with ribose nucleotides than with deoxyribose nucleotides i6. The enhancement of Tb(II1) luminescence by guanosine S-diphosphate (GDP) and guanosine 5’-triphosphate (GTP) is greater than that of guanosine 5’-monophosphate (GMP) at concentrations less than 100 pM16. At concentrations of GDP and GTP higher than 100 PM, the Tb(II1) luminescence is suppressed16. Only xanthine and guanine nucleotides exhibit sufficient energy transfer to permit useful LC detection. One concern in LC detection is the method of addition of the lanthanide chromophore. Both pre- and post-column modes have decided advantages. In post-column addition the chromophore will not influence retention properties. Postcolumn addition will dilute the column eluent, however, and cause a reduction in sensitivity. If a small-volume reciprocating pump is used for the post-column phase, the pulsing of the pump will increase the noise and further raise the detection limits. The viability of post-column addition of the lanthanide ions was tested by FIA using poly X and poly G. The same apparatus as shown in Fig. 1 was employed except the analytical column was removed. In one case, samples of the polynucleotide were prepared with Tb(II1). The polynucleotide-Tb(II1) solution was injected into a mobile phase containing Tb(II1) and acetate buffer. This was mixed with a “postcolumn” phase consisting only of the acetate buffer. In the second case, samples of the polynucleotide were prepared without Tb(II1) and injected into a mobile phase that contained no Tb(II1). The Tb(II1) was introduced in the “post-column” phase, and the only contact between the Tb(II1) and polynucleotide prior to detection occurred in the reaction coil. If the reaction coil provides enough time for bonding to occur, the response measured in the two experiments should be identical. The response we obtained in the two experiments were essentially the same for both poly X and poly G. Lanthanide ions can therefore be added in a post-column mode with nucleotides and nucleic acids. Pre-column introduction of the lanthanide ions is also suitable. Mixtures of the lanthanide ions and the homopolymers were found to be stable with no observable decomposition for at least 24 h. Mixtures of the lanthanide ions with nucleotides and RNA were stable with no observable decomposition for at least 2 h. The emission from Tb(II1) or Eu(II1) in these samples remained the same over these periods of time. The range of mobile phases and buffers with which our detection system is compatible are also of concern. In our previous work with aromatic aldehydes and ketones, which transfer energy to the lanthanide ions by an intermolecular process, we reported that water caused a significant quenching of the lanthanide luminescence’. This quenching could be reduced by adding a species that displaced water from the first coordination sphere of the lanthanide ion’. Nucleotides and nucleic acids transfer energy by an intramolecular process, and in bonding presumably displace some or all of the water in the first coordination sphere of the lanthanide ion. As a result, sufficient sensitivity of the lanthanide luminescence is observed when detecting nucleotides and nucleic acids in aqueous solvents. The intensity of lanthanide luminescence from a sample of poly X or poly G (2.5 . low4 A4) and terbium(II1) chloride (low4 A4) was essentially the same whether dissolved in aqueous buffer (see experimental section for description), aqueous buffer-acetonitrile (80:20),
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or aqueousbuffer-methanol (80:20). Mobile phases employing acetonitrile or methanol as organic modifiers are therefore suitable for use with lanthanide luminescence detection of nucleotides and nucleic acids. Not all buffers are compatible with our detection method. Complexes with the lanthanide ion that are insoluble may form and block the flow through the column or post-column reaction system. It is also possible that a buffer may disrupt the energy transfer process. Acetate*J4J5J7,1g, cacodylategJ-13J6J8~20-22, and Tris” buffers have been used with mixtures of nucleotides and lanthanide ions. Phosphate buffers, which are frequently employed with nucleic acids and nucleotides, form highly insoluble complexes with lanthanide ions. One means of circumventing this problem is to add a ligand that complexes with the lanthanide ion and prevents precipitation with the phosphate ion. This added ligand must bond in a way that permits the energy transfer to occur. Ethylenediaminetetraacetic acid (EDTA) was evaluated as one such ligand. Lanthanide complexes with EDTA are soluble in phosphate buffers, and it is known that the EDTA ligand does not fully encapsulate the metal ion30. As a result the nucleotide should be able to bond to the lanthanide ion. Lanthanide chelates of EDTA have been used as NMR shift reagents to assign the conformation of AMP3’. No transfer was observed, however, in solutions of S-GMP (2.5 . 10e4 M) and Tb(EDTA) (10e4 A4) at pH 6. The use of Tb(II1) as a detection chromophore was demonstrated on mixtures of the homopolymers of xanthine, guanine, adenine, cytosine, and uracil. An example is shown in Fig. 4 for the separation of a mixture of poly I and poly X by steric exclusion chromatography. The chromatogram obtained using UV detection at 254 nm is shown in Fig. 4a. Peaks are observed for both poly X and poly I. The response obtained using fluorescence detection with post-column addition of Tb(II1) is shown in Fig. 4b. The fluorescence instrument was set to an excitation wavelength of 290 nm and an emission wavelength of 545 nm. The poly I is not detected because it does not effectively transfer energy to Tb(II1). POLY
POLY
I
x
(a)
__/\
0
(b)
2
4
6
6
IO
12 min
Fig. 4. Chromatogram of a mixture of poly X and poly 1 with (a) UV detection at 254 mn, and (b) fluorescence detection at 545 nm with Tb(II1). Mobile phase: sodium chloride (0.1 A4)and sodium acetate (0.01 M) adjusted to pH 6,0.5 ml/min. Post-column phase: sodium chloride (0.1 M), sodium acetate (0.01 M), and terbium(II1) chloride (10m4M) adjusted to pH 6, 0.5 ml/min. Column: Synchropak GPC-500. Concentration of poly X and poly I based on nucleotide moieties: 2.5 . lo-4 hf.
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The Tb(II1) could not be added directly to the mobile phase when chromatographing the homopolymers. No peak was observed for poly X when Tb(II1) was added to the mobile phase. Apparently the Tb(II1) complexes with poly X in such a way as to cause irreversible adsorption on the column or an excessively long retention time. The selectivity of our detection method allows one to simplify liquid chromatograms. Using Tb(III), poly X or poly G may be detected in the presence of the other homopolymers. Detection limits of approximately 7 ng for poly X and 81 ng for poly G were obtained by our method using FIA and a 20-,nl injection volume. The ability of ribonucleic acids to transfer energy to the lanthanide ions has been demonstrateda*’ 6-Zl. Energy transfer will occur if the single-stranded portions of RNA contain bases capable of transferring energy. tRNA from Escherichia coli have been particularly well studied. The compounds tRNAPh”, tRNAWe’, tRNAG’“, and tRNA”” from E. coli are capable of transferring energy to Eu(III)‘~-~~ or Tb(III)17,1g through a thiouridine residue (excitation = 345 nm). This is usually a 4-thiouridine residue; however, in the case of tRNAG’“, the transfer occurs through a modified 2-thiouridine residue”. Yeast tRNAPh’, which does not have a thiouridine residue, will not transfer energy to Eu(II1) or Tb(II1) if an excitation wavelength of 345 nm is used17J8. An alternative is to excite the tRNA at 290 nm, which corresponds to absorption by guanine residues. Emission spectra of tRNAfM” (180 pg/ml) with either Eu(II1) ( lO-4 i%f)or Tb(II1) (IO” M) are shown in Fig. 5. In the spectra shown in Fig. 5a and b, the excitation wavelength was set at 345 nm. Emission from Eu(II1) and Tb(II1) are both observed and either ion can be used as an LC detection chromophore. The emission from Tb(II1) at 543 nm is more intense than that from Eu(III) at 590 or 615 nm. In the spectra shown in Fig. 5c and d, the excitation wavelength was 290 nm. Both Tb(II1) and Eu(II1) emission is observed, however, the emission from Tb(II1) is significantly larger than that from Eu(II1). The instrumental conditions are identical for all four spectra. A comparison of the emission intensities indicates that, in this example, detection based on transfer from 4-thiouridine is preferable to that from guanine. Transfer from guanine residues can be used to detect yeast tRNAPh” (ref. 17), yeast RNA18, and ribosomal RNA16s21. The applicability of our detection method for RNA was demonstrated using steric exclusion chromatography. Detection of tRNA from E. coli was successfully performed using either Tb(II1) or Eu(II1). As expected from cuvette studies, the response was larger with Tb(II1). The lanthanide ion was added to the mobile phase at a concentration of lop4 M. The retention times of tRNAG’“, tRNAr”“, and tRNAPh” were not altered by the presence of the lanthanide ion in the mobile phase. Detection limits were estimated to be 1.4 pg, 1.0 pg, and 1.6 pg for tRNAG’“, tRNAfM”‘, and tRNAPh’ respectively based on a 20-~1 injection volume. Mixtures of the tRNA compounds were not studied because of similar retention times. Since the LS-5 spectrofluorometer employed in our work uses a pulsed source., it is possible to incorporate a delay time between the lamp pulse and the start of data acquisition. In a previous study of biacetyl, which has a long excited state lifetime, it was found that the reduction in background scatter and fluorescence caused by the delay time, more than offset the reduction in signal intensity for biacety132. As a result, the signal-to-noise ratio was better in the phosphorescence mode. Since lanthanide ions have relatively long excited state lifetimes, the influence of a delay time
306
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T. J. WENZEL, L. M. COLLETTE
630
580
550
510
470
nm
Fig. 5. Emission spectrum from 470 to 670 run for tRNAfMc’ (180 pg/ml) in acetate buffer with (a) terbium(II1) chloride (10m4M), excitation wavelength, 345 nm; (b) europium(II1) chloride (lo-4 M), excitation wavelength, 345 nm; (c) terbium(II1) chloride (lo* M), excitation wavelength, 290 nm; (d) europium(II1) chloride (lo* M), excitation wavelength, 290 nm. The large peak at 580 nm in (c) and (d) is an artifact due to the second order band of the excitation grating.
on the sensitivity and detection limits for polynucleotides and nucleic acids was investigated. For samples of poly X and poly G with Tb(III), and tRNA with Tb(II1) and Eu(III), better sensitivity and detection limits were achieved in the phosphorescence mode. A range of delay and gate times were evaluated in an attempt to optimize the sensitivity. At a gate time of 1 ms, the signal-to-noise ratio remained almost constant for delay times from 10 to 140 ps. For delay times longer than 140 ,US,the signalto-noise ratio decreased. At a delay time of 30 ,US,the signal-to-noise ratio was comparable at gate times of 0.5 and 1 ms, and decreased at gates times greater than 1 ms. A delay time of 30 ps and gate time of 1 ms were selected for further measurements. At these settings the sensitivity and detection limits for poly X with Tb(II1) were improved by a factor of three, and poly G with Tb(II1) by a factor of two. A four-fold improvement in the sensitivity and detection limit was observed for tRNAfMe’ from E. coli with Tb(II1). A two-fold improvement was observed with Eu(II1). With the tRNA samples the excitation wavelength was set to that of the 4thiouridine residue. These improvements are considerably less than the 50-fold enhancement observed for biacetyl 32. It is possible that lower concentrations of fluorescent impurities, or less emission at the wavelengths of Tb(II1) and Eu(III), were present with our mobile phases. In the previous work, the sensitizing donor did exhibit a slight fluorescence at the emission wavelength of biacetyl (516 nm)j2.
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CONCLUSIONS
Sensitized lanthanide luminescence is a viable detection method for any single-stranded nucleic acid or polynucleotide containing guanine, thiouridine, or xanthine residues. It has been applied to homopolymers such as poly X and poly G, and transfer RNA from E. coli. It should be applicable as an LC detection method to single-stranded DNA and almost all types of RNA. The lanthanide ion can be added in a post-column mode. Improvements in sensitivity and detection limits are achieved if the fluorescence spectrofluorometer has a pulsed source and is used in the phosphorescence mode. ACKNOWLEDGEMENTS
We wish to thank Research Corporation, through a Leo H. Baekeland Grant, and the National Science Foundation (Biological Instrumentation Program, spectrofluorometer; College Science Instrumentation Program, liquid chromatograph) for supporting this work. REFERENCES 1 E. E. DiBella, J. B. Weissman, M. J. Joseph, J R. Schultz and T. J. Wenzel, J. Chromatogr., 328 (1985) 101. 2 J. J. Donkerbroek, C. Gooijer, N. H. Velthorst and R. W. Frei, Anal. Chem., 54 (1982) 891. 3 S. I. Weissman, J. Chem. Phys., 10 (1942) 214. 4 R. E. Whan and G. A. Crosby, J. Mol. Spectrosc., 8 (1962) 315. 5 G. A. Crosby, R. E. Whan and J. J. Freeman, J. Phys. Chem., 66 (1962) 2493. 6 A. P. B. Sinha, in C. N. R. Rao and J. R. Ferraro (Editors), Spectroscopy in Inorganic Chemistry,Vol. II, Academic Press, New York, 1971, p. 255. 7 A. A. Lamola and J. Eisinger, Biochim. Biophw. Acta, 240 (1971) 313. 8 C. Formoso, Biochem. Biophys. Res. Commun.. 53 (1973) 1084. 9 D. S. Gross and H. Simpkins, J. Biol. Chem., 256 (1981) 9593. 10 G. Yonuschot, G. Robey, G. W. Mushrush, D. Helman and G. van de Woude, Bioinorg. Chem., 8 (1978) 397. 11 D. P. Ringer, B. A. Howell and D. E. Kizer, Anal. Biochem., 103 (1980) 337. 12 M. D. Topal and J. R. Fresco, Biochemistry, 19 (1980) 5531. 13 T. Haertle, J. Augustyniak and W. Guschlbauer, Nucleic. Acids Res., 9 (1981) 6191. 14 G. Yonuschot and G. W. Mushrush, Biochemistry, 14 (1975) 1677. 15 C. Formoso, Photochem. Photobiol., 26 (1977) 159. 16 D. P. Ringer, S. Burchett and D E. Kizer, Biochemistry, 17 (1978) 4818. 17 M. S. Kayne and M. Cohn, Biochemistry, 13 (1974) 4159. 18 J. M. Wolfson and D. R. Keams, Biochemistry, 14 (1975) 1436. 19 D. Pavlick and C. Formoso, Biochemistry, 17 (1978) 1537. 20 J. M. Wolfson and D. R. Keams, J. Am. Chem. Sot., 96 (1974) 3653. 21 T. D. Barela, S. Burchett and D. E. Kizer, Biochemistry, 14 (1975) 4887. 22 D. P. Ringer, J. L. Etheredge and D. E. Kizer, J. Inorg. Biochem., 24 (1985) 137. 23 G. Yonuschot, D. Helman, G. Mushrush, G. van de Woude and G. Robey, Bioinorg. Chem., 8 (1978) 405. 24 A. Heller and E. Wasserman, J. Chem. Phys., 42 (1965) 949. 25 M. L. Bhaumik and M. A. El-Sayed, J. Phys. Chem., 69 (1965) 275. 26 W. J. McCarthy and J. D. Winefordner, Anal. Chem., 38 (1966) 848. 27 N. Filipescu and G. W. Mushrush, J. Phys. Chem., 72 (1968) 3516. 28 J. Eisinger and A. A. Lamola, Biochim. Biophys. Acta, 240 (1971) 299. 29 L. M. Hirschy, E. V. Dose and J. D. Winefordner, Anal. Chim. Acta, 147 (1983) 311. 30 M. D. Lind, B. Lee and J. L. Hoard, J. Am. Chem. Sot., 87 (1965) 1611. 31 P. Tanswell, E. W. Westhead and R. J. P. Williams, Biochem. Sot. Trans., 2 (1974) 79. 32 R. A. Baumann, C. Gooijer, N. H. Velthorst and R. W. Frei, Anal. Chem., 57 (1985) 1815.