Jun 12, 1978 - consequently brings the pKa values close to that of lin-benzo- adenosine. Fluorescence spectroscopy was used to investigate the interaction of ...
Proc. Natl. Acad. Sci. USA Vol. 75, No. 9, pp. 4204-4208, September 1978
Biochemistry
Spectroscopic sensitivity of linear-benzoadenine nucleotides to divalent metal counterions, side chain conformations, micelles, and enzymes (adenine binding sites/dimensional probes/fluorescence/phosphate-adenine interaction/pKa)
PIETER VANDERLIJN, JORGE R. BARRIO, AND NELSON J. LEONARD* Roger Adams Laboratory, School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801
Contribuied by N. J. Leonard, June 12, 1978
ABSTRACT From pKa data for lin-benzoadenosine 5'mono-, 5'-di-, and 5'-triphosphates, which are fluorescent "stretched-out" analogues of adenine nucleotides, it was possible to designate the cases of interaction of phosphate with the heteroaromatic moiety. The addition of divalent metal cations or quaternary ammonium micelles diminishes the direct intramolecular interaction between the phosphate(s) and base and consequently brings the pKa values close to that of lin-benzoadenosine. Fluorescence spectroscopy was used to investigate the interaction of Iin-benzoadenine nucleotides with Mg2+, Mn2+, and Co2+. The association constants for the formation of such complexes were obtained from measurements of steady-state fluorescence quenching. Phase and modulation measurements of the fluorescence lifetimes of lin-benzoadenine nucleotides as a function of Co2+ concentration permitted determination of the static component of the quenching due to intramolecular complex formation. The association constants of the lin-benzoadenine nucleotides with all of the divalent metal ions studied were greater than those observed for the corresponding adenine nucleotides and were in the order: linbenzo-ATP > lin-benzo-ADP > lin-benzo-AMP. Fourier transform IH NMR of lin-benzo-ATP in the presence of Co2+ showed broadening of the aromatic proton signals, the 2-H signal (corresponding to the 8-H in ATP) being the most affected. Models are proposed to'explain the phosphate-base interaction, the influence of metal ions on base protonation, and the intramothe intramolecular quenching observed in the complexes due to paramagnetic ion (Co2+, Mn2+) and base interaction.
ethylpiperazine-N'-2-ethanesulfonic acid (Hepes), Tris base, Tris-HCl, and the sodium salts of AMP, ADP, ATP, phosphoenolpyruvic acid, and pyruvic acid were purchased from Sigma Chemical Company; cetyltrimethylammonium bromide (CetNMe3Br) was purchased from Aldrich Chemical Company. Cetyltrimethylammonium chloride (CetNMe3Cl) was obtained from the bromide salt by ion exchange with Dowex 1-X8, C1form, followed by decolorization with charcoal and recrystallization from ethanol. 3-Cyclohexyl-lin-benzoadenine (1 f), lin-benzoadenosine, and derivatives were prepared as reported from this laboratory (1, 2, 6, 7). NH2 R: a, P-D-ribofuranosyl; b, 3-D-ribo81 furanosyl 5'-monophosphate; c, ,-Dribofuranosyl 5'-diphosphate; d,
1
P-D-ribofuranosyl 5'-triphosphate; e, P-D-ribofuranosyl 3',5'-cyclic monophosphate; f, cyclohexyl.
Micelle ultraviolet absorbance measurements Ultraviolet absorption spectra of solutions containing 10 mM Hepes (pH 6.5) and 10 mM CetNMe3Br, plus 1 M NaBr where noted, were obtained on a Beckman Acta MVI spectrophotometer from 380 to 240 nm to ensure a good baseline. Then, 2.5 ,u of a 10 mM solution of lin-benzo-ATP was combined with 1 ml of micellar solution and the spectra were determined again. Fluorescence Measurements. A Spex Fluorolog was used for fluorescence measurements at an excitation wavelength of 331 nm. No absorbance changes were observed upon addition of metal ions. Identical results in measuring the decrease in fluorescence emission were obtained by monitoring the emission at 371 nm or by integration of the recorded fluorescence spectrum. Calculations were aided by the use of a PDP 8 minicomputer. An SLM model 400 polarization fluorometer with 3-74 emission filters and exciting light of 331 nm was used for both micelle and enzyme studies.
The fluorescent lin-benzoadeninet nucleotides (1 b-e), defined by the formal insertion of a benzene ring (actually four carbons) into the center of the purine ring system, serve as dimensional probes to test the size restriction of enzyme-active sites specific for purine cofactors (1-4). The degree to which the biological activities of the lin-benzoadenine nucleotides approximate those of the corresponding adenine nucleotides makes it highly desirable and potentially useful to determine their environmental sensitivity. Within the physiological pH range optimal for most cellular enzymes, conformational changes in the nucleotide side chain and complex formation with metal ions (5) may affect both chemical and biological functions, including, inter alia, enzyme binding and phosphate and pyrophosphate reactivity. We have based our studies on acid-base, metal ion, and fluorometric titrations and on ultraviolet, fluorescence, and nuclear magnetic resonance spectroscopy.
Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; CetNMe3, cetyltrimethylammonium. * To whom reprint requests should be addressed. t The prefix lin refers to the linear disposition of the three rings in the "stretched-out" (by 2.4 A) version of the adenine nucleus; "benzo" in the trivial name refers to the additional ring which, only when central, contains no nitrogen. This terminology is adaptable to derivatives similarly related to adenosine (lin-benzoadenosine), adenylic
MATERIALS AND METHODS Chemicals and enzymes Rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40), yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), D-lyxose, N-2-hydroxy-
acid (lin-benzo-AMP), adenosine diphosphate (lin-benzo-ADP), adenosine triphosphate (lin-benzo ATP), etc. The other parts of the names follow accepted IUPAC-IUB nomenclature. The chemical name for lin-benzoadenosine is 8-amino-3-(3-D-ribofuranosyl)-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
imidazo[4,5-g]quinazoline.
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Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: VanDerLijn et al. Metal Ion Titrations. Solutions containing lin-benzoadenosine or one of its derivatives made up accurately at concentrations of less than 10 ,uM in 10 mM Hepes (pH 8.5) were titrated with metal ions. With Mn2+ and Co2+, a decrease in fluorescence was used to calculate an association constant. The data were analyzed by the Scatchard equation (8): K2+ K-P, -
[Me2+]
[1] [1
in which K is the association constant, [Me2+] is the free metal ion concentration, and i- is the concentraton of the bound form of the lin-benzoadenosine derivative divided by the total concentration of the lin-benzoadenosine derivative. The value is is obtained experimentally by dividing the decrease in fluorescence, Lf, for a particular metal ion concentration by the total decrease in fluorescence when all of the fluorescence probe is bound to the metal ion. The total decrease in fluorescence, AF, can be calculated by increasing the paramagnetic metal ion concentration until no further fluorescence decrease is observed. However, if the binding is weak and the paramagnetic metal ion affects the end point of titration by collisional quenching, the following equation can be used: 1 + 1, l1 -K AfAF [Me2+]total A
1
[21
[2
in which [Me2+]total is the total concentration of paramagnetic metal ion. This equation yields a straight line for weak binding only when [Me2+] _ [Me2+]total (9, 10). Kinetics. Kinetic runs were made in 100 mM KCI/20 mM Hepes, pH 8.0/1 mM phosphoenolpyruvate/40 ,M CoCl2 with 10 ,M lin-benzo-ADP and 10 ,uM lin-benzoadenosine or adenosine where noted. A Spex Fluorolog with a time drive was used; the excitation wavelength was 331 nm and the emission wavelength was 371 nm. The reaction was run in 2 ml of solution and was initiated by the addition of 1-6 units of pyruvate kinase. The end point was determined by allowing the reaction to go to completion. Polarization. The wavelength of the exciting light was 331 nm, and 3-74 emission and 7-54 excitation filters from Corning Glass were used. Solutions for micelle polarization studies contained 6.5 ,uM lin-benzo-ATP, 10 mM Hepes (pH 8.5), and 10 mM CetNMe3Cl, plus 1 M NaCl where noted. Measurements on 3-cyclohexyl-lin-benzoadenine (1 f) were made under the same conditions. The enzyme studies were done with 2 mg of pyruvate kinase (510 units/mg) and 0.8 mg of hexokinase (420 units/mg), each in 100,ul of commercial suspension and dialyzed against two changes of 100 mM KC1/10 mM Hepes, pH 8.0/1 mM MnCl2. After dialysis, D-lyxose was added to the hexokinase solution to make it 100 mM in D-lyxose. The resultant solution was then added to an equal volume of 0.1 mM lin-benzo-ADP in dialysis buffer and 100 mM D-lyxose. The pyruvate kinase experiment was performed in an identical manner except that lin-benzo-ATP was used instead of linbenzo-ADP and no lyxose was used. For enzyme studies, a 1mm-sq (internal dimensions) cuvette and brass adapter were used. The fluorescence polarization, P = (III - IL)/(II + Ii), was obtained by measurement of the intensity of emission in the same plane, 111, as the exciting light and that perpendicular, I , to the exciting light. All spectroscopic measurements were run at 23' unless otherwise specified.
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RESULTS AND DISCUSSION In the normal series of adenine nucleotides, titrimetric data obtained in aqueous solution have been used as evidence both for and against interaction between the ring and the phosphate side chain of ADP and ATP (11, 12).t Both electrostatic and hydrogen-bonded interactions (13), and accordingly the conformations that they help confer in nonaqueous media, would be expected to be modulated by water molecules in aqueous media. In an examination of the conformational possibilities, Sundaralingam (14) has ruled out, at least for transition metal ion complexes, the 6-amino group as a complexing site. The lin-benzoadenine nucleotides have the advantage that the nature of the environment of the lin-benzoadenine evokes spectroscopic responses that exhibit great sensitivity. For example, the spectroscopically determined pKa values of the series 1 a-e indicated a unique response to the presence and conformation of the phosphate side chain. The pKa values for aqueous solutions, which were determined spectroscopically because the ultraviolet absorption spectra provided the means of observing N-H deprotonation selectively, were as follows (1): 1 a, 5.6; 1 e, 5.6, 1 b, 7.6; 1 c, 7.3; 1 d, 7.1. Comparing the first two compounds, we observe that, when no intramolecular interaction can occur between phosphate and base, as in uin-benzoadenosine 3',5'-cyclic monophosphate (1 e), the pKa is the same as that for lin-benzoadenosine. By contrast, the pKa values of 1 b-d indicate that the phosphates in these molecules are involved in base protonation-deprotonation. The same results were obtained in phosphate buffer, showing that an intramolecular phenomenon was being observed for compounds 1 b-d. The addition of 5 mM Mg2+ to lin-benzoadenosine (1 a), the 5'-monophosphate (1 b), and the 3',5'-cyclic monophosphate (1 e) at less than 20 ,uM caused no change in the individual pKa values. The decrease of the pKa values of lin-benzo-ADP (1 c) and lin-benzo-ATP (1 d) to 6.9 and 6.6, respectively, in the presence of 5 mM Mg2+ was parallel to, but greater than, that observed for ADP and ATP (15). This decrease indicates the formation of Mg2+ complexes with the pyrophosphate unit (16) of lin-benzo-ADP and lin-benzo-ATP. The pKa value for linbenzo-ATP was lowered similarly in the presence of Mn2+ and to 6.0 in the presence of Co2+ (cf. ADP and ATP, ref. 16). With the Co2+, Mn2+, and Mg2+ results in hand, we anticipated that intramolecular interaction of the phosphate side chain with the lin-benzoadenine ring would also be disrupted by association with quaternary ammonium micelles. The pKa for lin-benzo-ATP (1 d) was observed to decrease from 7.1 to 5.6 in 10 mM CetNMe3Br, a concentration supporting the formation of micelles (the critical micelle concentration of CetNMe3Br is 0.78 mM) (17). The lower value was equal to that for lin-benzoadenosine (1 a), which was itself unaffected by CetNMe3Br in solution. With lin-benzo-ATP, the positively charged head groups of the micelles are breaking the interactions between the phosphate and the base. The addition of sodium bromide caused the pKa to return to the value of linbenzo-ATP in solution at the same ionic strength. The change is indicative of the breaking of ionic interactions between the micelles and lin-benzo-ATP by the added salt.§ Neutral micelles (e.g., Triton X-100) had no effect on the pKa of lin-benzo-ATP. The lin-benzoadenine moiety of the bound lin-benzo-ATP t In figure 5 of ref. 11, the legend should read '73 phosphate." § It has been observed previously that small changes in pKa, at low ionic strength and neutral pH, can be attributed to the electrostatic potential at the surface of the quaternary micelle (18). For the linbenzoadenine nucleotides, because the protonated base is attached to the micelle by its phosphates, the pKa differences due to the micellar electrostatic potential should be minor.
4206
Biochemistry: VanDerLijn et al.
remains outside the quaternary micelle, as shown by the observations that there is no appreciable change in its quantum yield in aqueous solution at pH 8.5 upon addition of CetNMe3Cl and that lin-benzoadenine derivatives show negligible fluorescence emission in aliphatic hydrocarbon solvents. The results with fluorescence polarization, P (19), observed for lin-benzo-ATP (1 d) in the presence of CetNMe3Cl micelles (Table 1) confirm the electrostatic binding. Binding of another sort, possibly by partial intercalation of 3-cyclohexyl-lin-benzoadenine (I f) (7) into the micelles, is shown by the second set of fluorescence polarization values in Table 1. In this case, the main effect of the added NaCl is to decrease the repulsion between the positively charged head groups of the micelle, which increases the size and the hydrophobic viscosity of the micelle (20), thus increasing the fluorescence polarization of the 3cyclohexyl-lin-benzoadenine (1 f). lin-Benzoadenosine and its derivatives exhibit satisfactory fluorescence properties in aqueous solution (quantum yield, 0.44; fluorescence lifetime, 3.7 nsec) (1), and the sensitivity of this fluorophore to environmental conditions can be examined in sufficiently dilute solution so that any changes observed can be ascribed to intramolecular rather than intermolecular interaction. In acidic aqueous solutions of lin-benzoadenosine triphosphate (1 d), the fluorescence emission maximum underwent a 13-nm red shift and the curve changed shape somewhat. The observed excited state pKa (21, 22) was close to that of the ground state value and there was little change in quantum yield. It should be noted that the fluorescence quantum yield of lin-benzo-ADP and of lin-benzo-ATP in aqueous solution is approximately 5% lower than that of linbenzoadenosine. The addition of Mg2+ up to a concentration at which the nucleotides are totally bound to the metal ion causes the quantum yields to increase to values almost equal to those at the nucleoside level. These results are indicative of Mg2+ ions' weakening the phosphate-base interaction. The quenching effect of a paramagnetic metal ion is shown in Fig. 1 in the fluorometric titration of lin-benzo-ATP with CoCl2, followed by restoration of fluorescence with MgSO4 in much higher concentration. The association constants of paramagnetic metal ions, such as Co2+ and Mn2+, to the lin-benzoadenine nucleotides can be determined by the decrease in fluorescence intensity of the lin-benzoadenine moiety with increasing concentration of the metal ion (Fig. 2 upper). The association constants of diaTable 1. Fluorescence polarization of lin-benzoadenine derivatives in the presence of micelles and enzymes
Fluorescence Solution polarization (P) lin-Benzo-ATP (1 d) 0.025*t + CetNMet3Cl 0.11* + CetNMet3Cl + NaCl 0.03* + NaCl 0.03* 0.01* 3-Cyclohexyl-lin-benzoadenine (1 f) + CetNMe3Cl 0.06* + CetNMe3Cl + NaCl 0.08* + NaCl 0.01* lin-Benzo-ATP (1 d) + pyruvate kinase 0.035 lin-Benzo-ADP (1 c) + lyxose 0.06 lin-Benzo-ADP + hexokinase + lyxose 0.10 * Determined for 6.5,uM lin-benzoadenine derivative in pH 8.5 or 8.0 buffer. t The limiting polarization of lin-benzo-ATP at 331 nm in propylene glycol at -50° was 0.42.
Proc. Natl. Acad. Sci. USA 75 (1978) Total Co2+ F-
x
,x/
80
x x
a) C
I
60
01)
K
40 C0
U[1
20
0
2
0
4 6 8 Total Mg2+, M X 102
10
FIG. 1. Fluorometric titration of lin-benzoADP > lin-benzo-AMP. The order is also shown graphically, for a separate experiment, in Fig. 2 upper. A higher concentration of Co2+ than shown in Fig. 1 was required to bring about significant quenching of lin-benzo-AMP fluorescence. The association constants for the analogue series were all greater than those for adenosine mono-, di-, and triphosphates (15, 23) determined by other methods and showed a greater AK between the respective di- and triphosphates. The same was true for the values in Table 1 compared with those for the fluorescent e-adenine nucleotide series (24, 25). For the binding of Co2+, Mn2+, and Mg2+ to lin-benzo-ADP and lin-benzo-ATP, 1:1 stoichiometry was observed. The presence of CetNMe3Cl micelles prevented the Co2+ quenching of 1 d from occurring appreciably. The addition of Co2+ or Mn2+ to a solution of
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: VanDerLijn et al.
lin-benzoadenine nucleotide fluorescence can be exploited as simple and efficient measurement of their association constants. More important, the nature of the nucleotide-Me2+ complexes can be determined spectrofluorometrically at the low concentrations used. The quenching effect of paramagnetic metal ions upon I c and d could be the result of either a static complex, in which the metal ion interacts with the phosphate(s) and base simultaneously, or a dynamic complex, in which the metal ion is bound to the phosphate(s) and is collisionally quenching the lin-benzoadenine moiety. With increasing Me2+ concentration, in the case of a static or "dark" complex, the fluorescence lifetime will remain constant; in the other case, the fluorescence lifetime will decrease (26). A graph of the fluorescence lifetimes of 1 a-d determined by phase vs. Co2+ concentration is shown in Fig. 2 lower. Before conclusions are drawn from these lifetimes, two factors must be kept in mind. First, the fluorescence lifetime of the N-protonated species of lin-benzoadenosine in aqueous solution at pH 1.5 is 5.3 nsec instead of the 3.5 to 3.7-nsec range reported for the base (1) and shown in Fig. 2. Second, as mentioned earlier, added Co2+ ions lower the pKa of lin-benzo-ADP and lin-benzo-ATP. We observed a fluorescence lifetime of ca 5.0 nsec for lin-benzo-AMP (Fig. 2), indicating the presence of N-protonated species, which is understandable, even at pH 8.5, because of the close pKa values for N+-H (7.6) and secondary phosphate 0-H (cf. AMP). The addition of Co2+ at the concentrations given in Fig. 2 does not alter the lifetime of the monophosphate (I b) because, like Mg2+ addition, it does not alter the pKa. By contrast, the binding of Co2+ to the di- and triphosphates (1 c and d), which lowers their pKa values, also lowers their fluorescence lifetimes, bringing them closer to the fluorescence lifetime of the unprotonated lin-benzoadenosine (1 a). An additional lifetime-lowering effect is observed, which is due to collisional quenching of the lin-benzoadenine moiety. That the lifetime-lowering effect is not due to free Co2+ in solution can be seen from the steady value of the fluorescence lifetime of lin-benzoadenosine over the same range of Co2+ concentration. Comparison of the two parts of Fig. 2 leads to the conclusion that, although there is some collisional quenching of the heteroaromatic ring by the bound Co2+, the major quenching effect is due to formation of a static complex. Additional evidence for the interaction of Co2+ and the heteroaromatic ring of lin-benzo-ATP in aqueous solution at pH 8.5 was obtained by means of Fourier transform 'H NMR. When lin-benzo-ATP was titrated with Co2±, a progressive broadening of the aromatic proton signals was observed, with the 2-H (corresponding to the 8-H of ATP) showing the greatest broadening. That this was not a collisional effect could be proved by the presence of an internal reference, acetone, for which the proton signal remained sharp and unchanged. UnBenzo-cAMP (1 e) was used as a control in this experiment because of the limited solubility of lin-benzoadenosine. No significant broadening of the signals for 1 e was observed even at higher Co2+/nucleotide concentration ratios than for the
a
a.
._
() 0
n
Q) u ._
G)
U
CD , a,
0)
c U lL C
0
0
1
2
4 6 5 3 Total Co2+, M X 105
7
8
9
FIG. 2. Change in fluorescence intensity (Upper) and fluoreslifetime (Lower) with increasing Co2+ concentration in aqueous solution at pH 8.5 and 230. Derivatives present at
