ANALYTICAL
BIOCHEMISTRY
96,70-76
(1979)
The influence KENNETH *Analytical TDepartment
OGAN*
of pH on Arsenazo AND ELIZABETH
Ill
R. SrMowt
Chemistry Department, The Perkin-Elmer Corporation, Norwalks of Biochemistry, Boston University School of Medicine, Boston,
Connecticut 06856, and Massachusetts 02218
Received August 17, 1978 We have investigated the effects of small changes in pH on the absorbance of arsenazo III, a calcium indicator dye. For physiological pH values, we find that arsenazo III is much more sensitive to changes in [H+] than to changes in [Ca2+]. Furthermore, for a dye solution containing calcium, the absorbance changes caused by pH increments cannot be distinguished from those caused by calcium additions. We also find that the response of arsenazo III to calcium is influenced by the other ionic constituents in the solution, such as Na+ and K+. Thus, arsenazo III must be calibrated in a solution which approximates the experimental system as closely as possible. Under such conditions, its response to changes in pH, at constant buffer and total calcium, or to small changes in [Ca2+], at constant buffer and pH, is linear.
of importance in biological systems. These ions cause different spectral changes in arsenazo III, and other investigators have shown that calcium ion can be determined in the presence of a constant magnesium concentration (7-9). The interaction of arsenazo III with various metal ions is also known to be strongly pH dependent, and Budesinsky (IO) has described the change of the extinction coefficient of the free dye with pH. Although most biological applications of arsenazo III to calcium concentration measurements are performed in buffered systems, pH sensitivity has, in general, not been taken into account in interpretations involving intracellular [Ca*+], e.g., in measurements of the effect of stimulation of single cells or of cells in suspension, even though cytoplasmic pH is known to change very markedly upon such stimulation. We have examined in more detail the effects of small changes in pH in the physiological range, and find that arsenazo III is more sensitive to changes in the concentration of hydrogen ion than of calcium ion. We also find that other ionic species in the
Micromolar levels of calcium ion play an important role in a number of physiological processes. This has created a need for a method of accurately measuring low calcium ion concentrations in biological systems. A spectroscopic approach utilizing an indicator dye responsive to changes in calcium would offer the potential of high sensitivity and selectivity. Arsenazo III (1), an arylazo derivative of chromotropic acid, is currently one of the most promising indicator dyes, and has been used in calcium measurements in giant axons (2,3), chromaffin granules (4), neurons (5), and hposomes (6). The characteristics which make arsenazo III most attractive are a large absorbance change upon binding calcium and a micromolar dissociation constant for this dye-calcium complex. Knowledge of the influence of other ions on the spectral behavior of arsenazo III is incomplete. Historically, arylazo derivatives of chromotropic acid have been used for spectroscopic determinations of ions of the alkaline- and rare-earth groups (1). Of these, only calcium and magnesium ions are
0003-2697/79KWO70-07$02.00/O Copyright 0 lW9 by Academic F’ress. Inc. All rigbts of reproduction in any form reserved.
70
THE INFLUENCE TABLE
OF pH ON ARSENAZO
1
COMPOSITION OF SOLUTIONS TO DILUTE DYE
METHODS USED
Solution No.
Composition
1 2 3 4 5 6 7
6.0 rnr.4 CaC& 100 mbr KHzPOa + 50 mM NaCl 100 mM NaHzPOe + 50 mM NaCl 10 mM NaHzP04 + 140 mM NaCl 100 mbr NaOH + 50 mM NaCl 50 mM MOPS’ + 100 mrvr NaCl 50 mbr NaOH + 100 mM NaCl
u Mops, morpholinopropanesulfonic
acid.
solution affect the dye-calcium complex equilibrium, in particular, that Na+ and K+ are not equivalent counterions.
71
III
AND MATERIALS
Reagents. Arsenazo III was purchased from Sigma Chemical Company (Grade I, purified, A-8891). The hydration value from Sigma was used in calculations of dye concentration. Demineralized water was passed through Chelex 100 (Bio-Rad Laboratories) to remove Caz+. All glassware was acid washed, rinsed with a 0.1 M EGTA-0.1 M EDTA solution, then thoroughly rinsed with Ca2+-free water and dried. A stock solution of 1.OOrnM arsenazo III and solutions of the compositions listed in Table 1 were made with Ca2+-free water. An appropriate volume of the 1 rnM arsenazo III solution was added to portions of each of the solutions in Table 1 to obtain
0.2 A
FIG. 1. Arsenazo III difference spectra resulting from successive additions of CaC& to final concentrations indicated; 50 J.LM arsenazo III, 100 mM KHzPO*, 50 mbt NaCl, pH 7.38.
72
OGAN AND SIMONS
a final dye concentration of 50 PM. Solution 5, 100 rnM NaOH, was used for pH adjustments on solutions 2-4, and solution 7, 50 rnM NaOH, was used with solution 6. Znstrumentution. Solution pH values were read with either a Corning Model 12 or an Orion Model 701/A, using a glass electrode with a calomel reference electrode. Absorbance spectra were recorded on either a Perkin-Elmer Model 576 or a PerkinElmer Model 340 spectrophotometer.
A 0
0.60 A 0 0.70 0.60 0.50 z
0.40 0.30 -
RESULTS
Figure 1 gives the difference spectra resulting from successive additions of CaC& to a 50 PM solution of arsenazo III. (The CaCIZ solution also contained 50 PM arsenazo III in order to eliminate dilution effects.) The dye-calcium dissociation constant can be determined from the slope of a double reciprocal plot, utilizing the absorbance and calcium concentration data (9,ll):
0
20
40
[co-j
60
60
FIG. 2. Absorbance change (A = 654 nm) of 50 pM arsenazo III as a function of free calcium concentration (for K,, = 30 PM) for different pH buffer systems. (0 --- 0) 50 rnM Mops, 100 rnM NaCl, pH 7.31. (A A) 100 rnM KHzPOd, 50 rnM NaCL pH 7.38.
shoulder at 655 nm, in contrast to the twin peaks at 603 and 654 nm resulting from calKll cium addition (Fig. I). There is an isosbestic &++ 1, Ul point at 582 nm for pH changes around pH [Caz+] 7.3, while the calcium-induced changes in with [Caz+] given by Fig. I give an isosbestic point at 569 nm. As shown in Fig. 4, the absorbance changes [Caz+]=C~-[D~Cal=C~--~. [21 in Fig. 3 are linear with the pH change over this range of pH values. Here A is the observed absorbance change, The pH experiment was repeated with C,, and CA are the total concentrations of both reference and sample solutions having dye and added calcium, respectively, KD equal amounts of added calcium. A typical is the dye-calcium dissociation constant, set of difference spectra is shown in Fig. 5. and Ae is the difference between the dyeAlthough these spectra resemble those obcalcium and the free dye extinction coefficients, l Dca-eD.With the literature value of tained by calcium additions, only the pH 2.80 x 104 for E~,-~(8,12), we calculate KD was altered. The spectral changes therefore imply an alteration in KD and a concomitant = 30 pM. This value is consistent with Kenincrease in the concentration of the dyedrick et al. (9), who reported values ranging calcium complex. from 0.7 to 52 PM, depending on the ionic It has been reported that phosphate does composition of the solutions. Using our value for KD, the apparent free calcium con- not interfere with the analysis for Ca2+ using arsenazo-III (8,12). In order to verify centration can be calculated (Fig. 2). The effect of changes in the hydrogen ion this question, we replaced the phosphate concentration on the absorbance of the free with an organic buffer, Mops1 (morphohnodye is shown in Fig. 3. These spectra ex* Abbreviation used: Mops, morpholinopropanesulhibit a single peak at 605 nm and a minor fonic acid.
THE INFLUENCE
OF pH ON ARSENAZO
III
FIG. 3. Arsenazo III difference spectra obtained by increasing solution pH. Reference is pH 6.50; (- - -) pH 7.00; (-.-.-) pH 7.20; (. . .) pH 7.42; (-) pH 7.70.
propanesulfonic acid; see soluGon 6 in Table l), and repeated the experiments. A value of KD = 30 PM was again obtained. The absorbance changes are plotted as a function of calculated free calcium in Fig. 2. The initial slopes of the phosphate and Mops curves are identical, and the absorbance changes for larger amounts of added calcium are asymptotic to the same value. The only detectable difference is a shift at low calcium levels, suggesting that a very small quantity of calcium, approximately 1 PM, is unavailable in the phosphate buffer system and is presumably complexed to phosphate. The pH difference spectra were also remeasured in the Mops buffer system (Fig. 6). The absorbance change at 603 nm, near the absorbance peak, is linear with pH and identical to that in the phosphate system (Fig. 4), although the absorbance change at
654 nm is no longer linear with pH. The isosbestic point at 582 nm can no longer be detected. In order to evaluate the effect of monovalent ions on the concen&aGon of Caz+arsenazo III complex, KHZPOd was replaced with NaHZPOd. The baseline difference spectrum between the sodium solution and the potassium solution shows trace amounts of calcium in the salts. Addition of an equal amount of CaC& soluGon to both solutions to a final concentration of 59 PM led to a large spectral change (Fig. 7), indicative of an alteration in the concentration of the absorbing species. DISCUSSION
The absorbance change of is linear with change in pH, or logarithmic with the hydrogen tration (Fig. 4). Thus, the
arsenazo III equivalently, ion concenabsorbance
74
OGAN AND SIMONS
change caused concentration, the logarithmic the initial [H+]
by a small change in H+ i.e., the tangential slope of curve, is dependent upon value:
dA = m d(pH),
0.22 0.20 0.16 -
r31
from which
0.16 -
dA -=-ET ciPI+1
m
0.14 -
[41
where m is the slope from Fig. 4. At physiological pH, 7.4, this is 0.74 absorbance units per PM change in [H+] (A = 654 nm). In comparison, the absorbance change attributable to [Caz+] change (Fig. 3) is 0.024 absorbance units per PM [Caz+] change. Thus, at physiological pH, arsenazo III is 30 times more responsive to a change in [H+] than to an equal change in [Caz+]. This fact must be carefully borne in mind in Ca2+ measurements, particularly since arsenazo III releases a proton upon binding calcium (8). Even in cell-free systems, the pH of calibration solutions must be strongly buffered, and the pH of experimental systems must be accurately monitored. Figure 5 clearly demonstrates the importance of pH buffering: Even though there is a clear change in the amount of dye-calcium complex, it reflects an apparent change in KD and not a change in the level of calcium in the system. In addition, the buffer cation plays a sizeable and as yet not understood
0.12 2 0.10 0.06 0.06 -
0
0.5
10
1.5
APH
FIG. 4. Absorbance change of 50 pM arsenazo III at 603 and 654 nm as function of pH increase. (0 - - - 0) 50 rnM Mops, 100 mM NaCl, 50 rnM EGTA, (A A) 100 rnM KH2PO+ 50 rnM NaCl.
role in the stability of the dye-calcium complex, and hence in the measurement of free calcium, For example, replacement of 100 rnM K+ with 100 rnM Na+, in the presence of a constant total calcium concentration of 59 PM, leads to an absorbance change at 654 nm that is equivalent to adding half again as much calcium to the K+ system.
FIG. 5. Arsenazo III difference spectra obtained from increasing pH of solutions containing rnM Car& in addition to 50 PM arsenazo III, 100 rnM KH2P04, and 50 rnM NaCl.
89
THE INFLUENCE
OF pH ON ARSENAZO
III
75
FIG. 6. Arsenazo III difference spectra for increased solution pH, relative to pH 6.56: 50 @r arsenazo III, 50 rnM Mops, 100 rnM NaCl, 50 pM EGTA. (- - -) pH 7.00; (-.-) pH 7.25; (. . .) pH 7.44; (-) pH 7.62; (- - -) pH 7.82.
Therefore, if comparison between experiments is to be made, not only the pH but also the buffer ions and their concentrations must be carefully controlled. Complications in intracellular applications arise because the cytoplasmic buffering capacity cannot be evaluated, but has been demonstrated to be inadequate to cope with intracellular
pH changes associated with cell stimulation (13,14). Our preliminary attempts to separate the spectral contribution due to pH changes from those to calcium changes have been unsuccessful. This is probably due to the complexity of the problem. It has been shown that free
FIG. 7. Arsenazo III difference spectra for Na+ buffer compared to K+ buffer. Botb solutions contained SO PM arsenazo III and 50 rnM NaCl. The reference solution contains 100 rnM KHzP04 (pH 74, while the sample solution contains 100 rnM NaHzP04 (pH 7.44). Spectra were recorded for no added CaC& (- - -) and for 59 p,~ CaC& in both solutions (-).
76
OGAN AND SIMONS
arsenazo III exists in solution in a multiplicity of states of protonation, each exhibiting a different molar absorptivity at any given pH (10,12). It is also probable that the dye can exist not only in the 1: 1 dye:Caz+ stoichiometry (9) but also in the 2: 1 stoichiometry under some conditions (15). The reasons for the complexity of arsenazo III-ligand structures remain unelucidated. It is possible that the quinonehydrazone form of arsenazo III, responsible for the free dye absorbance at 650-660 nm (16), plays a role. The appearance of this band in the absorbance spectrum of the dye-calcium complex, suggests that the perkdioxy group is evidently intimately involved in the binding of calcium. The charge on this oxygen would greatly enhance the binding of Caz+. The acidity of the first naphthalene hydroxyl is also sensitive to the ionic components of the solution, and may explain the pH sensitivity of the system. Therefore, not only does the dye structure vary with pH, but the exact structure of the dye-calcium complex is simultaneously dependent on H+ and Caz+ concentrations, with the apparent KD being a function of both variables (at constant concentrations of all other cations such as K+, Na+, Mg*+, etc). The importance of these other cations is illustrated by the observation, reported here (Fig. 7), that mere changes of the buffer cation from K+ to Na+, with no other changes, give rise to a sizeable change in the absorbance of the solution. In summary, we find that for physiological pH values, the absorbance of arsenazo III is much more sensitive to changes in hydrogen ion concentration than to changes in calcium ion concentration. Furthermore, the effects of small pH changes on the absorbance of a system containing calcium are similar to the effects of changing the calcium content, because the apparent dissociation constant of the complex is a function of both pH and [Ca*+]. The individual contributions cannot be distinguished at present; quantitation of calcium changes
must hence be performed at constant pH. The response of arsenazo III to calcium changes is also influenced by the buffer cations (K+, Na+). It is thus vital not only that calibration curves for cell-free systems be performed under identical conditions to those of eventual measurement, but also that the inability to exert such control intracellularly be considered when interpretations of intracellular (cytoplasmic) [Ca*+] measurements via arsenazo III are attempted. ACKNOWLEDGMENT The partial support of NIH grants HL 15335 and HL 16375 is gratefully acknowledged.
REFERENCES 1. Budesinsky, B. (1%9) in Chelates in Analytical Chemistry. (Flaschka, H. A., and Barnard, A. J., Jr., eds.), Vol. 2, p. 1, Dekker, New York. 2. Brown, J. E., Cohen, L. B., de Weer, P., Pinto, L. H., Ross, W. H., and Salzberg, B. M. (1975) Bi&ys. J. 15, 1155-l 160. 3. Dipole, R., Requena, J., Brinley, F. J., Jr., MulIins, L. J., Scarpa, A., and Tiffert, T. (1976) .I. Gen.
Physiol.
67, 433-467.
4. Johnson, R. G., and Scarpa, A. (1976) j. Gen. Physio/. 68, 601-631. 5. Thomas, M. V., and German, A. L. F. (1977) Science 196, 531-533. 6. Weissman, G., Collins, T., Evers, A., and Dunham, P. (1976) Proc. Nat. Acad. Sri. USA 73, 510-514. 7. Michaylova, V., and llkova, P. ( 197 1) Anal. Chim. Acta 53, 194- 198. 8. Michaylova, V., and Kouleva, N. (1974) Talantu 2t, 523-532. 9. Kendrick, N. C., At&&f, R. W. R., and Blaustein, M. P. (1977) Anal. Biochem. 83, 433-450. 10. Budesinsky, B. (1969) Talanta 16, 1277- 1288. 11. Gratzer, W. B., and Beaven, G. H. (1977) Anal. Biochem. 81, 118- 129. 12. Michaylova, V., and Kouleva, N. (1973) Talunta 20, 453-458. 13. Johnson, J. D., Epel, D., and Paul, M. (1976) Nature (London) 262, 661-664. 14. Shen, S. S., and Steinhardt, R. A. (1978) Nature (London)
272,
253-254.
15. Thomas, M. V., and German, A. L. F. (1978) Biophys. J. 21, 53a. 16. Savvin, S. B., and Kuzin, E. L. (1968) Talanta 15, 913-921.
BIOCHEMISTRY
96,70-76
(1979)
The influence KENNETH *Analytical TDepartment
OGAN*
of pH on Arsenazo AND ELIZABETH
Ill
R. SrMowt
Chemistry Department, The Perkin-Elmer Corporation, Norwalks of Biochemistry, Boston University School of Medicine, Boston,
Connecticut 06856, and Massachusetts 02218
Received August 17, 1978 We have investigated the effects of small changes in pH on the absorbance of arsenazo III, a calcium indicator dye. For physiological pH values, we find that arsenazo III is much more sensitive to changes in [H+] than to changes in [Ca2+]. Furthermore, for a dye solution containing calcium, the absorbance changes caused by pH increments cannot be distinguished from those caused by calcium additions. We also find that the response of arsenazo III to calcium is influenced by the other ionic constituents in the solution, such as Na+ and K+. Thus, arsenazo III must be calibrated in a solution which approximates the experimental system as closely as possible. Under such conditions, its response to changes in pH, at constant buffer and total calcium, or to small changes in [Ca2+], at constant buffer and pH, is linear.
of importance in biological systems. These ions cause different spectral changes in arsenazo III, and other investigators have shown that calcium ion can be determined in the presence of a constant magnesium concentration (7-9). The interaction of arsenazo III with various metal ions is also known to be strongly pH dependent, and Budesinsky (IO) has described the change of the extinction coefficient of the free dye with pH. Although most biological applications of arsenazo III to calcium concentration measurements are performed in buffered systems, pH sensitivity has, in general, not been taken into account in interpretations involving intracellular [Ca*+], e.g., in measurements of the effect of stimulation of single cells or of cells in suspension, even though cytoplasmic pH is known to change very markedly upon such stimulation. We have examined in more detail the effects of small changes in pH in the physiological range, and find that arsenazo III is more sensitive to changes in the concentration of hydrogen ion than of calcium ion. We also find that other ionic species in the
Micromolar levels of calcium ion play an important role in a number of physiological processes. This has created a need for a method of accurately measuring low calcium ion concentrations in biological systems. A spectroscopic approach utilizing an indicator dye responsive to changes in calcium would offer the potential of high sensitivity and selectivity. Arsenazo III (1), an arylazo derivative of chromotropic acid, is currently one of the most promising indicator dyes, and has been used in calcium measurements in giant axons (2,3), chromaffin granules (4), neurons (5), and hposomes (6). The characteristics which make arsenazo III most attractive are a large absorbance change upon binding calcium and a micromolar dissociation constant for this dye-calcium complex. Knowledge of the influence of other ions on the spectral behavior of arsenazo III is incomplete. Historically, arylazo derivatives of chromotropic acid have been used for spectroscopic determinations of ions of the alkaline- and rare-earth groups (1). Of these, only calcium and magnesium ions are
0003-2697/79KWO70-07$02.00/O Copyright 0 lW9 by Academic F’ress. Inc. All rigbts of reproduction in any form reserved.
70
THE INFLUENCE TABLE
OF pH ON ARSENAZO
1
COMPOSITION OF SOLUTIONS TO DILUTE DYE
METHODS USED
Solution No.
Composition
1 2 3 4 5 6 7
6.0 rnr.4 CaC& 100 mbr KHzPOa + 50 mM NaCl 100 mM NaHzPOe + 50 mM NaCl 10 mM NaHzP04 + 140 mM NaCl 100 mbr NaOH + 50 mM NaCl 50 mM MOPS’ + 100 mrvr NaCl 50 mbr NaOH + 100 mM NaCl
u Mops, morpholinopropanesulfonic
acid.
solution affect the dye-calcium complex equilibrium, in particular, that Na+ and K+ are not equivalent counterions.
71
III
AND MATERIALS
Reagents. Arsenazo III was purchased from Sigma Chemical Company (Grade I, purified, A-8891). The hydration value from Sigma was used in calculations of dye concentration. Demineralized water was passed through Chelex 100 (Bio-Rad Laboratories) to remove Caz+. All glassware was acid washed, rinsed with a 0.1 M EGTA-0.1 M EDTA solution, then thoroughly rinsed with Ca2+-free water and dried. A stock solution of 1.OOrnM arsenazo III and solutions of the compositions listed in Table 1 were made with Ca2+-free water. An appropriate volume of the 1 rnM arsenazo III solution was added to portions of each of the solutions in Table 1 to obtain
0.2 A
FIG. 1. Arsenazo III difference spectra resulting from successive additions of CaC& to final concentrations indicated; 50 J.LM arsenazo III, 100 mM KHzPO*, 50 mbt NaCl, pH 7.38.
72
OGAN AND SIMONS
a final dye concentration of 50 PM. Solution 5, 100 rnM NaOH, was used for pH adjustments on solutions 2-4, and solution 7, 50 rnM NaOH, was used with solution 6. Znstrumentution. Solution pH values were read with either a Corning Model 12 or an Orion Model 701/A, using a glass electrode with a calomel reference electrode. Absorbance spectra were recorded on either a Perkin-Elmer Model 576 or a PerkinElmer Model 340 spectrophotometer.
A 0
0.60 A 0 0.70 0.60 0.50 z
0.40 0.30 -
RESULTS
Figure 1 gives the difference spectra resulting from successive additions of CaC& to a 50 PM solution of arsenazo III. (The CaCIZ solution also contained 50 PM arsenazo III in order to eliminate dilution effects.) The dye-calcium dissociation constant can be determined from the slope of a double reciprocal plot, utilizing the absorbance and calcium concentration data (9,ll):
0
20
40
[co-j
60
60
FIG. 2. Absorbance change (A = 654 nm) of 50 pM arsenazo III as a function of free calcium concentration (for K,, = 30 PM) for different pH buffer systems. (0 --- 0) 50 rnM Mops, 100 rnM NaCl, pH 7.31. (A A) 100 rnM KHzPOd, 50 rnM NaCL pH 7.38.
shoulder at 655 nm, in contrast to the twin peaks at 603 and 654 nm resulting from calKll cium addition (Fig. I). There is an isosbestic &++ 1, Ul point at 582 nm for pH changes around pH [Caz+] 7.3, while the calcium-induced changes in with [Caz+] given by Fig. I give an isosbestic point at 569 nm. As shown in Fig. 4, the absorbance changes [Caz+]=C~-[D~Cal=C~--~. [21 in Fig. 3 are linear with the pH change over this range of pH values. Here A is the observed absorbance change, The pH experiment was repeated with C,, and CA are the total concentrations of both reference and sample solutions having dye and added calcium, respectively, KD equal amounts of added calcium. A typical is the dye-calcium dissociation constant, set of difference spectra is shown in Fig. 5. and Ae is the difference between the dyeAlthough these spectra resemble those obcalcium and the free dye extinction coefficients, l Dca-eD.With the literature value of tained by calcium additions, only the pH 2.80 x 104 for E~,-~(8,12), we calculate KD was altered. The spectral changes therefore imply an alteration in KD and a concomitant = 30 pM. This value is consistent with Kenincrease in the concentration of the dyedrick et al. (9), who reported values ranging calcium complex. from 0.7 to 52 PM, depending on the ionic It has been reported that phosphate does composition of the solutions. Using our value for KD, the apparent free calcium con- not interfere with the analysis for Ca2+ using arsenazo-III (8,12). In order to verify centration can be calculated (Fig. 2). The effect of changes in the hydrogen ion this question, we replaced the phosphate concentration on the absorbance of the free with an organic buffer, Mops1 (morphohnodye is shown in Fig. 3. These spectra ex* Abbreviation used: Mops, morpholinopropanesulhibit a single peak at 605 nm and a minor fonic acid.
THE INFLUENCE
OF pH ON ARSENAZO
III
FIG. 3. Arsenazo III difference spectra obtained by increasing solution pH. Reference is pH 6.50; (- - -) pH 7.00; (-.-.-) pH 7.20; (. . .) pH 7.42; (-) pH 7.70.
propanesulfonic acid; see soluGon 6 in Table l), and repeated the experiments. A value of KD = 30 PM was again obtained. The absorbance changes are plotted as a function of calculated free calcium in Fig. 2. The initial slopes of the phosphate and Mops curves are identical, and the absorbance changes for larger amounts of added calcium are asymptotic to the same value. The only detectable difference is a shift at low calcium levels, suggesting that a very small quantity of calcium, approximately 1 PM, is unavailable in the phosphate buffer system and is presumably complexed to phosphate. The pH difference spectra were also remeasured in the Mops buffer system (Fig. 6). The absorbance change at 603 nm, near the absorbance peak, is linear with pH and identical to that in the phosphate system (Fig. 4), although the absorbance change at
654 nm is no longer linear with pH. The isosbestic point at 582 nm can no longer be detected. In order to evaluate the effect of monovalent ions on the concen&aGon of Caz+arsenazo III complex, KHZPOd was replaced with NaHZPOd. The baseline difference spectrum between the sodium solution and the potassium solution shows trace amounts of calcium in the salts. Addition of an equal amount of CaC& soluGon to both solutions to a final concentration of 59 PM led to a large spectral change (Fig. 7), indicative of an alteration in the concentration of the absorbing species. DISCUSSION
The absorbance change of is linear with change in pH, or logarithmic with the hydrogen tration (Fig. 4). Thus, the
arsenazo III equivalently, ion concenabsorbance
74
OGAN AND SIMONS
change caused concentration, the logarithmic the initial [H+]
by a small change in H+ i.e., the tangential slope of curve, is dependent upon value:
dA = m d(pH),
0.22 0.20 0.16 -
r31
from which
0.16 -
dA -=-ET ciPI+1
m
0.14 -
[41
where m is the slope from Fig. 4. At physiological pH, 7.4, this is 0.74 absorbance units per PM change in [H+] (A = 654 nm). In comparison, the absorbance change attributable to [Caz+] change (Fig. 3) is 0.024 absorbance units per PM [Caz+] change. Thus, at physiological pH, arsenazo III is 30 times more responsive to a change in [H+] than to an equal change in [Caz+]. This fact must be carefully borne in mind in Ca2+ measurements, particularly since arsenazo III releases a proton upon binding calcium (8). Even in cell-free systems, the pH of calibration solutions must be strongly buffered, and the pH of experimental systems must be accurately monitored. Figure 5 clearly demonstrates the importance of pH buffering: Even though there is a clear change in the amount of dye-calcium complex, it reflects an apparent change in KD and not a change in the level of calcium in the system. In addition, the buffer cation plays a sizeable and as yet not understood
0.12 2 0.10 0.06 0.06 -
0
0.5
10
1.5
APH
FIG. 4. Absorbance change of 50 pM arsenazo III at 603 and 654 nm as function of pH increase. (0 - - - 0) 50 rnM Mops, 100 mM NaCl, 50 rnM EGTA, (A A) 100 rnM KH2PO+ 50 rnM NaCl.
role in the stability of the dye-calcium complex, and hence in the measurement of free calcium, For example, replacement of 100 rnM K+ with 100 rnM Na+, in the presence of a constant total calcium concentration of 59 PM, leads to an absorbance change at 654 nm that is equivalent to adding half again as much calcium to the K+ system.
FIG. 5. Arsenazo III difference spectra obtained from increasing pH of solutions containing rnM Car& in addition to 50 PM arsenazo III, 100 rnM KH2P04, and 50 rnM NaCl.
89
THE INFLUENCE
OF pH ON ARSENAZO
III
75
FIG. 6. Arsenazo III difference spectra for increased solution pH, relative to pH 6.56: 50 @r arsenazo III, 50 rnM Mops, 100 rnM NaCl, 50 pM EGTA. (- - -) pH 7.00; (-.-) pH 7.25; (. . .) pH 7.44; (-) pH 7.62; (- - -) pH 7.82.
Therefore, if comparison between experiments is to be made, not only the pH but also the buffer ions and their concentrations must be carefully controlled. Complications in intracellular applications arise because the cytoplasmic buffering capacity cannot be evaluated, but has been demonstrated to be inadequate to cope with intracellular
pH changes associated with cell stimulation (13,14). Our preliminary attempts to separate the spectral contribution due to pH changes from those to calcium changes have been unsuccessful. This is probably due to the complexity of the problem. It has been shown that free
FIG. 7. Arsenazo III difference spectra for Na+ buffer compared to K+ buffer. Botb solutions contained SO PM arsenazo III and 50 rnM NaCl. The reference solution contains 100 rnM KHzP04 (pH 74, while the sample solution contains 100 rnM NaHzP04 (pH 7.44). Spectra were recorded for no added CaC& (- - -) and for 59 p,~ CaC& in both solutions (-).
76
OGAN AND SIMONS
arsenazo III exists in solution in a multiplicity of states of protonation, each exhibiting a different molar absorptivity at any given pH (10,12). It is also probable that the dye can exist not only in the 1: 1 dye:Caz+ stoichiometry (9) but also in the 2: 1 stoichiometry under some conditions (15). The reasons for the complexity of arsenazo III-ligand structures remain unelucidated. It is possible that the quinonehydrazone form of arsenazo III, responsible for the free dye absorbance at 650-660 nm (16), plays a role. The appearance of this band in the absorbance spectrum of the dye-calcium complex, suggests that the perkdioxy group is evidently intimately involved in the binding of calcium. The charge on this oxygen would greatly enhance the binding of Caz+. The acidity of the first naphthalene hydroxyl is also sensitive to the ionic components of the solution, and may explain the pH sensitivity of the system. Therefore, not only does the dye structure vary with pH, but the exact structure of the dye-calcium complex is simultaneously dependent on H+ and Caz+ concentrations, with the apparent KD being a function of both variables (at constant concentrations of all other cations such as K+, Na+, Mg*+, etc). The importance of these other cations is illustrated by the observation, reported here (Fig. 7), that mere changes of the buffer cation from K+ to Na+, with no other changes, give rise to a sizeable change in the absorbance of the solution. In summary, we find that for physiological pH values, the absorbance of arsenazo III is much more sensitive to changes in hydrogen ion concentration than to changes in calcium ion concentration. Furthermore, the effects of small pH changes on the absorbance of a system containing calcium are similar to the effects of changing the calcium content, because the apparent dissociation constant of the complex is a function of both pH and [Ca*+]. The individual contributions cannot be distinguished at present; quantitation of calcium changes
must hence be performed at constant pH. The response of arsenazo III to calcium changes is also influenced by the buffer cations (K+, Na+). It is thus vital not only that calibration curves for cell-free systems be performed under identical conditions to those of eventual measurement, but also that the inability to exert such control intracellularly be considered when interpretations of intracellular (cytoplasmic) [Ca*+] measurements via arsenazo III are attempted. ACKNOWLEDGMENT The partial support of NIH grants HL 15335 and HL 16375 is gratefully acknowledged.
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