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Cation Complexation by Antipyrylazo III: Chelate Spectral Behavior Correlated with Divalent Cation Properties
181
NOTES & TIPS 10. Miyazaki, J., Takaki, S., Araki, K., Tashiro, F., Tominaga, A., Takatsu, K., and Yamamura, K. (1989) Gene 79, 269–277. 11. Foecking, M. K., and Hofstetter, H. (1986) Gene 45, 101–105. 12. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene 108, 193–200. 13. Altelt, P., Grannemann, R., Stocking, C., Friel, J., Bartsch, J., and Hauser, H. (1991) Gene 99, 249–254. 14. Mizushima, S., and Nagata, S. (1990) Nucleic Acids Res. 18, 5322.
Cation Complexation by Antipyrylazo III: Chelate Spectral Behavior Correlated with Divalent Cation Properties Scott E. Pattison,1 Diana Maniak, and Steve Friar Department of Chemistry, Ball State University, Muncie, Indiana 47306 Received January 9, 1997
Antipyrylazo III (APIII)2 is a metallochromic indicator which is commonly used to monitor metal ion 1 2
To whom correspondence should be addressed. Fax: (317) 285-2351. Abbreviations used: APIII, antipyrylazo III; ARIII, arsenazo III.
movement in biological systems. It is closely related to arsenazo III (ARIII); both are relatively rigid structures and have a limited number of binding site conformations. We report for the first time the existence of a 1 APIII:2 Me2/ complex under conditions used to study biological metal transport and provide a structural analysis to predict APIII chelating behavior. Unlike ARIII, APIII forms two distinct complexes with calcium ion (1): (a) a 2 APIII:1 Ca2/ complex yields a characteristic difference spectra with a broad positive peak centered between 650 and 700 nm; (b) a 1 APIII:1 Ca2/ complex produces a difference spectra with a negative peak at about 600 nm. The 1:1 APIII complexes with Mg 2/ or Mn2/ are characterized by a negative peak at about 600 nm (2,3). We have extended this information to include six biologically important metals using conditions which are common in the study of biological transport (see legend to Fig. 1). Figure 1 shows the spectral change for each chelate. Where successive changes occur, the first change is shown with a solid line and the second with a dashed line. Table 1 reports the chelate characteristics (both spectral changes and dissociation constants) for each metal complex. As shown in Fig. 2, APIII (like ARIII) is a chelator
TABLE 1
Comparison of APIII:Metal Chelates Cation
Ionic radius (pm)
Spectral D
Ca2/b
99
Mn2/
80
Mg2/c
65
Positive, 650 nm, 720 nm Negative, 600 nm Negative, 600 nm Positive, 580 nm, 620 nm Negative, 600 nm
Apparent dissociation constanta
Hard Lewis acids — 2200 mM 12 { 4 mM 1600 { 320 mM2 1200 mM
Intermediate (between hard and soft) Lewis acids Zn2/
74
Ni2/
69
Cu2/
68
Negative, 610 nm Positive, 570 nm, 600 nm Negative, 610 nm Positive, 600 nm, 650 nm Negative, 600 nm Positive, 610 nm, 650 nm
14 185 10 171 39
{ 11 mM { 46 mM2 { 3 mM { 32 mM2 —d { 5 mM2
Soft Lewis acids Pb2/ Cd2/
121 97
Positive, 600 nm, 650 nm Positive, 590 nm, 650 nm
a
0.6 { 1.0 {
0.1 mM2 0.2 mM2
e
Titrations were fit with an iterative program to two equilibria (KaleidaGraph, Synergy Software Inc.). Taken from Hollingworth et al. (1). c Taken from Gavin et al. (3). d Equilibrium for the first complex was not measurable. e Although only one complex was observed during titration, some evidence for a second complex was noted while measuring Cd2/-vesicle diffusion kinetics. b
ANALYTICAL BIOCHEMISTRY ARTICLE NO. AB972038
247, 181–183 (1997)
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
03-17-97 15:09:05
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NOTES & TIPS
FIG. 1. Spectral changes due to the reaction of APIII with selected divalent cations under experimental conditions used for membrane transport studies. 4 mM APIII (0.05 M Hepes buffer, pH 7.4, 0.1 M NaNO3) was enclosed in small unilamellar vesicles (composed of phosphatidyl choline) by sonication. The vesicles were treated with the ionophore A23187, known amounts of metal ion were added, and the spectral change was recorded by a Hewlett–Packard diode array spectrophotometer. Following titration of the APIII, difference spectra were selected at the saturation point for each complex, first complex (—) and second complex (---). Titrations were conducted for the following cations: a, Zn2/; b, Cu2/ (the spectrum of the first complex was not completely isolated); c, Ni2/; d, Mn2/; e, Cd2/ (although the titration showed only one complex, Cd2/ –vesicle diffusion kinetic studies showed some evidence for a second complex); f, Pb2/.
with limited conformational possibilities. In order to maximize overlap between p systems, a planar conformation is favored. The most stable form of the chelator has a trans configuration at both azo links. Thus, we consider two chelating structures, a ‘‘condensed’’ form (Fig. 2a) and an ‘‘open’’ form (Fig. 2b). For ARIII these two forms have markedly different chelating properties because of the bulky and negatively charged benzenearsonic acid groups (4): (a) In the open ARIII conformation, smaller cations can complex with the ligands in the side ‘‘sockets’’ adjacent to the azo links; (b) The condensed ARIII form provides a central site which can bind well to a large cation such as calcium. Thus, ARIII yields a 1:1 complex with larger cations and a 1:2 complex with smaller cations. In contrast for APIII, smaller pyrazol derivatives replace the bulky benzenearsonic acid groups. Whether APIII is in the condensed or open conformation, (a)
smaller cations can bind at the side sockets (given bond distances of about 200 pm) and (b) there is ample room for larger cations to bind in the middle of APIII but to only a small number of ligands (given bond distances of 200–300 pm). Thus, (a) small cations form two APIII complexes binding to either or both side sockets. The first complex is characterized by a negative spectral change around 600 nm and the second complex shows a positive spectral change between 600 and about 650 nm. The titration curves are fit well using a 1:1 and 1:2 binding stoichiometry. (i) Over a relatively large concentration range only a 1:1 complex is observed for the hard Lewis acids. Hence, these cations form a 1:1 complex much more easily than a 1:2 complex. (ii) The cations that are intermediate between hard and soft Lewis acids are more likely to form a 1:2 complex at low metal concentrations. In several cases (Cu2/, Zn2/), the second metal ion binds more
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mM and K2 Å 4.42 { 0.76 m M. We suggest that cations with a higher affinity for nitrogen ligands favor the 1:2 complex by shifting the APIII conformation toward the open form as they bind with the azo link. (b) Large cations fall into two categories based on their reaction with APIII. (i) The soft Lewis acids form almost exclusively a 1:2 complex. (ii) Hard Lewis acids may not be completely coordinated by one APIII. Thus, these cations can bind two APIII (e.g., Ca2/) as well as forming a 1:1 complex. APIII spectral signals vary predictably depending on the metal ion to be studied. The large, hard Lewis acids (e.g., Ca2/) have the potential to form both 2:1 and 1:1 complexes. Small, hard Lewis acids (e.g., Mg 2/ and Mn2/) will form predominately 1:1 complexes and will provide the simplest of spectral signals with APIII (a decrease in absorbance at 600 nm). Other small divalent cations will show more complex spectral changes because both the 1:1 and 1:2 complexes easily coexist. Divalent zinc is a case in point where the second zinc actually binds more tightly than the first. This has proved advantageous for membrane transport studies where APIII provides a spectral signal for the first zinc transported and, then, a different signal for transport of the second zinc ion. Finally, the large, soft Lewis acids (e.g., Pb2/, Cd2/) tend to form a 1:2 complex almost exclusively. FIG. 2. Two stable conformations of APIII: a, condensed; b, open. APIII is shown in a stick formula with the atomic volumes shown for the azo nitrogens and the phenolic oxygens in gray dots. The phenolic oxygens are shown without their hydrogen atoms.
REFERENCES
tightly than the first (cooperative binding). We have examined the APIII reaction with zinc in the absence of lipid vesicles as well. For a range of [APIII] from 5 to 76 mM (9 determinations), K1 Å 29.2 { 5.4
1. Hollingworth, S., Aldrich, R. W., and Baylor, S. M. (1987) Biophys. J. 51, 383–393. 2. Salama, G., and Scarpa, A. (1985) J. Biol. Chem. 260, 11697– 11705. 3. Gavin, C. E., Gunter, K. K., and Gunter, T. E. (1991) Anal. Biochem. 192, 44–48. 4. Rowatt, E., and Williams, R. J. P. (1989) Biochem. J. 259, 295– 298.
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NOTES & TIPS 10. Miyazaki, J., Takaki, S., Araki, K., Tashiro, F., Tominaga, A., Takatsu, K., and Yamamura, K. (1989) Gene 79, 269–277. 11. Foecking, M. K., and Hofstetter, H. (1986) Gene 45, 101–105. 12. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene 108, 193–200. 13. Altelt, P., Grannemann, R., Stocking, C., Friel, J., Bartsch, J., and Hauser, H. (1991) Gene 99, 249–254. 14. Mizushima, S., and Nagata, S. (1990) Nucleic Acids Res. 18, 5322.
Cation Complexation by Antipyrylazo III: Chelate Spectral Behavior Correlated with Divalent Cation Properties Scott E. Pattison,1 Diana Maniak, and Steve Friar Department of Chemistry, Ball State University, Muncie, Indiana 47306 Received January 9, 1997
Antipyrylazo III (APIII)2 is a metallochromic indicator which is commonly used to monitor metal ion 1 2
To whom correspondence should be addressed. Fax: (317) 285-2351. Abbreviations used: APIII, antipyrylazo III; ARIII, arsenazo III.
movement in biological systems. It is closely related to arsenazo III (ARIII); both are relatively rigid structures and have a limited number of binding site conformations. We report for the first time the existence of a 1 APIII:2 Me2/ complex under conditions used to study biological metal transport and provide a structural analysis to predict APIII chelating behavior. Unlike ARIII, APIII forms two distinct complexes with calcium ion (1): (a) a 2 APIII:1 Ca2/ complex yields a characteristic difference spectra with a broad positive peak centered between 650 and 700 nm; (b) a 1 APIII:1 Ca2/ complex produces a difference spectra with a negative peak at about 600 nm. The 1:1 APIII complexes with Mg 2/ or Mn2/ are characterized by a negative peak at about 600 nm (2,3). We have extended this information to include six biologically important metals using conditions which are common in the study of biological transport (see legend to Fig. 1). Figure 1 shows the spectral change for each chelate. Where successive changes occur, the first change is shown with a solid line and the second with a dashed line. Table 1 reports the chelate characteristics (both spectral changes and dissociation constants) for each metal complex. As shown in Fig. 2, APIII (like ARIII) is a chelator
TABLE 1
Comparison of APIII:Metal Chelates Cation
Ionic radius (pm)
Spectral D
Ca2/b
99
Mn2/
80
Mg2/c
65
Positive, 650 nm, 720 nm Negative, 600 nm Negative, 600 nm Positive, 580 nm, 620 nm Negative, 600 nm
Apparent dissociation constanta
Hard Lewis acids — 2200 mM 12 { 4 mM 1600 { 320 mM2 1200 mM
Intermediate (between hard and soft) Lewis acids Zn2/
74
Ni2/
69
Cu2/
68
Negative, 610 nm Positive, 570 nm, 600 nm Negative, 610 nm Positive, 600 nm, 650 nm Negative, 600 nm Positive, 610 nm, 650 nm
14 185 10 171 39
{ 11 mM { 46 mM2 { 3 mM { 32 mM2 —d { 5 mM2
Soft Lewis acids Pb2/ Cd2/
121 97
Positive, 600 nm, 650 nm Positive, 590 nm, 650 nm
a
0.6 { 1.0 {
0.1 mM2 0.2 mM2
e
Titrations were fit with an iterative program to two equilibria (KaleidaGraph, Synergy Software Inc.). Taken from Hollingworth et al. (1). c Taken from Gavin et al. (3). d Equilibrium for the first complex was not measurable. e Although only one complex was observed during titration, some evidence for a second complex was noted while measuring Cd2/-vesicle diffusion kinetics. b
ANALYTICAL BIOCHEMISTRY ARTICLE NO. AB972038
247, 181–183 (1997)
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
03-17-97 15:09:05
abnt
182
NOTES & TIPS
FIG. 1. Spectral changes due to the reaction of APIII with selected divalent cations under experimental conditions used for membrane transport studies. 4 mM APIII (0.05 M Hepes buffer, pH 7.4, 0.1 M NaNO3) was enclosed in small unilamellar vesicles (composed of phosphatidyl choline) by sonication. The vesicles were treated with the ionophore A23187, known amounts of metal ion were added, and the spectral change was recorded by a Hewlett–Packard diode array spectrophotometer. Following titration of the APIII, difference spectra were selected at the saturation point for each complex, first complex (—) and second complex (---). Titrations were conducted for the following cations: a, Zn2/; b, Cu2/ (the spectrum of the first complex was not completely isolated); c, Ni2/; d, Mn2/; e, Cd2/ (although the titration showed only one complex, Cd2/ –vesicle diffusion kinetic studies showed some evidence for a second complex); f, Pb2/.
with limited conformational possibilities. In order to maximize overlap between p systems, a planar conformation is favored. The most stable form of the chelator has a trans configuration at both azo links. Thus, we consider two chelating structures, a ‘‘condensed’’ form (Fig. 2a) and an ‘‘open’’ form (Fig. 2b). For ARIII these two forms have markedly different chelating properties because of the bulky and negatively charged benzenearsonic acid groups (4): (a) In the open ARIII conformation, smaller cations can complex with the ligands in the side ‘‘sockets’’ adjacent to the azo links; (b) The condensed ARIII form provides a central site which can bind well to a large cation such as calcium. Thus, ARIII yields a 1:1 complex with larger cations and a 1:2 complex with smaller cations. In contrast for APIII, smaller pyrazol derivatives replace the bulky benzenearsonic acid groups. Whether APIII is in the condensed or open conformation, (a)
smaller cations can bind at the side sockets (given bond distances of about 200 pm) and (b) there is ample room for larger cations to bind in the middle of APIII but to only a small number of ligands (given bond distances of 200–300 pm). Thus, (a) small cations form two APIII complexes binding to either or both side sockets. The first complex is characterized by a negative spectral change around 600 nm and the second complex shows a positive spectral change between 600 and about 650 nm. The titration curves are fit well using a 1:1 and 1:2 binding stoichiometry. (i) Over a relatively large concentration range only a 1:1 complex is observed for the hard Lewis acids. Hence, these cations form a 1:1 complex much more easily than a 1:2 complex. (ii) The cations that are intermediate between hard and soft Lewis acids are more likely to form a 1:2 complex at low metal concentrations. In several cases (Cu2/, Zn2/), the second metal ion binds more
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mM and K2 Å 4.42 { 0.76 m M. We suggest that cations with a higher affinity for nitrogen ligands favor the 1:2 complex by shifting the APIII conformation toward the open form as they bind with the azo link. (b) Large cations fall into two categories based on their reaction with APIII. (i) The soft Lewis acids form almost exclusively a 1:2 complex. (ii) Hard Lewis acids may not be completely coordinated by one APIII. Thus, these cations can bind two APIII (e.g., Ca2/) as well as forming a 1:1 complex. APIII spectral signals vary predictably depending on the metal ion to be studied. The large, hard Lewis acids (e.g., Ca2/) have the potential to form both 2:1 and 1:1 complexes. Small, hard Lewis acids (e.g., Mg 2/ and Mn2/) will form predominately 1:1 complexes and will provide the simplest of spectral signals with APIII (a decrease in absorbance at 600 nm). Other small divalent cations will show more complex spectral changes because both the 1:1 and 1:2 complexes easily coexist. Divalent zinc is a case in point where the second zinc actually binds more tightly than the first. This has proved advantageous for membrane transport studies where APIII provides a spectral signal for the first zinc transported and, then, a different signal for transport of the second zinc ion. Finally, the large, soft Lewis acids (e.g., Pb2/, Cd2/) tend to form a 1:2 complex almost exclusively. FIG. 2. Two stable conformations of APIII: a, condensed; b, open. APIII is shown in a stick formula with the atomic volumes shown for the azo nitrogens and the phenolic oxygens in gray dots. The phenolic oxygens are shown without their hydrogen atoms.
REFERENCES
tightly than the first (cooperative binding). We have examined the APIII reaction with zinc in the absence of lipid vesicles as well. For a range of [APIII] from 5 to 76 mM (9 determinations), K1 Å 29.2 { 5.4
1. Hollingworth, S., Aldrich, R. W., and Baylor, S. M. (1987) Biophys. J. 51, 383–393. 2. Salama, G., and Scarpa, A. (1985) J. Biol. Chem. 260, 11697– 11705. 3. Gavin, C. E., Gunter, K. K., and Gunter, T. E. (1991) Anal. Biochem. 192, 44–48. 4. Rowatt, E., and Williams, R. J. P. (1989) Biochem. J. 259, 295– 298.
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