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The apomyoglobin-arylaminonaphthalenesulfonate system: Insights into fluorescent behavior
Journal of Luminescence 18/19 (1979) 495—499 © North-Holland Publishing Company
THE APOMYOGLOBIN-ARYLAMINONAPHTHALENESULFONATE SYSTEM: INSIGHTS INTO FLUORESCENT BEHAVIOR H. DODIUK, H. KANETY and E.M. KOSOWER Department of Chemistry, Tel-Aviv University, Ramat Aviv, Tel Aviv. Israel
Binding site “polarity” of apomyoglobin has been further probed through a study of substituent effects on complexes with ANS derivatives. The p values, absence of heavy atom effects and the formation of “photoproduct” imply that the excited state behavior is mainly determined by the restrictions on motion of the ANS molecule, and cannot be described only by solvent polarity parameters.
1. Introduction Among the fluorescent probes which have been often used for biochemical and biological systems are 8-N-arylamino-l-naphthalenesulfonates (8, 1ANS (1)), 6-N-arylamino-2-naphthalenesulfonates (6, 2-ANS (2)) and 6-N-methylN-arylamino-2-naphthalenesulfonates (N-Me-6, 2-ANS (3)), see fig. 1. Weber and Laurence [1] discovered that ANS derivatives, essentially nonfluorescent in water, became highly fluorescent on binding to serum albumin. In addition, the emission maximum shifted to shorter wavelengths and the quantum yields increased greatly as the solvent used was varied from polar to non-polar. The changes in the fluorescence of the 8, 1-ANS on binding (higher cbF and blue H
_03s
~OSH~N~~X
H CH
3
4~
R
Fig. 1. 1.(X=3-Cl, 4-Cl, 4-F,H,3-CH3, 3,5-(CH3)2,3,4-(OCH3)2,4-CH3,4-OCH3); 2.(R—H,X 3,5-Cl2, 3-Br, 3-F, 4-Cl, 4-Br, 3-OCH3, 4-F, H, 3-CH3, 4-OCH3); 3. (R - CH3, X — 4-OCH3, 4-CH3, H, 3-OCH3, 4-Cl). 495
496 H. Dodiuk et a!. / Apom yoglobin AR YLAMINONAPHTHALENESULFONATE system
shift of emission maximum) were attributed to the non-polar environment of the binding site. The excited state behavior of ANS derivatives developed by Kosower and coworkers in the past few years (2—7) showed that there were two emitting states, S1~, and S1~, [np = nonpianar, Ct charge-transfer (eq. (1)] which respond differently to intramolecular changes (polarity and size of substituents, heavy atom effects) and extramolecular changes (solvent polarity and viscosity). One of the most interesting and useful distinctions between the emissions from different excited states was via the Hammett correlation coefficient (the p-value) which revealed the substantial differences in electron demand upon the substituent for the S1,~1,and S1,~states. Of particular importance is our demonstration that the viscous solvent glycerol inhibits (a) the formation of the charge-transfer state from the “naphthalene-centered” local excited state, S1~,,and (b) the promoted intersystem crossing induced by spin—orbit coupling in ANS deriva~ tives substituted with heavy atoms. T1~~+— Sj,M,
/
hi.’~
~
S0,~ hv~~ hVECE
(I)
We report in this article on a new approach to the problem of interpreting fluorescent probe behavior.
Results and discussion (I) All of the ANS derivatives tested thus far form 1: 1 complexes with apomyoglogin with relatively little variation in the dissociation constant for either 8, 1-ANS 2.7—S.4) X 10 6 derivatives M] (table 1). [KD(3.0—5.O)X10 6 Ml or 6,2-ANS derivatives [KD(The emission maxima for the ANS within the apomyoglogin complex vary (2) considerably with the nature of the substituent. For 8, 1-ANS derivatives, the maximum varies from 447 nm for 1, X 3—Cl to 490 nm for 1, X 4—OCH 3. For 6,2-ANS derivatives, the maximum varies from 404 nm (2, X 3, 5-Cl2) to 427 nm (2, X 4-C H3), (table I). (3) Correlation of the emission energies (which correspond to the maxima), after proper scaling by dividing by 2.303RT, with the Hammett substituent constants (cr) leads to correlation coefficients (p-values, ordinarily called “reaction constants”) (fig. 2). The p-values arise from rather good correlations and are different for the two series: 8,1-ANS, p —5.3 and 6, 2-ANS, p 3.8. These p-values establish the emissions for both 8,1-ANS and 6, 2-AN S derivatives as arising from S1,~,states. (4) The linear relationship between solvent polarity and the p-value (and on the position of the emission maximum) is well established for ANS derivatives (5). Starting with the p-values, we can thus estimate an approximate polarity for the —
—
H. Dodiuk et al./Apomyoglobin-ARYLAMINONAPHTHALENESULFONATE
system
497
Table 1 Emission data for ANS: apomyoglobin complexes Substituent (X)
Dissociation constant
8, 1 ANS (1)
Quantum yield qS~
Substituent constant
0.69 0.58 0.52 0.50
0.37 0.23 0.06 0.0
0.37 0.030
0.17 0.27
0.17 0.23 0.30 0.12 0.12 0.10 0.07 0.09 0.09 0.10 0.11
0.74 0.39 0.37 0.34 0.23 0.23 0.12 0.06 0.0 0.07 0.17
M 3-Cl 4-Cl 4-F H 4-CH
3 4-OCH3
447 458 464 465
3.7 3.0 3.0 3.5
x x x x
10 10 10 10
6
478 490
5.Ox 10 3.Ox 10
6
404 410 410 410 418 420 418 421 421 422 427
2.7 x 10 5.3 x 10 4.9x 10 5.ox 10 5.Ox 10 4.5 x 10 5.1 x 10 4.7 x 10 5.4x 10 5.2 x 10 5.Ox 10
6
6 6 6
6
6,2ANS(2) 3,5-Cl2 3-Br 3-Cl 3-F 4-Cl 4-Br 3-OCH3 4-F H 3-CH3 4-CH3
6 6 6 6 6 6 6 6 6 6
4F~
0.55. ±2nm; All measurements ‘At temperature were(22±1)°C; made twice ‘ ‘20%, in two different quinine sulfate concentrations in 0.1 N of H2S04, ANS.
binding site of apomyoglobin, in terms of the parameter, E 1(30). For 8, 1-ANS derivatives, the ET(30)-value is 34 kcal/mol. For 6, 2-ANS derivatives, the ET(30)-value is 42 kcal/mol. The EF versus ET(30) relationship for each compound in dioxane-water mixtures leads to the same conclusions about the “equivalent” polarity for the apomyoglobin active site. (5) Quantum yield of halogenated 6, 2 ANS were almost equal to H or greater as found in glycerol (4) and in contrast to what was found in fluid non-polar solvents. No heavy atom effect upon quantum yield is noted indicating the low intrinsic isc rates for the states involved in emission as in viscous solvents [7]. (6) N-methyl-6, 2-ANS derivatives bound to apomyoglohin give rise, to an “unusual” emission (405 nm). Further irradiation produces an emission assigned to a C-protonated photoproduct 4 [3]. The behavior of N-methyl-6, 2-ANS derivatives complexed with apoxyoglobin is similar in many respects to that of the same materials dissolved in the viscous solvent, glycerol [3].
498 H. Dodiuk et al./Apornvog!obin ARYLAMINONAPHTHALENESULFONATE system I I
S
3
F
AN
APCMY~ L
F -~
F
F N 3 ñCH
37
50H
H3.•~ ,74F
Br
br
~
4
:73/ 3 CH3.~./S
~
3~1CH~2/.
3,4 (OCH3) •4
OCH3
S
AN
APOMYOGLOBIN
A
~°I0
06
02
SUBSTTUENT
02
06
0
CONSTANTIa-)
Fig. 2. A plot of emission energies divided by 2.3OIRT (position of emission maximum expressed as kcal/mol) versus Hammett substituent constants (a-) for apomyoglobin complexes with 6-N aryl amino 2-naphthalenesulfonate derivatives (• and with 8 N arylamino I naphthalenesulfonate derivatives (•). The correlation coefficients (p values) for the lines are 3.8 for the 6. 2 ANS complexes (•) and S.3 for the 8, 1-ANS complexes (s).
3. Conclusions The important new conclusion which arises from the foregoing points is that probes are an unreliable measure for the polarity of a protein binding site. The specific interactions between the binding site and the ANS determine the “polarity” of the microenvironment. The restrictions imposed by the covalent linkages between the various components of the binding site limit the nature of the interactions with the probe. It is the combination of protein binding site and probe which determines the observed photophysical characteristics. Thus, no useful unique “polarity” can be measured, and it seems likely that the same rule will apply to the binding sites of all molecules of sufficient size. Using structurally different probes and absorption spectroscopy, Ainsworth and Flanagan
H. Dodiuk et al./Apomyoglobin-ARYLAMINONAPHTHALENESULFONATE
system
499
[8] reached a similar conclusion from measurements designed to measure the binding site “polarity” of albumin.
References [II G. Weber and D.J.R. Laurence, Biochem. J. 56 (1954) xxxi. [2] EM. Kosower, H. Dodiuk, K. Tanizawa, M. Ottolenghi and N. Orbach, J. Am. Chem. Soc. 97 (1975) 2167. [31H. Dodiuk and E.M. Kosower, J. Am. Chem. Soc. 99 (1977) 859. [4] H. Dodiuk and E.M. Kosower, Chem. Phys. Lett. 26 (1974) 545. [5] E.M. Kosower and H. Dodiuk (IDA III), J. Am. Chem. Soc. 100 (1978) 4173. [6] E.M. Kosower, H. Dodiuk and H. Kanety (IDA IV), J. Am. Chem. Soc. 100 (1978) 4179. [7] E.M. Kosower and H. Dodiuk (IDA V), J. Phys. Chem. 82 (1978) in press. [81 S. Ainsworth and M.F. Flanagan, Biochim. Biophys. Acta 194 (1969) 213.
THE APOMYOGLOBIN-ARYLAMINONAPHTHALENESULFONATE SYSTEM: INSIGHTS INTO FLUORESCENT BEHAVIOR H. DODIUK, H. KANETY and E.M. KOSOWER Department of Chemistry, Tel-Aviv University, Ramat Aviv, Tel Aviv. Israel
Binding site “polarity” of apomyoglobin has been further probed through a study of substituent effects on complexes with ANS derivatives. The p values, absence of heavy atom effects and the formation of “photoproduct” imply that the excited state behavior is mainly determined by the restrictions on motion of the ANS molecule, and cannot be described only by solvent polarity parameters.
1. Introduction Among the fluorescent probes which have been often used for biochemical and biological systems are 8-N-arylamino-l-naphthalenesulfonates (8, 1ANS (1)), 6-N-arylamino-2-naphthalenesulfonates (6, 2-ANS (2)) and 6-N-methylN-arylamino-2-naphthalenesulfonates (N-Me-6, 2-ANS (3)), see fig. 1. Weber and Laurence [1] discovered that ANS derivatives, essentially nonfluorescent in water, became highly fluorescent on binding to serum albumin. In addition, the emission maximum shifted to shorter wavelengths and the quantum yields increased greatly as the solvent used was varied from polar to non-polar. The changes in the fluorescence of the 8, 1-ANS on binding (higher cbF and blue H
_03s
~OSH~N~~X
H CH
3
4~
R
Fig. 1. 1.(X=3-Cl, 4-Cl, 4-F,H,3-CH3, 3,5-(CH3)2,3,4-(OCH3)2,4-CH3,4-OCH3); 2.(R—H,X 3,5-Cl2, 3-Br, 3-F, 4-Cl, 4-Br, 3-OCH3, 4-F, H, 3-CH3, 4-OCH3); 3. (R - CH3, X — 4-OCH3, 4-CH3, H, 3-OCH3, 4-Cl). 495
496 H. Dodiuk et a!. / Apom yoglobin AR YLAMINONAPHTHALENESULFONATE system
shift of emission maximum) were attributed to the non-polar environment of the binding site. The excited state behavior of ANS derivatives developed by Kosower and coworkers in the past few years (2—7) showed that there were two emitting states, S1~, and S1~, [np = nonpianar, Ct charge-transfer (eq. (1)] which respond differently to intramolecular changes (polarity and size of substituents, heavy atom effects) and extramolecular changes (solvent polarity and viscosity). One of the most interesting and useful distinctions between the emissions from different excited states was via the Hammett correlation coefficient (the p-value) which revealed the substantial differences in electron demand upon the substituent for the S1,~1,and S1,~states. Of particular importance is our demonstration that the viscous solvent glycerol inhibits (a) the formation of the charge-transfer state from the “naphthalene-centered” local excited state, S1~,,and (b) the promoted intersystem crossing induced by spin—orbit coupling in ANS deriva~ tives substituted with heavy atoms. T1~~+— Sj,M,
/
hi.’~
~
S0,~ hv~~ hVECE
(I)
We report in this article on a new approach to the problem of interpreting fluorescent probe behavior.
Results and discussion (I) All of the ANS derivatives tested thus far form 1: 1 complexes with apomyoglogin with relatively little variation in the dissociation constant for either 8, 1-ANS 2.7—S.4) X 10 6 derivatives M] (table 1). [KD(3.0—5.O)X10 6 Ml or 6,2-ANS derivatives [KD(The emission maxima for the ANS within the apomyoglogin complex vary (2) considerably with the nature of the substituent. For 8, 1-ANS derivatives, the maximum varies from 447 nm for 1, X 3—Cl to 490 nm for 1, X 4—OCH 3. For 6,2-ANS derivatives, the maximum varies from 404 nm (2, X 3, 5-Cl2) to 427 nm (2, X 4-C H3), (table I). (3) Correlation of the emission energies (which correspond to the maxima), after proper scaling by dividing by 2.303RT, with the Hammett substituent constants (cr) leads to correlation coefficients (p-values, ordinarily called “reaction constants”) (fig. 2). The p-values arise from rather good correlations and are different for the two series: 8,1-ANS, p —5.3 and 6, 2-ANS, p 3.8. These p-values establish the emissions for both 8,1-ANS and 6, 2-AN S derivatives as arising from S1,~,states. (4) The linear relationship between solvent polarity and the p-value (and on the position of the emission maximum) is well established for ANS derivatives (5). Starting with the p-values, we can thus estimate an approximate polarity for the —
—
H. Dodiuk et al./Apomyoglobin-ARYLAMINONAPHTHALENESULFONATE
system
497
Table 1 Emission data for ANS: apomyoglobin complexes Substituent (X)
Dissociation constant
8, 1 ANS (1)
Quantum yield qS~
Substituent constant
0.69 0.58 0.52 0.50
0.37 0.23 0.06 0.0
0.37 0.030
0.17 0.27
0.17 0.23 0.30 0.12 0.12 0.10 0.07 0.09 0.09 0.10 0.11
0.74 0.39 0.37 0.34 0.23 0.23 0.12 0.06 0.0 0.07 0.17
M 3-Cl 4-Cl 4-F H 4-CH
3 4-OCH3
447 458 464 465
3.7 3.0 3.0 3.5
x x x x
10 10 10 10
6
478 490
5.Ox 10 3.Ox 10
6
404 410 410 410 418 420 418 421 421 422 427
2.7 x 10 5.3 x 10 4.9x 10 5.ox 10 5.Ox 10 4.5 x 10 5.1 x 10 4.7 x 10 5.4x 10 5.2 x 10 5.Ox 10
6
6 6 6
6
6,2ANS(2) 3,5-Cl2 3-Br 3-Cl 3-F 4-Cl 4-Br 3-OCH3 4-F H 3-CH3 4-CH3
6 6 6 6 6 6 6 6 6 6
4F~
0.55. ±2nm; All measurements ‘At temperature were(22±1)°C; made twice ‘ ‘20%, in two different quinine sulfate concentrations in 0.1 N of H2S04, ANS.
binding site of apomyoglobin, in terms of the parameter, E 1(30). For 8, 1-ANS derivatives, the ET(30)-value is 34 kcal/mol. For 6, 2-ANS derivatives, the ET(30)-value is 42 kcal/mol. The EF versus ET(30) relationship for each compound in dioxane-water mixtures leads to the same conclusions about the “equivalent” polarity for the apomyoglobin active site. (5) Quantum yield of halogenated 6, 2 ANS were almost equal to H or greater as found in glycerol (4) and in contrast to what was found in fluid non-polar solvents. No heavy atom effect upon quantum yield is noted indicating the low intrinsic isc rates for the states involved in emission as in viscous solvents [7]. (6) N-methyl-6, 2-ANS derivatives bound to apomyoglohin give rise, to an “unusual” emission (405 nm). Further irradiation produces an emission assigned to a C-protonated photoproduct 4 [3]. The behavior of N-methyl-6, 2-ANS derivatives complexed with apoxyoglobin is similar in many respects to that of the same materials dissolved in the viscous solvent, glycerol [3].
498 H. Dodiuk et al./Apornvog!obin ARYLAMINONAPHTHALENESULFONATE system I I
S
3
F
AN
APCMY~ L
F -~
F
F N 3 ñCH
37
50H
H3.•~ ,74F
Br
br
~
4
:73/ 3 CH3.~./S
~
3~1CH~2/.
3,4 (OCH3) •4
OCH3
S
AN
APOMYOGLOBIN
A
~°I0
06
02
SUBSTTUENT
02
06
0
CONSTANTIa-)
Fig. 2. A plot of emission energies divided by 2.3OIRT (position of emission maximum expressed as kcal/mol) versus Hammett substituent constants (a-) for apomyoglobin complexes with 6-N aryl amino 2-naphthalenesulfonate derivatives (• and with 8 N arylamino I naphthalenesulfonate derivatives (•). The correlation coefficients (p values) for the lines are 3.8 for the 6. 2 ANS complexes (•) and S.3 for the 8, 1-ANS complexes (s).
3. Conclusions The important new conclusion which arises from the foregoing points is that probes are an unreliable measure for the polarity of a protein binding site. The specific interactions between the binding site and the ANS determine the “polarity” of the microenvironment. The restrictions imposed by the covalent linkages between the various components of the binding site limit the nature of the interactions with the probe. It is the combination of protein binding site and probe which determines the observed photophysical characteristics. Thus, no useful unique “polarity” can be measured, and it seems likely that the same rule will apply to the binding sites of all molecules of sufficient size. Using structurally different probes and absorption spectroscopy, Ainsworth and Flanagan
H. Dodiuk et al./Apomyoglobin-ARYLAMINONAPHTHALENESULFONATE
system
499
[8] reached a similar conclusion from measurements designed to measure the binding site “polarity” of albumin.
References [II G. Weber and D.J.R. Laurence, Biochem. J. 56 (1954) xxxi. [2] EM. Kosower, H. Dodiuk, K. Tanizawa, M. Ottolenghi and N. Orbach, J. Am. Chem. Soc. 97 (1975) 2167. [31H. Dodiuk and E.M. Kosower, J. Am. Chem. Soc. 99 (1977) 859. [4] H. Dodiuk and E.M. Kosower, Chem. Phys. Lett. 26 (1974) 545. [5] E.M. Kosower and H. Dodiuk (IDA III), J. Am. Chem. Soc. 100 (1978) 4173. [6] E.M. Kosower, H. Dodiuk and H. Kanety (IDA IV), J. Am. Chem. Soc. 100 (1978) 4179. [7] E.M. Kosower and H. Dodiuk (IDA V), J. Phys. Chem. 82 (1978) in press. [81 S. Ainsworth and M.F. Flanagan, Biochim. Biophys. Acta 194 (1969) 213.