J. Phys. Chem. 1983,87, 1329-1337
Since the decomposition temperatures for KN03 and KzO are 400 and 350 "C, respectively,the added potassium (treated by evacuation at 400 "C) is likely to be in a state which can serve as a good electron donor for Pt. We
i-...
4
90A
90A /
1329
80A
\
" '
1
5
1
10 x10-3
1
2
x10-1
K l P t ATOMIC RATIO
Flgure 1. H2 uptake as a function of potassium added to Pt/TiO, samples. The of upper curve is the total amount of hydrogen uptake at 40 torr of H., The lower curve is the amount of strongly chemisorbed hydrogen that can not be removed by evacuation at 300 K of a sample exposed to 40 torr of H,. The numbers along the lower curve are the average Pt particle sizes determhed by analysis of XRD peak broadening.
particle size marked along the lower curve is also nearly constant (95 f 15 A) while the bulk K / P t atomic ratio varies from 0.00 to 0.20 as indicated along the x axis of Figure 1. Therefore, the observed suppression of H2 chemisorption ability by Pt is not due to metal sintering.
suggest that the suppression of Hzchemisorption ability in these Pt/Ti02/K systems is consistent with a scheme involving electron transfer from potassium to the platinum. Samples 8 and 9 in Table I were exposed to air for extended periods after preparation and, by XPS, showed no surface Ti3+.* Upon reduction and evacuation at 200 "C, no increment in Ti3+was found (compare 8a with 8c and 9a with 9c). This is consistent with there being very little Ti3+a t the surfaces of these materials but relatively large concentrations in the bulk. In summary, we have shown that small amounts of potassium convey SMSI-like properties to Pt/TiO,. When K is added, no additional Ti3+is formed. We attribute the loss of Hz chemisorption capacity to negative charge transfer from K to Pt. ESR measurements show that surface Ti3+ is not required for SMSI behavior. SMSI (suppression of H2 chemisorption) appears to be a very general phenomenon in Pt/TiO, samples requiring only that a source of electrons be available that will transfer small amounts of charge to Pt. Acknowledgment. This work was supported in part by the Office of Naval Research. Registry No. K, 7440-09-7; Pt, 7440-06-4; TiOz, 13463-67-7; HP,1333-74-0; Ti3+,22541-75-9.
Molecular Orbital Calculations of the Optical Properties of Congo Red and Cibacron Blue and Their Complexes with Proteins Robert A. Edwardst and Robert W. Woody" Department of Biochemistry. cdoredo State Universw, Fort Collins, Colorado 80523 (Received:Ju& 3 I, 198 1; In Final Form: November 22, 1982)
The dyes congo red and cibacron blue have been used as spectroscopic probes of protein conformation. We have performed semiempirical molecular orbital calculations in the s-electron approximation on these dyes to provide information about their spectroscopicproperties. These calculations account well for the position and intensity of the absorption bands of congo red. For cibacron blue, the intensities are predicted well, but the wavelengths show significant discrepancies, with the calculated long-wavelengthtransitions being blue shifted by about 120 nm. Calculations of rotational strengthsfor chiral conformations, selected to mimic the conformation of reduced nicotinamide adenine dinucleotide bound to dehydrogenases,yield values compatible with the observed CD spectrum of the enzyme-bound dyes, indicating that the intrinsic contributions could be responsible for the observed CD. Calculations were also performed on the coupling of the dye transitions with the ss* transitions of an indole group, using a large range of relative geometries of the dye and the indole group. These calculations showed that coupling with protein groups is not likely to be a significant factor in the induced CD of congo red but may be comparable to the intrinsic contributions for cibacron blue. The implications of these results for interpreting the induced CD spectra of dye-dehydrogenase complexes are discussed. The effects of dye aggregation on the spectral properties of these dyes are also discussed, using the results of the MO calculations.
The circular dichroism induced in the spectrum of a ligand upon binding to a macromolecule is determined by the stereoisomeric geometry of the ligand and its interaction with the surrounding environment.' It is only possible to obtain information about the conformation of
the bound ligand after distinguishing between intrinsic optical activity, which results from a dissymetric conformation of the bound molecule, and extrinsic optical activity, which results from the interaction of the bound molecule with a dissymetric environment. This distinction
'Present address: Department of Chemistry, University College of Swaziland, P / B Kwaluseni, Swaziland.
(1) G. Blauer in "Structure and Bonding", Vol. 18,J. D. Dunitz e t al., Ed., New York, 1974, p 69.
0022-365418312087-1329$01.50/0
0 1983 American Chemical Society
The Journal of Physical Chemistty, Vol. 87, No. 8, 1983
1330 Chart I
.f I
so; 43
w
6
s6;
CONGO R E D
CI
CIBACRON
BLUE
is important because it differentiates between the weak bonding and steric interactions of the macromolecule with the ligand, which determine the conformation of the bound molecule, and the effect of interactions involving macromolecule and ligand, which do not influence the ligand conformation but do cause spectral perturbation^.^-^ In two previous papers4v5we have reported the CD spectra induced when congo red or cibacron blue (Chart I) are bound to dehydrogenasesand kinases. In the present paper, we describe a theoretical approach to the analysis of the induced CD in such systems, especially to resolving the relative importance of intrinsic and extrinsic sources of optical activity. The intrinsic contributions to the optical activity are calculated by using semiempirical molecular orbital theory. The extrinsic contributions are estimated by performing calculations of the coupled-oscillator interactions between the dye molecule and the indole chromophore of a tryptophan side chain in a variety of different relative orientations. A tryptophan side chain was chosen as representative of the aromatic side chains occurring in proteins. Such side chains will, in all probability be the dominant contributor to the extrinsic optical activity of bound dye chromophores, as they are in the case of the heme groups in myoglobin and hemoglobin.6 Furthermore, because of its low symmetry and the low energy of its allowed mr* transitions, the contributions of the indole chromophore can be expected to be larger than those of the other aromatic chromophores which occur in proteins. Since we do not initially know either the conformation of the bound dyes or their orientation relative to the protein groups, we sample possible conformations and orientations. The conformations were found by compar(2)J. F. Towell, 111, Ph.D. Thesis, Colorado State University, 1977. (3)R. A. Edwards, Ph.D. Thesis, Colorado State University, 1979. (4)R. A. Edwards and R. W. Woody, Biochemistry, 18,5197 (1979). (5)R.A. Edwards and R. W. Woody, Biochem. Biophys. Res. Commun., 79,470 (1977). (6)M.-C. Hsu and R. W. Woody, J.Am. Chem. Soc., 93,3515(1971).
Edwards and Woody
isons to molecular modeh3s4 The orientations were sampled by systematically rotating an indole chromophore around centers located near spectrally significant regions of the dye chromophores. The semiempirical molecular orbital (MO) calculations which we use treat only the r-electrons, in what is referred to as the PPP method (after Pariser, Parr, and P ~ p l e ~ , ~ ) . This method has been used extensively for calculations of absorption spectra for aromatic system^.^^^ From the MO wave functions, we calculate both oscillator strengths ( f ) and rotational strengths ( R ) in the dipole-velocity appro~imation.~J~ After calculating the spectral properties intrinsic to a chromophore, we couple the chromophores together using a monopole-monopole approximation,ll in order to calculate extrinsic effects. This approximation, which distributes the transition charge density among individual atomic centers on the molecule, is considered to be superior to the dipole-dipole approximation, which treats the transition charge density as a property of the chromophore as a whole. A more accurate description of the transition, as it depends on the size and geometry of the coupled chromophores, is achieved. The monopoles are also useful in conveying information about the localization of transitions. The transition monopole charges are readily obtainable from the wave functions. This study has provided considerable insight into the spectral properties of the two specific dyes under consideration. It also illustrates the potential of this approach for the analysis of other chromophore systems.
Methods The computer program SCF CIO written by Bloor and Gilson12was modified to calculate rotational strengths13 and to incorporate the variable$ approximation of Nishimoto and Forster14J5for heteroaromatic systems. Nishimoto and Forster's parameterization did not extend to azo compounds. By analogy, we use PN-N
= - 0 . 5 6 ~- 1.81
(1)
where 6 is the resonance integral and p the density matrix element for the bonded nitrogens. For a full N-N double bond ( p = 1.01, eq 1 gives P = -2.37 eV. This value was derived from p, = -2.379 eV for ethylene, using the Wolfsberg-Helmholz16 relationship. Self-consistency in the density matrix was considered to have been obtained when the largest deviation in ,f3 values between successive iterations was less than 0.01 eV. Two-center repulsion integrals were calculated by the Nishimoto-Mataga formula.17 Overlap integrals, required in the calculation of rotational strengths, were interpolated from the numerical tables of Mulliken et a1.18 (7)J. N. Murrell and A. J. Harget, "Semi-Empirical Self Consistent Field Molecular Orbital Theory of Molecules", Wiley-Interscience, London, 1972,Chapter 2,p 11. (8)R. G.Parr, *Quantum Theory of Molecular Electronic Structure", Benjamin, New York, 1963. (9)J. N.Murrell, "The Theory of the Electronic Spectra of Organic Molecules", Wiley, New York, 1963. (10) W. Moffitt, J. Chem. Phys., 25, 467 (1956). (11)I. Tinoco, Jr., Adu. Chem. Phys., 4, 113 (1962). (12)J. E. Bloor and B. R. Gilson,Quantum Chem. Program Exchange, 10,71 (1965). (13)J. C. Smith and R. W. Woody, J. Phys. Chem., 80,1094 (1976). (14)K.Nishimoto and L. S. Forster, Theor. Chim. Acta (Bed.), 3,407 (1965). (15)K. Nishimoto and L. S. Forster, Theor. Chim. Acta (Bed.), 4,155 (1966). (16)M. Wolfsberg and L. Helmholz, J. Chem. Phys., 20,837 (1952). (17)K. Nishimoto and N. Mataga, Z. Phys. Chem. (Frankfurt am Main), 12, 335 (1957).
MO Calculations for Congo Red and Cibacron Blue
The Journal of Physical Chemistry, Vol. 87,No. 8, 1983 1331
B
Flgure 1. Stereodrawings of (A) congo red in conformation 1 of Table I V and B cibacron blue in the conformation proposed by Thompson et
Configuration interaction was considered, including the singly excited configurations resulting from transitions from the six highest occupied orbitals to the six lowest virtual orbitals. Those configurations in which initial and final MO energy levels are separated by more than 6 MO levels were excluded. Thus 21 configurations were included in the configuration interaction. The geometries of the dye molecules were calculated by assuming the bond angles, bond lengths, and dihedral angles shown in Table I. Dihedral angles were derived by twisting Corey-Pauling-Koltun models of the dyes to match the photographs of Thompson et al.19or Stellwagen,2o or to mimic a model of the nicotinamide adenine dinucleotide (NAD) based on the conformation of the bound coenzyme.21,22Figure 1shows stereoviews of two of the dye conformations considered in the calculation. Twisting was predominantly around the formal single bonds, although it was necessary to introduce 15-25' twists around the nitrogen-nitrogen bonds of congo red in order to bring the terminal aromatic rings into conformity with the bases of the bound coenzyme. Since twisting around these nitrogen-nitrogen double bonds is certainly unfavorable, the absorption spectrum for the free dye was calculated for a conformation lacking these twists. The geometry used for indole was taken from the crystal (18)R. S.Mulliken, C. A. Rieke, D. Orloff, and H. Orloff, J. C h e n . Phys., 17, 1248 (1949). (19)S.T. Thompson, K. H. Caw,and E. Stellwagen, R o c . Natl. Acad. Sci. U.S.A.72,669 (1975). (20)E.Stellwagen, Acc. Chem. Res., 10,92 (1977). (21)M. J. Adams. A. McPherson. Jr.. M. G. Rossmann. R. W. Schevitz, and A. J. Wonacott, J. Mol. Biol., 51, 31 (1970). (22)K. Chandrasekhar, A. McPherson, Jr., M. J. Adams, and M. J. Rossman, J. Mol. Biol., 76,503 (1973).
structure of 3-indolylacetic acidsz3 Coupled-oscillator calculations were based on a transition monopole-monopole interaction potentiall' and coupling with the permanent dipole (one-electron effect) has been neglected. The monopoles were taken from the Telectron calculations in the dipole-length approximation but were improved by scaling them to experimental oscillator strengths and energies (for indole) or to the geometric mean of the oscillator strengths calculated by the dipole-velocity and dipole-length methods. (This geometric mean provides the best approximation to observed oscillator strengths for most electronic t r a n ~ i t i o n s , ~even ~J~ though the long-wavelength band of aromatic systems is best approximated by the dipole-velocity m e t h ~ d . ~The ) interaction between these sets of scaled monopoles gives an interaction potential matrix which describes how the individual oscillators (transitions) are mixed to yield a final series of transitions for the whole system. The final oscillator strengths resulting from the coupled-oscillator calculation are in the dipole-velocity approximation to make them comparable to the monomer results from the T-electron calculations. In order to conveniently compare calculated and experimental spectra, we reduced the oscillator and rotational strengths to graphic form by assuming the spectra to be composed of Gaussian bands centered at the calculated energies. The bandwidth for the long-wavelength band was taken from experimental spectra. For the higher en(23)I. L.Karle, K. Britts, and P.Gum, Acta Crystallogr., 17,496 (1964). (24)R. Harris, J . Chem. Phys., 50,3947 (1969). (25)T. Yoshinaga, H. Hiratsuka, and Y. Tanizaki, Bull. Chem. SOC. Jpn., 50,2548 (1977).
Edwards and Woody
The Journal of Physical Chemistty, Vol. 87, No. 8, 1983
1332
8
TABLE I : Bond Angles, Bond Lengths, and Dihedral Angles Used To Derive Molecular Geometries for Congo Red and Cibacron Blue congo red
cibacron blue
all are 120"
Bond Angles all are 120" except in the central anthraquinone ring C-Cqmone-C intraring, 117.5
C-C in aromatic rings C-C central bond C-N ring to azo
C-N ring to amine nitrogens N = N azo
Bond Lengths, A 1.39 C-C in aromatic rings 1.48 C-C quinone C to outer rings 1.41 C-N ring to amine nitrogens 1.38 C-N in triazine ring 1.23 C = O quinone
X
- 2
1.39 1.50 1.39
300
200
400
500
600
700
WAVELENGTH ( n m )
1.34 1.23 1.5
Dihedral Angles, deg conformation bond
la
dihedral angles bond
26
(1)C-N naphthyl t o azo ( 2 ) N = N azo
-15 -10 (1)anthraquinone t o nitrogen t 1 5 -25 ( 2 ) nitrogen to phenyl ( 3 ) N-C azo to - 1 5 -30 ( 3 ) phenyl to phenyl nitrogen ( 4 ) central bond -30 -30 ( 4 ) nitrogen to triazine ( 5 ) phenyl to azo 0 0 ( 5 ) triazine to nitrogen ( 6 ) azo -15 -25 ( 6 ) nitrogen t o phenyl ( 7 ) azo to naphthyl 0 0
conformation 1c
-40
1.0
X
0.5
45
+ 5 -45 200
300
t 4 5 +45 -50 + 4 5 t 80 -45
--45
0
ergy bands, the bandwidth was found by using the empirical relationship26 (2) where 6 is the bandwidth, k is an empirical constant taken from the long-wavelength band, and X is the wavelength of the transition. Absorption and CD spectra were recorded at 25 f 1 "C in 10 mM Tris-HC1buffer as described previo~sly.~!~ k~312
Results Spectroscopic Properties of the Free Dyes. The absorption spectrum of congo red (Figure 2A) has peaks at 500,342, and 235 nm, which undergo blue shifts and exhibit hypochromism at increased dye concentration. The insert shows that the hypochromism becomes significant at dye concentrations above M in 10 mM Tris-HC1 buffer (pH 7.5). In buffers of higher ionic strength, both the blue shift and hypochromism can be observed at lower concentrations. The energies of the calculated absorption bands agree with the experimental energies very well and the intensities agree to within a factor of 1.5. This qualitative agreement was expected from our previous calculations on azobenzene compound^.^ The long-wavelength band is actually composed of two transitions (Table 11). The lowest energy transition is allowed while the higher energy one is forbidden. Several transitions are involved in the band at 342 nm, and there
400 500 WAVELENGTH ( r r r )
73 0
600
Figure 2. (A) Absorption spectra of congo red. Experimental spectra for 1.3 pM (-) and 0.62 mM (- -). Calculated spectra for monomer (- - -) and dimer (. .). The insert shows the extinction at 500 nm as a function of the negative log of the total congo red concentration. The solid line of the insert is for K,D = 100 pM and KAD= 16 pM (see text). (B) Absorption spectra of cibacron blue. Experimental spectra for 1.5 pM (-) and 0.26 mM (- -). Calculated spectra for monomer (---) and dimer (. .). The insert shows the extinction at 610 nm as a function of the negative log of the total cibacron blue concentration.
-
-
-
TABLE 11: Calculated Spectral Properties" of Congo Red and Cibacron Blue congo red
cibacron blue
oscillator oscillator wavelengthb strengthC wavelengthb strengthC 489 442 339 332 314 310 299 286 258 256 24 9 244
1.24 0.00 0.15 0.00 0.35 0.01 0.01 0.21 0.00 0.06 0.07 0.00
490 34 1 314 294 281 277 274 26 9 26 2 256 24 8 24 4
0.17 0.12 0.08 0.02 0.00 0.26 0.04 0.07 0.03 0.10 0.01 0.00
a These calculations are for an* transitions only. A nn* transition is expected in the 420-480-nm region for congo red. n a * transitions are also anticipated in cibacron blue, but their location is not known a t present. Wavelength in nm. Oscillator strength by dipole velocity approximation.
is a multitude of higher energy transitions (not included in Figure 2A because of the limits of our configuration interaction). In the 420-480-nm range there are n r * transitions involving nonbonding orbitals composed of the u lone pairs on the azo nitrogens. These nr* transitions are observed at approximately 450 nm in several aromatic azo compounds, and their energy is not drastically influenced by substitution on the aromatic rings.27 However, hydrogen
(26) A. Brown, C. M. Kemp, and S. F. Mason, J . Chem. SOC.A, 751 (1971).
5
0
2d
a First conformation designed to mimic N A D H bound t o A second conformation which lactic dehydrogenase. Conformamore closely mimics the NADH base planes. tion of Thompson e t al." Conformation of Stellwagen.2q
6=
UI
( 2 7 ) H. Rau, Angew. Chem., Int. E d . Engl., 12, 224 (1973).
The Journal of Physical Chemistry, Vol. 87, No. 8, 1983
MO Calculations for Congo Red and Cibacron Blue n
1333
TABLE 111: Frequency Shifts of Amino-Substituted 9,lO-Anthraquinones Relative to
1-Amino-9,lO-anthraquinone AV
compd
theoP
exptlb
1,4-diamino 1,5-diamino 1,B-diamino 2-amino 2,6-diamino
-3500 -400 -900 1600 3900
-3300,c -2900 -3720e -500 -600,d-500e + 200) 2600: 1300,d 900e 4000,h 2500d
a Calculated frequency of the long-wavelength band relative t o that for 1-aminoanthraquinone. In cm-'. Experimental frequency of the long-wavelength band relative t o that of 1-aminoanthraquinone in the same solvent (see, however, footnotes f and h ) . In some systems vibrational fine structure is observed, generally with two distinct maxima and a shoulder a t higher frequency. In such cases, the higher frequency discrete band is used, which is the most intense in nearly all cases. In cm-'. Dioxanehexane mixture ( 1 : 4 ) ,ref 29. Ethanol, ref 29. e 2Propanol, ref 30. N o direct comparisons could be found. The value given is based on data for 1,B-diaminoanthraquinone in dioxane vs. 1-aminoanthraquinone in 1:4 dioxane-hexane, ref 29. g Heptane, ref 29. 2,6Diaminoanthraquinone in hexane vs. 1-aminoanthraquinone in heptane, ref 29.
Flgure 3. Transtin monopoles of congo red. The transitions are listed in Table 11. The magnitude of the transition charge density is proportional to the diameter of the octagon on each atom. Open and closed symbols indicate transition charge density of opposite signs. The wavelengths and oscillator strengths identify the transition for each set of monopoles: (A) 489 nm, f = 1.24; (8)442 nm, f = 0.00; (C) 339 nm, f = 0.15; (D) 332 nm, f = 0.00 (E) 314 nm, f = 0.35.
bonding between these orbitals and the amine hydrogens on the naphthyl rings may have a larger effect than is observed with most substituents. These n r * transitions will have small oscillator strengths but may have significant rotational strengths. The transition monopoles calculated for congo red are shown in Figure 3. Strong transition density is observed on atoms throughout the chromophore, except the carbons on the outermost ring of the naphthyls. The nitrogens show particularly large monopoles. The longest wavelength transition (Figure 3A) has a high transition dipole because the symmetrically placed monopoles on opposite sides of the molecular center have opposite signs and thus they contribute additively to a large transition dipole, with its positive end on one side of the molecule and its negative end on the other. In contrast, the second longest wavelength transition (Figure 3B) is forbidden electrically, because the symmetry-related monopoles on opposite sides of the molecular center have the same sign, thus cancelling. Although large monopoles are present for most of the transitions, symmetrical cancellation gives rise to the zero oscillator strengths reported for many of the transitions in Table 11. For conformations in which the exact C2 symmetry axis through the molecule center (perpendicular to the page in Figure 3) is destroyed, the transitions which are forbidden in the symmetrical molecule have low oscillator strengths. Less symmetric conformations are more realistic models of the conformational space sampled by the molecule in solution, but cancellation across the molecular center is still predominant, with the weak transitions polarized predominantly in a plane perpendicular to the long axis of the molecule. In general, the twisted conformations
give absorption properties similar to those of the conformation with the C2symmetry axis (results not shown). The absorption spectrum of cibacron blue (Figure 2B) shows a broad band with its maximum at 610-615 nm and another band at 257 nm which has shoulders a t approximately 280 and 375 nm. At concentrations above 5 X lo4 M, cibacron blue also shows a hypochromic deviation from Beer's law, but the absorption bands are red shifted. The energies of the calculated absorption bands for cibacron blue are larger than those observed experimentally, but the intensities are calculated very well. Only one transition is involved in the longest wavelength band (Table 11). There are several shorter wavelength transitions contributing to the 257-nm band with one strong transition calculated at 341 nm giving the shoulder on the long-wavelength side. a-electron calculations in the variable-@appr~ximation'~ overestimate the aa*-transition energies for 1,4-diaminoanthraquinone and N-phenyl-1,4-diaminoanthraq~inone.~ The discrepancy is ca. 4000 cm-l which, for these transitions near 600 nm, corresponds to nearly a 100-nm blue shift of the calculated absorption maximum relative to the experimental spectrum. The present results agree well with those reported by Inoue et a1.,28who applied the variable-@approximation to 1,4-diaminoanthraquinone.Inoue et al. also reported calculations on 1-aminoanthraquinone and 1,4,5,8-tetraaminoanthraquinone, and the energy of the long wavelength band was calculated to be ca. 4000 cm-l higher than that observed in all cases. Calculations on various mono- and diaminoanthraquinones have been performed. The 1-amino, 2-amino, 1,5-diamino, l,&diamino, and 2,6-diamino derivatives of 9,lO-anthraquinone all show a discrepancy comparable to that found for the 1,4-diamino derivative. Nevertheless, the calculations accurately reproduce the observed trends within this group of chromophores. This is illustrated in Table 111, where the observed frequencies of the longwavelength band for the various derivatives relative to that for the 1-amino derivative are compared with the calculated frequency differences. With the possible exception (28) H.Inoue, T. Hoshi, J. Yoshino, and Y.Tanizaki, Bull. Chem. SOC. Jpn., 45, 1018 (1972).
1334
The Journal of Physical Chemistty, Vol. 87,No. 8, 1983
TABLE IV: Calculated Rotational Strengths for Congo Reda n-electron calculations
Edwards and Woody
_ I -
I
conformationb
0,0,45'
I_
h
1
2
h
490
-1.78 0.81 1.00
2.92 --4.32 0.79 0.50 3.64 -0.45 -4.79 0.89
500 440 340 330 3 20 310 300 290 280e 270e 260 260
440
340 330 310 310 300 290 260 260
0.22
4.02 --1.53 -4.21 1.02 0.26 0.02
0.43 -0.05
-0.11 0.00 0.10 0.00
0.11 0.00 0.00 0.14 --0.06 0.21 -0.41
0.00
dye coupled t o indole 0,0,45
(2) 0.56 -0.02 0.09 0.00 0.24 -0.01 -0.01 0.17 0.14
0.00 -0.27 0.00
-90,0,45 (3) -0.62 0.12 -0.26
0.02 -0.17 0.01 0.1 2
-0.04 -0.02 -0.02 0.05 0.00
-90,45,45 (4)
-0.29 0.00 -0.11 -0.04
-0.10 -0.04 -0.58 -0.08 0.01 0.89 -0.02 0.00
-90,45,45 (5) -0.30 0.01 -0.13 -0.03 -0.12 -0.04 -
0.50
-0.06
0.02 0.67 0.01 0.00
0,90,90 (6) -0.31 -0.14 --0.04
0.02 0.07 -0.01 0.03 0.04 0.02
-0.01 -0.05 0.00
These calculations are for m* transitions only. Rotational strengths in Debye-Bohr magnetons. Wavelengths ( h ) t o the Indole rotation in degrees around three axes, in the See Table I for information on these conformations. closest 10 nm. order given. (1)The long axis of the molecule. ( 2 ) An axis perpendicular to the first axis in the plane of the molecule. ( 3 ) An axis defined by the cross product of the first two. The directions of the first two axes are shown on the congo red structural formula. The long axis of the indole was initially parallel to the first axis. The indole plane was in the plane defined by the first two axes with the indole nitrogen having a positive coordinate along the second axis. A positive rotation is clockwise Location of the indole center a t van der Waals distance as viewed with the axis of interest pointing toward the observer. from the dye atoms in the numbered locations on the congo red structural formula. (Location 4 is in the plane of the molecule and location 5 is 1.5 X directly above location 4.) e These coupled transitions are over 99% indole transitions. a
of the l,&diamino derivative, for which adequate data are not available, the agreement between theoretical and observed frequency differences is very satisfactory. From this table it is clear that whatever the source of the discrepancy, it is characteristic for all of the derivatives considered. The finding that the 2-amino and 2,6-diamino derivatives behave similarly to the 1-amino compounds indicates that intramolecular hydrogen bonding is not responsible for the anomalous results. We have assumed that the CO bond is like a standard carbonyl with a bond length of 1.23 8, while the CN bond is taken to be typical of aromatic amines with a bond length of 1.39 A. Effectively, these bond lengths imply a neglect of charge transfer in the ground state. On the other hand, if one assumes charge transfer in the ground state, the CO bond length should be like that in phenolate ions &e., 1.36A) and the CN bond length like that in iminium ions (ca. 1.30 A). Alternative geometries are currently being investigated to determine whether this factor may account for the apparent shortcomings of the variable-@approximation in these systems. All of the visible and near-UV transitions are localized on the anthraquinone and the first phenyl ring, as is shown in Figure 4. This is consistent with the spectra of phenyl derivatives of 1,4-diaminoanthraquinone,which also show absorbance peaks near 600 and 390 nmS3g3l Thus the dihedral angles around the bonds linking the nitrogen to the anthraquinone and the first phenyl ring are the determining factors for spectral perturbations of the cibacron blue visible spectrum. The most probable conformations of cibacron blue in solution have smaller twists around the formal single bonds than the conformations which mimic the coenzyme, for which the results of Figure 2B, Figure 4B, and Table I11 were calculated. However, there is no cancellation of the cibacron blue monopoles due to symmetry or pseudospymmetry, and calculations on a planar molecule (results not shown) gave essentially the same results for absorption spectra as those shown.
Figure 4. Transition monopoles of cibacron blue. The transitions are listed in Table 11. Open and closed octagons indicate transition charge density of opposite signs, with magnitudes proportional to the diameter of the symbol. (A) 490 nm, f = 0.17; (B) 341 nm, f = 0.12; (C)314 nm, f = 0.08; (D) 294 nm, f = 0.02; (E) nm, f = 0.00:(F) 277 nm, f
(29) N. A. Shcheglova, D. N. Shigorin, and N. S. Dokunikhin, Russ. J . Phys. Chem., 42, 1449 (1968). (30) G. S. Egerton and A. G. Roach, J. SOC.Dyers. Colour.. 74. 401
= 0.26.
(1958).
The intrinsic rotational strengths calculated for two congo red conformations and the rotational strengths due to
(31)E. Gurr. "Synthetic Dyes in Biology, Medicine and Chemistry". Academic Press, Nevi York, 1971.
Spectroscopic Properties of Dye-Enzyme Complexes.
The Journal of Physical Chemlstry, Vol. 87, No. 8, 1983
MO Calculations for Congo Red and Cibacron Blue 34
IA
400
300
600
500
WAVELENGTH
(nm)
11 4
300
I
I
I
400
500
600
WAVELENGTH
(nm)
Flgwe 5. (A) CD spectra of congo red. Experimental spectra for heart lactic dehydrogenase (36 pM)-congo red (10.4 pM) (-). Calculated spectra of the two congo red conformations of Table I V : first conformation (.* .); second conformation (---). (6)CD spectra of cibacron blue. Experimental spectra for heart lactic dehydrogenase (50 pM)-cibacron blue (23 pM) (-) and yeast alcohol dehydrogenase (43 pM)-cibacron blue (20 pM) (- -). Calculated spectra for cibacron blue conformation of Thompson and co-worker~'~ (. ..) and of StellwagenZ0 (---) for 10 pM concentration.
.
coupling are given in Table IV. In Figure 5a the CD spectrum which would result from the calculated intrinsic optical activity is compared to the experimental CD spectrum which results from binding congo red to beef heart lactic dehydrogenase. (Experimental CD spectra similar to the one shown are observed for a number of related dehydrogenases which bind congo red in competition with coenzyme NADH.*5) The calculated spectrum for the first conformation reproduces the sign and magnitude of the experimental spectrum throughout the visible region. However, calculations using several other conformations designed to mimic the coenzyme give a variety of calculated curves. This variability is illustrated by the predicted spectrum for the second conformation, also shown in Figure 5A. This spectrum reproduces the short-wavelength couplet of the experimental spectrum, but it gives bands of incorrect sign at long wavelengths.
1335
In a small fraction of the 120 different orientations of indole relative to congo red which were used for the coupled-oscillator calculations, the long-wavelengthband approaches 113 of the magnitude observed or calculated by the a-electron method. This is illustrated in Table IV, where we have selected the orientation with the largest rotational strength at each center as indicative of the largest possible contribution of coupling. The coupledoscillator mechanism contributions at shorter wavelengths (300-400 nm) are at most 10% of those calculated for the twisted chromophore. In addition, the coupled-oscillator results do not predict the near-ultraviolet couplet. Intrinsic rotational strengths calculated for two cibacron blue conformations and the rotational strength contributions from coupling are given in Table V. In Figure 5B the CD spectrum which would result from the calculated intrinsic optical activity is compared to the experimental CD spectra which are observed for cibacron blue bound to lactic dehydrogenase and to alcohol dehydrogenase. The extrema of the calculated spectra are substantially blue shifted relative to the experimental spectra, as discussed for the absorption spectra. The shift has been enhanced by the red shift of the observed transitions caused by the nonpolar environment on the enzymes to which the dye is bound. Two different CD spectra are observed for the two dehydrogenases. In fact, these spectra have bands of opposite sign in each of the spectral regions-the longwavelength band, the band just above 400 nm, and that in the 300-400-nm region. After allowing for the large blue shift, the spectrum calculated for the dye conformation of Thompson and co-workerslg qualitatively reproduces the experimental visible spectrum of cibacron blue bound to lactic dehydrogenase (Figure 5B). The longest wavelength bands (630 nm observed, 489 nm calculated) are positive, and the intermediate-wavelengthbands (415 nm observed, 341 nm calculated) have negative ellipticities. Differences between the calculated and observed spectra are evident in the 300-400-nm region, since the positive rotational strengths calculated at 310 and 290 nm (Table V) are overwhelmed by the negative rotational strength at 280 nm. Thus the observed positive ellipticity is not present in the theoretical spectrum. Also, if one allows for the large blue shift, the spectrum calculated for Stellwagen'sm conformation of cibacron blue qualitatively reproduces the experimental spectrum of cibacron blue bound to alcohol dehydrogenase (Figure 5B). The long-wavelengthbands (600 nm observed, 478 calculated) are negative, the intermediate-wavelength bands (430 nm observed, 346 calculated) are positive, and three transitions with negative rotational strengths at 304, 282, and 275 nm correspond to the observed negative bands between 300 and 400 nm (see Table V). The conformations of cibacron blue presented by Thompson et al.19and Stellwagen20differ in the dihedral angles around the bonds linking the anthraquinone to the first phenyl ring. These two rings are the site of localization for all of the transitions above 270 nm. In order to obtain conformations of the dye which appear to be equally good mimics of the bound NADH and which have very different dihedral angles in the spectroscopically important region, the dihedral angles for the bonds which link the cyanuric ring to the two phenyl rings are also very different for the two conformations. The rotational strengths of most of the eighty orientations of indole relative to cibacron blue, which were used for the coupled-oscillator calculations, approach the magnitude observed and calculated for the twisted chro-
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The Journal of Physical Chemistry, Val. 87, No. 8, 1983
Edwards and Woody
TABLE V : Calculated Coupled-Oscillator Rotational Strengths" isolated chromophores cibacron blueb 490 34 1 314 294
R 0.29 -0.57 0.21 0.18
281 277 2 74 269 262
0.00 -1.77 0.83 -0.03 -0.24
256
1.29
h
rotational strengths for coupled chroniophores
indole h
R -0.01 -0.51 0.21 0.27
286
26 1
0.00
0.00
0.05e
0.00 -1.25 0.50 0.92f -1.17f -0.18 1.28
(2) 0.58 -0.71 0.21 0.31 --0.13 e
0.00 0.43f -0.12 -0.43 -0.791 -0.19 1.28
45,45,13 5 (3) 0.48 -0.64 0.22 0.18 0.10e
0.00 -0.09 0.22 -2.07f 0.84f -0.15 1.29
45,45,13 5 (4 1 0.4 1 -0.56 0.19 0.22 0.03e 0.01 -1.91 -0.04f 1.29f -0.01f -0.22 1.27
a Results for one orientation of indole a t each of the centers in Chart I. The orientation with the largest change in longwavelength rotational strength compared t o the isolated chromophore was selected for inclusion in the table. Wavelengths in nm; rotational strengths in Debye-Bohr magnetons. The conformation is that given by Thompson e t al.," specified as conIndole rotation in degrees around three axes, in the order given. (1)An Axis defined by the nitroformation 1 in Table I. gens attached to the anthraquinone and pointing t o the right in Chart I. ( 2 ) An axis perpendicular to the first axis in the plane of the page in Chart I and which points toward the top of the page. ( 3 ) An axis defined by the cross product of the first two. The long axis of the indole was initially parallel to the first axis. The indole plane was in the plane defined by the first two axes with the indole nitrogen having a positive coordinate along the second axis. A positive rotation is clockwise as viewed with the axis of the interest pointing toward the observer. Location of the indole center as designated o n the structure of cibacron blue in Chart I. e These coupled transitions are 99% indole transitions. f These coupled transitions are a mixture of dye and indole transitions.
'
mophore. This is illustrated in Table V, where we have selected the orientation with the largest rotational strength a t each center as representative of the largest possible contribution of coupling. In contrast to Table IV, where a planar dye geometry was used for the coupling calculations, the twisted (dissymmetric) conformation of Thompson et al.19 was used for cibacron blue. For the transitions which are nearly pure dye transitions, the rotational strengths listed for the coupling calculations must be compared to the r-calculation results to find the calculated extrinsic optical activity. The transitions above 280 nm are nearly pure indole or dye transitions, while several of the transitions at shorter wavelengths involve substantial mixing of the dye and indole transitions, as noted in the table. Dye Aggregation. We attribute the changes in absorption at high dye concentrations to self-association. Both dyes have large aromatic nonpolar regions which would tend to stack as a result of hydrophobic and van der Waals interactions. The strong tendency of congo red to aggregate has been reported by other investigator^.^^^^^ The negatively charged sulfonate groups can stack over the amino group, which is electron deficient, thus decreasing the repulsion between sulfonates. The behavior at higher ionic strengths is also consistent with aggregation of species with like charges. In order to get a quantitative measure of the congo red self-association, we have used a simple model to fit the absorbance at 500 nm over a iO4-fold range in dye concentration. This model assumes that the dyes aggregate into a linear stacked polymer with a dissociation constant KIDfor the dimer and a second dissociation constant KAD which describes the dissociation of monomers from all higher aggregates (see ref 3 for details of the model and the fitting procedure). An excellent fit to the experimental data is obtained for KID= 500 pM and KAD= 16 pM as is shown in the insert of Figure 2A. (32) S. R. S. Iyer and G. S. Singh, Kolloid-2. 2. Polyn., 242, 1196 (1970). (33) T. Yasunaga and S.Nishikawa, Bull. Chem. SOC.Jpn., 45, 1262 (1972).
Aggregation would affect the spectra in two ways: through the coupling of the transitions in one molecule with those in another associated molecule, and by the decrease in polarity of the chromophoric environment. In order to assess the influence of coupling we have carried out coupled-oscillator calculations on two dye chromophores stacked in van der Waals contact in geometries which would maximize the surface contact, yet minimize the repulsion from the sulfonate groups. As is shown on the dotted curve of Figure 2A the effect of coupling is large for the congo red spectra and accounts for both the blue shift and hypochromism. For cibacron blue, however, the effect of coupling is small (Figure 2B) and decreased polarity and increased polarizability of the chromophoric environment must be the principal causes of the red shift. There are no simple rules for treating the effect of decreased environmental polarity on intensity, but this effect may be acting in concert with the small effect calculated for the coupled-oscillator mechanism and polarizability changes to give the observed hypochromism. Discussion The semiempirical r-electron method which we have used in this work gives a good account of the visible and near-ultraviolet absorption spectra of the complex chromophores which constitute congo red and cibacron blue. In agreement with experiment, theory predicts that the long-wavelength band of congo red consists of a dominant strongly allowed transition at lower energies and a forbidden or weakly allowed transition at higher energies. The predicted wavelengths and intensities of the congo red transitions are in good agreement with experiment. In the case of cibacron blue, the calculated wavelengths do not agree well with experiment, but are shifted to the blue, in agreement with previous calculation^^^ on anthraquinone derivatives. Despite this discrepancy, the calculations are successful in predicting a single isolated long-wavelength band. The predicted intensities are generally satisfactory. Finally, the long-wavelength transitions are predicted to be almost completely localized in the phenylaminoanthraquinone moiety of the dye, in agreement with ex~eriment.~'
MO Calculatlons for Congo Red and Cibacron Blue
The validity of the rotational strength calculations is more difficult to assess since comparisonswith experiment are restricted to dyes of uncertain conformation bound to proteins. The a-electron approximation we have used in this work does yield results for the long-wavelength CD bands of dissymmetric chromophores, such as helicenes and 2,2'-diaminobiphenyl, which agree qualitatively with e ~ p e r i m e n t . Other ~ workers, using somewhat different parameterizations, have also successfully treated these kinds of molecule^.^^^^^ In the case of biphenyl itself, corresponding agreement is not obtained? This is probably attributable to the dihedral angle of 45O, which leads to a breakdown of the u-a separability assumption inherent in the a-electron approximation. Mixing of u and T orbitals is especially significant for calculations of rotational strength, because u a* and ?r u* excitations have large magnetic dipole transition moments. In the molecules with which we are dealing here, the dihedral angles about formal single bonds never exceed 30' in the case of congo red. For cibacron blue, some of the conformations considered have some formal single bonds (ring-N bonds) with dihedral angles exceeding 45O. Several converging lines of evidence indicate that the CD of congo red bound to dehydrogenases is caused primarily by the chiral conformation of the bound chromophore, rather than by interaction with protein groups. The strength of the observed CD argues for this, since the calculations of coupled-oscillator contributions indicate that only moderate rotational strengths can result from coupling. The similarity of the CD induced in congo red by various dehydrogenases3v4supports the intrinsic origin. A combination of coupling contributions which are all of the same sign could account for the observed CD, but would require a rather unlikely combination of perturbing groups which are additive for several congo red transitions and which are nearly identical for several dehydrogenases with significantly different amino acid sequences. In the case of cibacron blue bound to dehydrogenases, the same arguments suggest that extrinsic and intrinsic
-
-+
(34) W. S. Brickell, A. Brown, C. M. Kemp, and S. F.Mason, J. Chem. SOC.A, 756 (1971).
The Journal of Physical Chemistry, Vol. 87, No. 8, 1983 1337
contributions to the induced CD are probably of comparable magnitude. Coupling with a single aromatic side chain can lead to rotational strengths comparable to, or even exceeding, the intrinsic contributions. Experimentally, a variety of CD patterns is observed for cibacron blue bound to a series of dehydrogenase^.^,^ This observation may be due to a single dye conformation interacting with different protein conformations. This interpretation would fit Stellwagen's h y p ~ t h e s i s that ' ~ ~ ~cibacron ~ blue binds tightly to dehydrogenases because it mimics the conformation of the NADH coenzyme, which shows little variation among the dehydrogenases for which detailed structures are known.22 However, in the complex of cibacron blue with liver alcohol dehydrogenase, the dye mimics only the ADP-ribose portion of NADH and shows substantial deviations from the nicotinamide portion of the coenzyme.35 In addition, N-phenyldiaminoanthraquinone binds to various enzymes nearly as tightly as does the complete cibacron blue molecule.36 The methods described here are also useful for examining the effects of aggregation which are common in dyes. Calculations of coupling among identical dye molecules, using a hypothetical but plausible geometry, show that such coupling can account for the observed blue shift and hypochromism on aggregation of congo red. Similar calculations on cibacron blue, however, indicate that coupling among dye molecules is not the major factor in aggregation-induced spectral shifts. In this case, more generalized environmental effects must be involved.
Acknowledgment. This work was supported by a grant from the U.S. Public Health Service, GM22994. We thank Mr. John Schock for his assistance in generating the stereodrawings of the dyes in Figure 1. Registry No. Congo red, 573-58-0; cibacron blue, 12236-82-7; l-amino-9,10-anthraquinone, 82-45-1;1,4-diamino-9,10-anthraquinone, 128-95-0;1,5-diamino-9,10-anthraquinone, 129-44-2; 1,8-diamino-9,10-anthraquinone, 129-42-0; 2-amino-9,lO-anthraquinone, 117-79-3;2,6-diamino-9,10-anthraquinone, 131-14-6. (35) J.-F.Biellemann, J.-P. Samama, C. I. Branden, and H. Eklund, Eur. J. Biochem., 102, 107 (1979). (36) R. S. Beissner and F.B. Rudolph, Arch. Biochem. Biophys., 189, 76 (1978).
