VISIBLECIRCTJLAR DICHROISM OF PLANAR NICKELIONCOMPLEXES
4277
Visible Circular Dichroism of Planar Nickel Ion Complexes of Peptides and Cysteine and Derivatives’ by Joyce Wen Chang and R. Bruce Martin Department of Chemistry, University of Virginia, Charlottesville, Virginia $2901
(Received M a y 90,1969)
Planar, diamagnetic nickel ion complexes of peptides composed of &amino acid residues typically exhibit negative circular dichroism extrema from 450 to 490 mp. These minima appear about 50 mp to longer wavelengths than the absorption maxima at 410-450 mp due to d-d transitions on the nickel ion. Positive CD extrema also appear near the absorption maxima if histidyl residues are involved in the chelate rings or when bulky amino acid side chains are present in the amino terminal residue. The identity of CD sign for adjacent positions about a chelate ring observed in nickel ion complexes of Gly-Gly-L-Ala and Gly-L-Ala-Gly cannot be accounted for by any octant rule. A hesadecant rule accommodates the results for planar complexes by dividing the COordination plane perpendicularly into eight sectors centering on the metal ion and assigning opposite signs to adjacent sectors. Small side chains for I,-amino acid residues then fall into sectors of the same sign regardless of position in a peptide. Four d-d transitions are identified in the circular dichroism for planar complexes of nickel ion and cysteine and derivatives.
Introduction I n a solution containing equimolar amounts of nickel ion and a tripeptide such as triglycine, the addition of 3 equiv of base results in nickel ion promoted amide-hydrogen ionizations yielding a yellow complex containing one amino, two ionized amide nitrogen, and one carboxylate donor atoms arranged in a planar array about nickel ion. For a solution containing equimolar amounts of nickel ion and a tetrapeptide, the addition of 4 equiv of base results in three amide-hydrogen ionizations and yields one amino and three amide nitrogen donor atoms about the nickel ion in a planar c ~ m p l e x . ~ JThis paper reports the circular dichroism (CD) occurring in the d-d transitions of diamagnetic4 nickel ion complexes with optically active derivatives of triglycine and tetraglycine. Ionization of the pep tide hydrogens yields planar, trans amide bonds which provide a relatively rigid chelate framework for amino acid side chains. As a result of knowledge of side chain disposition, conclusions may be drawn about features of optical properties in planar nickel ion complexes. Only planar nickel ion complexes yield significant CD in the visible region of the spectrum, and of the common amino acids, only cysteine induces this configuration. This paper also presents the CD results for diamagnetic5 nickel ion complexes of cysteine and derivatives. The research reported here furnishes model compound studies for the interactions of nickel ion at the N-terminal and sulfhydryl groups of proteins.
Experimental Section Ligands employed in this research were obtained from Cyclo Chemical Corporation, Mann Research Laboratories, and Cnlbiochem. Cary 11 and Cary 14R
spectrophotometers were used for absorption measurements. Circular dichroism was measured on a Durrum-Jasco ORD/UV-5 recording spectropolarimeter with a CD attachment. The long wavelength limit of CD measurements is about 700 mp. The absorbance of solutions for CD measurements was 1 to 1.5, made by varying concentrations and cell lengths from 1 to 50 mm. For each CD curve, a base line was obtained by using the identical cell and distilled water under the same conditions as the sample. The CD scale was calibrated periodically by setting the scale deflection at 0.0097 absorption unit for a 1 mg/ml aqueous solution of d-10-camphorsulfonic acid in a 10-mm cell. All CD magnitudes are reported as the difference in molar absorptivity based on nickel ion concentration between left and right circularly polarized light. These differential molar absorptivities are designated as A€. Areas under CD curves were evaluated with the aid of a
(1) This paper is abstracted from the Ph.D. Thesis (1967) of Joyce Wen Chang, and the research was supported by a grant from the National Science Foundation. (2) R. B. Martin, M. Chamberlin. and J. T. Edsall, J. Amer. Chem. Soc., 82,495 (1960). (3) M. K. Kim and A. E. Martell, &id., 89,5138 (1967). This paper confirms the results and conclusions of our earlier study in ref 2. Their statement that we have drawn opposite conclusions about the “simultaneous” ionizations in nickel complexes of triglycine and tetraglycine is incorrect. Determination of equilibrium constants for ionizations that occur in a less than statistical manner is P legitimate procedure, and their relative values present a quantitative expression of the cooperative nature of the transition from octahedral to square planar geometry about nickel ion. These authors also report a second formation constant for nickel ion and diglycine more than five times greater than the first formation constant. This unlikely interpretation may be modified by allowing for amide hydrogen ionization, as has been done.* (4) R. Mathur and R. B. Martin, J . Phy8. Chem., 69, 068 (1965). (5) R. B. Martin and R. Mathur, J . Amer. Chem. Soc., 87, 1005 (1965). Volume 73, Number 12 December 1969
JOYCE WEN CHANGAND R. BRUCEMARTIN
4278
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Table I: Circular Dichroism of Nickel Ion Complexes of Peptides Composed of L-Amino Acid Residues Peptide
Alaninamide Prolinamide Gly- Ala-NHz Gly-Phe-NH2 Gly-His Gly-Gly- Ala Gly- Ala-Gly Ala-Gly-Gly Ala-Ala-Ala Gly- Ala-Leu Gly-Gly-Leu-NHz Gly-Gly-Leu Gly-Leu-Gly Leu-Gly-Gly Leu-Leu-Leu Val-Gly-Gly Pro-Gly-Gly Gly-Gly-Phe-NHz Gly-Gly-Phe Gly-Phe-Gly Gly-Phe-Phe Phe-Gly-Gly Phe-Phe-Phe
---_mfi
Ciroular dichroism-----
A€
416 410
-0.50 -0.10
430
$2.08
420
+0.16
420 422
$0.98
$0.15
400
-1.18
425
-1.62
432
A€
510 496 480 460 488 475 475 490 470 470 454 465 475 490 478 486 490 470
-0.09 -1.10 -0.67 -0.84 -1.92 -0.85 -1.12 -0.11 .-2.10 -2.20 -1.62 -1.38 -1.30 -0.29 -2.28 -0.30 -0.38 -0.88
Shoulder 475 475
-0.90 -2.84
480
-3.04
$1.22
planimeter. All experiments were performed at room temperature, near 23".
Results The differential molar absorptivities obtained for the
CD of square-planar nickel ion complexes of L-amino acid amides and peptides are recorded in Table I. The first two entries refer to 2 : 1 amide-nickel ion complexes of net zero charge. Solutions containing amide and nickel ion in equimolar amounts show disproportionation to give the 2: 1 complexes. All other entries of Table I contain peptide and nickel ion in a 1: 1 ratio. The two dipeptide amide chelates bear a net zero charge. I n order to induce the yellow color indicative of a square-planar nickel ion coInplex in a solution containing equimolar amounts of glycylhistidine and nickel ion, sufficient base must be added to yield a complex of net - 1 charge.6 All tripeptide and tripeptide amide chelates in Table I refer to complexes bearing one unit of net negative charge. Circular dichroism curves have been presented for some of the complexes in Table I.' A striking feature of the nickel-peptide complexes of Table I is the narrow range of wavelengths appearing in each column. Wavelengths of extrema in CD in the second column extend only from 400 to 430 mp, of minima in the third column, from 450 to 520 mp or from450 to 490 mp if the first two entries are excluded, and of absorption maxima in the third column, from 410 to 450 mp. It is also possible to conclude that a weaker absorption band occurs near 500 mp in the alaninamide complex. The low molar absorptivities for absorption (all less than 300) indicate that only d-d transitions occur in this The Journal of Physical Chem6stTy
mr
-
-----Absorption-mfi
430 434 448 453 450 430 429 428 427 428 408 425 430 425 426 428 430 410 430 430 430 432
c
60 65 40 60 110 180 190 228 275 178 162 95
110 110 144 240 172 126 225 156 196 205
wavelength region. No charge-transfer transitions of higher absorptivities occur above 270 mp in these nickel ion-pep tide complexes. With but one exception, all the chelates in Table I exhibit a negative CD extremum listed in the third column a t a wavelength about 50 mp longer than the absorption maximum tabulated in the last column. This negative CD may be taken as indicative of L-amino acid residues in nickel ion-peptide chelates. A negative sign is also observed in the square-planar Ni(~-1,2diaminopropane)z complex in nitromethane solvent where,8 a t the 460 mp minimum, Ae = -0.15, less than the value for the alaninamide complex where the chelate rings should be less puckered. Rather infrequently, a CD extremum of variable sign, listed in the second column of Table I, appears at a wavelength slightly less than the absorption maximum. Except for the glycylhistidine complex, which possesses a unique structure,6 positive values appear in the second column of Table I only when bulky side chains occur in N terminal amino acid residues. A similar sign reversal to positive CD with bulky groups takes place in some of the corresponding cupric ion ~ h e l a t e s . ~The tetrahedral nitrogen at the N terminus of peptides is much less rigid than the nickel ion bound, planar, trigonal nitrogens in the deprotonated peptide bonds. (6) R. B. Martin and J. T. Edsall, J . Amer. Chem. SOC.,82, 1107 (1960). (7) G. F. Bryce and F. R. N. Gurd, J . Biol. Chem., 241, 1439 (1966). (8) B. Bosnich, J. H. Dunlop, and R. D. Gillard, Chcm. Commun., 274 (1965). (9) J. M. Tsangaris and R. B. Martin, in preparation.
VISIBLECIRCULAR DICHROISM OF PLAXAR NICKELIONCOMPLEXES I n addition to the Ae values calculated a t the CD extrema and reported in Table I, areas under many of the curves were also evaluated. The aieas may be scaled so as to be directly comparable to the AE values in Table I. The scaled areas for the 6th (Gly-Gly-Ala) through 13th (Gly-Leu-Gly) entries in Table I are -0.74, -1.16, -0.10, -2.11, -2.25, -1.58, -1.33, and - 1.38, respectively. The agreement between these scaled areas and the Ae values recorded in Table I is excellent and usually within experimental error of the measurements. We conclude that for a relatively uniform CD peak of one sign, calculation of the differential molar absorptivity, Ae, a t the extrema is equivalent to evaluating the area. I n order to compare areas with A € for partially cancelling, oppositely signed CD peaks, scaled areas for the nickel ion complexes of Leu-Gly-Gly, Val-Gly-Gly, and Pro-Gly-Gly were calculated employing the same scale factor used above. The respective pairs of scaled areas obtained are +0.08, -0.19; +0.07, -0.20; and +0.57, -0.19. These scaled areas are only 50 to 65% of the Ae values recorded in Table I for the same complexes. It is apparent that the partial cancellation of positive and negative portions of a CD curve results in greater diminution of areas than of Ae values read a t extrema. We conclude that for a partially cancelling, oppositely signed CD curve, the differential molar absorptivities, Ae, read at each extremum, are better indicators of the rotational strengths of the transitions than are the areas under each signed curve. Only for two transitions yielding a skewed curve with CD of the same sign would the area appear to be a better measurement of total rotational strength than Ae, though the problem of resolution remains. For the reasons advanced in this and the preceding paragraph, Ae values rather than areas will be employed in discussing CD results. No conclusions in the Discussion section would be altered, however, if areas instead of Ae values were utilized. I n Table I1 are recorded the results for the deep red, divalent nickel ion complexes of L-cysteine and derivatives. I n all cases, a sufficient amount of base has been added so that all carboxylate, amino, and sulfhydryl groups are in their basic forms and all complexes are fully formed. The molar ratio of ligands to nickel ion is either 2 : 1, if only one kind of ligand molecule is present, or 1 :1 :1 for the mixed complexes of optically active ligand and either ethylenediamine or pmercaptoethylamine, and as specified in Table I1 for the glutathione (y-glutamylcysteinylglycine) complexes. All molar absorptivities and differential molar absorptivities are quoted on the basis of nickel ion concentration. Absorption spectra results for cysteine and cysteine ester complexes may be summarized by noting that the molar absorptivity per sulfhydryl group a t absorption maxima or a t shoulders on absorption curves in the complexes ranges from 15 to 18 a t 580-590 mp, and
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Table 11: Circular Dichroism of Nickel Ion Complexes of L-Cysteine and Derivatives' Circular dichroism
mr
Cysteine, 2: 1
Cysteine and mercaptoethylamine Cysteine and ethylenediamine Cysteine ethyl ester, 2: 1 Cysteine methyl ester, 2: 1 Cysteine methyl ester and mercaptoethylamine Cysteine methyl ester and ethylenediamine N-Acetylcysteine, 2: 1
Glutathione, 1: 1
mfi
f
+0.45 -0.62 -0.11 $0.20 -0.30 -0.07 +0.20 -0.24 -0.05 +0.21 $0.14 -0.04 $0.22 $0.19 -0.03 +O.ll +0.23 -0.09
590s 475 390s 590s 470 390s
30 116 90 35 109 270
470 385s 580s 480 390s
57 75 35 104 150
484
110
485 380
170 380
478
57
535 455 390 525 455 395 525 450 390 580 490 410 580 500 420 570 505 425 585 490 610 525 460 400 620 540 470
$0.06 $0.04 -0.20 -0.07 $0.35 $0.16 f0.50 -0.40
495 415 s 620 490 380
f0.32 -0.18 $0.25 +0.18 -0.72
+0.08
1.5:1
2: I
a
-Absorption-
A€
525 415
630s 520s 410
55 100 300
560s 420
90 285
Small s designates shoulder.
from 51 to 58 at 470-485 mp. It is more variable at 380-390 mp as this last absorption band is underlaid by charge-transfer absorption with maxima further into the ultraviolet region. The only exception is the cysteine methyl ester-mercaptoethylamine mixed complex which exhibits greater absorption intensity in the visible region. Though the sulfhydryl groups determine the nature of the absorption band, substitution of oxygen for nitrogen donors displaces absorption maxima to longer wavelengths as shown by the complexes of Nacetylcysteine in Table 11. The longest wavelength absorption maximum for the N-acetylcysteine and some other complexes in Table I1 is not easily resolved though another band appears to be present in the absorption spectra. The CD results for the three L-cysteine complexes of Table I1 may be summarized by noting that the Ahel mol of optically active ligand is $0.20 to $0.22 a t 525-535 mp; -0.24 to -0.31 a t 450-455 mp, and -0.05 to -0.07 a t 390-395 mp. Corresponding to the long Volume 73, Number 12 December 1969
4280 wavelength absorption band, an irresolvable, weak, positive CD band seems to appear at about 600 mp in the cysteine complexes. For the cysteine ester complexes, A€/mol of optically active ligand is $0.08 to +0.11 at 570-585 mp and $0.06 to +0.10 at 490-505 mp. The exception, as in the absorption spectra summary, is the cysteine methyl ester-mercaptoethylamine mixed complex, which again yields higher values. Excluding the last complex, the nearly constant) values of Ae/mol of optically active ligand in the pure 2: 1 and mixed 1:l:l nickelioncomplexes of cysteine and its esters indicate that the contributions to the optical activity of two chelate rings with asymmetric centers are independent and additive. The sign of the CD for the first charge-transfer band a t
