VOL. 16, 199-206 (1977)

BIOPOLYMERS

ESR Investigation of the Binding of Some Neutral Polyamino Acids to Synthetic Apatite* R. A. PECKAUSKASt and I. PULLMAN, Department of Radiology, New York Medical College, N e w York, New York 10029; and J. D. TERMINE, Laboratory of Biological Structure, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 2001 4 Synopsis Interaction between synthetic crystalline apatitic calcium phosphate and neutral polypeptides [poly(DL-alanine), poly(L-proline), poly(~-hydroxyproline)] was investigated by electron-spin-resonance spectroscopy of mineral-macromolecule complexes doped with vanadyl ion (VO++)as a paramagnetic probe. Changes in magnetic parameters were interpreted in terms of polypeptide-induced axial interactions with VO++ ions that have mineral surface phosphate oxygens as their primary ligands. This implies very close proximity between mineral surface and macromolecular peptide bond dipolar substituents.

INTRODUCTION

A series of reports reviewed by Urry' indicated that calcium ions can bind to the carbonyl oxygen of the peptide bond. It was then postulated that similar interaction between mineral calcium and protein carbonyls might also be implicated in biological calcifications, especially those involving apolar molecules.' We report here the use of electron-spin-resonance (ESR) measurements on vanadyl ion (VO++) probes in an investigation of neutral polypeptide-calcium phosphate complexes that might act as models for such mineral calcium-protein carbonyl binding. METHODS AND MATERIALS Sample Preparation Basic calcium phosphate (apatite) crystals were prepared as described previously2 after mixing solutions of CaC12 (12.0 mM) and NaZHP04 (10.8 mM) in 0.15 M Tris buffer, pH 7.5 at 25OC. All precipitates were recovered by centrifugation, washed three times with distilled water, and lyophilized. * Based in part on a thesis submitted by R.A.P., in partial fulfillment of the requirements for the Ph.D. degree, to the Graduate School of Medical Sciences, Cornell University, Ithaca, N.Y. The work was carried out in the Biophysics Division, Sloan-Kettering Institute, New York, N.Y., and in the Department of Radiology, New York Medical College, New York, N.Y. T o whom correspondence should be addressed. 199

'

0 1977 hy John Wiley & Sons,Inc

200

PECKAUSKAS, PULLMAN, AND TERMINE

The resulting dry powders were characterized as crystalline apatite by ir spectroscopy3 and X-ray diffra~tion.~ Apatite-macromolecule complexes (coprecipitates) were obtained in similar f a ~ h i o neach , ~ macromolecule being first dissolved in phosphate solution so that a final concentration of 1mg/ml would be reached just prior to precipitation. The ir spectra from the freeze-dried coprecipitate powders revealed the presence of substantial amounts of poly(amino acid) in each case. VO++was incorporated into mineral, poly(amino acid), and coprecipitate specimens as described previously.6 X-ray diffraction4and ir spectroscopy3 showed no differences between VO++-dopedand standard precipitates. The water soluble poly(amino acids) [poly(DL-alanine),poly(L-proline) (type 11) and poly(~-hydroxyproline)]were obtained from the Sigma Chemical Company, vanadyl acetylacetonate (VO(AcAc)z)from Alfa Inorganics, Ventron, while N-methyl acetamide (AcNHMe) was obtained from Eastman Co.

ESR Spectroscopy ESR spectra were taken with an x -band superheterodyne spectrometer as described previously.6 The resultant VO++ probe ESR spectra were all axially symmetric powder patterns spread over the range 2,500 to 4,000 G a t 8,810 MHz with an average line width of 20-50 G. The method used to extract g-values and hyperfine splitting constants ( A-values) from such spectra is described elsewhere.6 In addition, however, the extrapolation normally used for determining the lowest field line of the VO++ perpendicular component6 was also applied to estimating the highest field line of the parallel component for this study’s VO++-polyproline I1 sample. (This was necessary since instrumental limitations made direct measurement of this high field line unreliable.) In spectra from solutions of vanadyl acetylacetonate (VO(AcAc)z) anisotropy was averaged out due to rapid tumbling. Thus, isotropic ESR values were obtained directly using lines 4 and 5 of the total 8 line spectrum followed by application of those corrections discussed by Kuska and Roge r ~ .Typical ~ examples of both isotropic and anisotropic VO++ spectra are shown in Figures 1and 2, respectively.

Vanadium Bonding Parameters VO++ complexes generally exhibit a square pyramidal fivefold coordination about the central axial V=O group. However, sixfold coordination is also possible where the square pyramidal complex weakly adds a sixth ligand in the axial position trans to the oxygen atom; this weak interaction is usually considered only a perturbation on the square pyramidal system.8 The symbols below refer to molecular orbitals of vanadium complexes which are oriented so as to lie either in the ligand plane or normal to it. (All of

ESR INVESTIGATION

201

005R.l VOS04 Aqueous Solution

t 3144 gauss

q

H

=

2.0023

100 gauss frequency

i

8810MHz

Fig. 1. Typical isotropic ESR spectrum a t room temperature of vanadyl ion in solution. First derivative of the absorption vs. magnetic field increasing to the right.

+ 3144 gauss g = 2.0023 H

100 gauss frequency 8810MHZ

.

.

: : : : :, :. : : 9, transitions Hakes

1 Ho,ve,.7

.

.

I

I

-1-5.3-1 I 3 5 7

1

1

-5

-3

1

_, 1 /

I 911 7transitionq

Fig. 2. Typical axially symmetric ESR spectrum at room temperature of a synthetic apatitic calcium phosphate precipitated in the presence of vanadyl ion and lyophilized. First derivative of the absorption vs. magnetic field increasing to the right. The nuclear spins of the parallel and perpendicular components are indicated.

1.962

1.963

192.9

1.961

191.3

80

60.5

64.2

193.9

-i2

s

107.8

102.1

103.1

103.5

8

58.8

189.4

1.956

1.953

v

"

cn

2

a(DL-Ala), :poly(DL-alanine); (Pro-II), :poly(L-proline-11); (Hyp), :poly(L-hydroxyproline); A ~ / ( D-Ala), L :coprecipitate of (DL-Ala), with Ap; Ap/(Pro-II), :coprecipitate of (Pro-II), with Ap; Ap/(Hyp), :coprecipitate of (Hyp), with Ap; Apsynthetic precipitation apatite. Errors: g-values = ?- 0.001 G . A-values = f 0.6 G.

59.9

191.6

186.4

1.970

1.929

AP

1.964

1.932

AP/(HYP),

5U

108.8

107.1

A0

67.6

188.1

1.952

1.961

1.966 1.958

1.934

Ap/( Pro-I1),

1.942

Ap/( DL-Ala),

107.4

66.8

65.1

A1

All

1.971

1.975

1.975

1.944

1.938

1.934

gll gl

(HYP),

(Pro-I1,)

(D L -Ala ),

TABLE I Effect of Neutral Polypeptides on the ESR Parameters of VO++ in Synthetic Apatitesa

M

n

ESR INVESTIGATION 1.990

I.96 0

1

203

“IS4

L 60

70

80 90 100 110 120

A,

(gauss)

Fig. 3. Plot of ESR parameters go vs. A. for bound vanadyl ion with different equatorial ligands as indicated. Data from Boucher e t al.8

these were described previously6 but are restated here to provide a convenient frame of reference.) In-plane orbitals are described by which relates to sigma bonding and by P Z * ~which concerns Pi bonding. Outof-plane axial interactions are described by er*2. These coefficients can vary between 0.5, which indicates complete covalency, and 1.0, which implies complete ionicity. / 3 ~ may * ~ be calculated solely from ESR data while exact calculation of P1*2 and e,*2 would require data concerning optical-energy-level differences. In the case of p1*2,an empirical relation between energy-level differences and the Fermi contact term, K (a parameter easily calculable from ESR data), exists which relates to the interaction between the unpaired electron of the vanadium atom and its nucleus, thereby allowing a fair estimate of the sigma bonding coefficient. No similar method was available for er*2 so instead we introduced the function f(g.l&*2) which is directly proportional to e,*2/AE, LIE being the unknown optical-energy-level difference.6 Changes in f imply either sixth ligand addition on the V=O axis andlor hydrogen bonding to the vanadyl oxygen. More detailed information on the electronic structure of the vanadyl ion can be found in Ballhausen and Gray: while a fuller theoretical discussion for the relation between ESR and vanadyl orbital coefficients is given by Kivelson and Lee.lo The calculation of these parameters was discussed previously.6

RESULTS AND DISCUSSION The isotropic g-factor (go) observed in vanadyl complexes depends on ligand types and comparison of the values observed in the pure neutral polypeptides (Table I) with those in the literature (Fig. 3) indicated that the vanadium was most likely coordinated to polypeptide oxygen ligands. This indicates that VO++, like calcium, can bind to carbonyl oxygens of peptide bonds. In synthetic apatite (Table I), the go-value is significantly lower than that reported for most vanadyl complexes. This has been interpreted as oxygen ligands with increased ionic character in their bonding.6 The go-values

204

PECKAUSKAS, PULLMAN, AND TERMINE

we observed in the mineral-poly(amino acid) coprecipitates (Table I) were also consistent with liganding of vanadium to mineral oxygens. An even greater ionicity was observed, however, in the case of the imino polypeptide coprecipitates, both of which had exceptionally low go-values (Table I). It can be calculated6 that elevated f-values (indicating enhanced axial interactions and seen in all the coprecipitates, Table 11)would lead to lower go-values. However, in the case of poly(DL-alanine), this seems to have been compensated by a significant decrease (relative to apatite) in in-plane sigma bonding ionicity as reflected in the p1*2 value of the complex (Table 11). This apparently resulted then in an average coprecipitate go-value not significantly different from that of apatite. The imino polymer coprecipitates, on the other hand, showed no significant shifts in However, the poly(L-proline)-apatite complex showed a relative increase in ionicity via p ~ *suggesting ~, that in-plane pi bonding might play a role in the observed depression of this imino polymer coprecipitate’s go-values relative to that observed in pure apatite. Turning to the hyperfine splitting, a sharp decrease in the value of A0 relative to that seen in apatite was noted in all the coprecipitates (Table I). This drop in Ao is most probably not a result of changes in equatorial ligands for two reasons. First, the lowered go-values strongly argue for the in-plane ligands to be mineral oxygens, as noted above. Second, the AoTABLE I1 Calculated ESR Bonding Parameters for VO++ in Apatite-Poly(Amino Acid) Coprecipitatesa AP

Ap/(DL-Ala),

Ap/(Pro-11),

AP/(HYP )n

P,*’ P,

1.04b

1.03b

1.08b

1.04b

0.92

0.81

0.87

0.92

f

0.031

0.035

0.038

0.037

*2

o,*’

adbbreviations: As in Table I. Errors: = r O . O 1 . & * ’ = i0.03. f = *0.001. bVa1ues f o r can be somewhat greater than unity because several constants used in their calculations are chosen arbitrarily.* Therefore, these parameters should be considered o n a relative basis only.

o*’

TABLE I11 Effects of Peptide Models and Calcium Ions o n the ESR Parameters of Vanadyl Acetoacetonate in Aqueous Solution __

Additive a

go

none Ca++ AcNHMe Ca++ + AcNHMe

1.967 1.968 1.966 1.970

- ~ _An_ _ _ _ 102.6 100.7 101.2 95.0

a AcNHME = 1:l ( v/ v) N-methyl acetamide:water. CaCI, added to make total solution Ca++ = 0.25 M. Errors: go i 0.001 G. A n i 0.6 G.

ESR INVESTIGATION

205

values one might obtain by mixing equatorially both mineral and poly(amino) ligands could not be lowered since the Ao-valuesfor VO++bound to all the pure polymers were in the same range as that for the mineral alone. In order to determine how axial interactions act on the A0 of VO++ independently of potential equatorial ligand changes, solutions of VO(AcAc)z,a compound having fixed VO++-equatorial ligands, were prepared using added AcNHMe as a model for peptides. The literatures shows that the value of VO(AcAc)z has considerable solvent dependence, ranging from 102.3 (methanol) to 108.3 (carbon disulfide) G. Table I11 shows that the Ao-value for VO++ in water is comparable to that reported for methanol. Upon addition of calcium, the value of A” was significantly lowered, thereby indicating axial interactions between Ca++ and the vanadyl moiety. AcNHMe also lowered A. significantly and acted synergistically with Ca++ in this regard. Thus, it is quite probable that in the case of the coprecipitates, polypeptide-induced axial interactions accounted for the lowered Ao-values seen in Table I. The general thrust of these ESR data is that in the neutral poly(amino acid)-calcium phosphate complexes described above, probe VO++ ions appear to sit at mineral crystal surfaces utilizing mineral phosphate oxygens as their primary equatorial ligands. The attached polypeptides interact axially with the VO++ probe and, in addition, slightly change the ionic character of its main equatorial ligands. These data imply then a very close proximity between mineral surfaces and peptide bond dipolar substituents. It should be pointed out, however, that if acidic sites such as carboxyls andlor serine phosphate groups exist on a polymer or protein they also would act as potential mineral-binding site in competition with or perhaps even in preference to peptide bond sites, as suggested by our previous results with VO++ complexes of acidic biopolymers.6 This work was supported in part by USHEW-NCI Grant CA-08748 and AEC Contract AT-(30-1)-910(Sloan-Kettering Institute), in part by USHEW-NIH-NIDR Grant DE-01945 (Hospital for Special Surgery),and in part by USHEW-NIH-NIDR Contract N01-DE-32418 (New York Medical College).

References Urry, D. W. (1974) Biol. Med. 18,68-84. Termine, J. D. & Posner, A. S. (1970) Arch. Biochern. Riophys. 140,307-317. Termine, J. D. & Posner, A. S. (1966) Science 153,1523-1525. Termine, J. D. & Posner, A. S. (1967) Calcif. Tissue Res. ‘ ,8-23. Termine, J. D. & Posner, A. S. (1970) Arch. Biochern. Biophys. 140,318-325. 6. Peckauskas, R. A., Termine, J. D. & Pullman, I. (1976) Biopolymers 15,569-581. 7. Kuska, H. A. & Rogers, M. T. (1968) “ESR of first row transition metal complex ions,” in Radical Ions, Kaiser, E. T. & Kevan, L., Eds., Wiley-Interscience, New York, ch. 13. 1. 2. 3. 4. 5.

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PECKAUSKAS, PULLMAN, AND TERMINE

8. Boucher, L. J., Tynan, E. C. & Yen, T. F. (1969) “Spectral properties of oxovanadium (IV) complexes. (IV) correlation of ESR spectra with ligand type,” in Electron Spin Resonance of Metal Complexes, Yen, T. F., Ed., Plenum, New York, pp. 111-130. 9. Ballhausen, L. J. & Gray, H. H. (1961) Znorg. Chem. I, 111-122. 10. Kivelson, D. & Lee, S. (1964) J.Chem. Phys. 41,1896-1903.

Received May 17,1976 Accepted August 11,1976