Interaction of polypeptides with metal ions

3040

M. P A L ~ O

eJ al.

5. N. M. t - ~ R K O V a n d P. Ye. MATKOVSKII, Sopolimerizatsiya na kompleksnykh katalizatorakh (Copolymerization on Complex Catalysts). Izd. " N a u k a " , 1974 6. K. F U K U I and J. SHIMITZU, J. Polymer Sci. 37: 341, 356, 1959; 55: 321, 1961 7. P. Ire. MATKOVSKII, G. P. BELOV and L. N. RUSSIYAN, Vysokomol. soyed. A I 2 : 2286, 1970 (Translated in Polymer Sci. U.S.S.R. 12: 10, 2590, 1970) 8. G. A. BEIKHOLD, P. Ire. MATKOVSKII, Yu. V. KISSIN, Kh.-M. A. BRIKENSHTEL~ a n d F. S. DYACHKOVSKH~ Vysokomol. soyed. A13: 131, 1971 (Translated in P o l y m e r Sci. U.S.S.R. 13: 1, 149, 1971) 9. V. N. BELOVA, M. P. GERASINA, G. A. BEIKHALD, L. N. RUSSIYAN, P. Ye. MATKOVSKII~ Kh.-M. A. BRIKENSHTEIN, F. S. DYACHKOVSKII and N. M. CHIRKOV, Plast. massy, No. 3, 7, 1972 10. G. A. BEEgHOLD, L. N. RUSSIYAN, P. Ye. MATKOVS]glT, F. S. DYACHKOVSKII and Kh.-M. A. BRIKENSHTEIN, Vysokomo]. soyed. A I 8 : 384, 1976 (Translated in Polymer Sci. U.S.S.R. 18: 2, 439, 1976)

Polymer ScienceU.S.S.R. Vol. 21, pp. 3040-3045. Pergamon Press Ltd. 1980. Printed in Poland

0032=3950/79/1201-3040507.50/0

INTERACTION OF POLYPEPTIDES WITH METAL IONS* ]~. PALUMBO, A . COSANI, ~/~. TERBOJEVICH a n d

El PEGGIOlg

Organic Chemistry Institute, Padova, I t a l y

(Received 17 November 1978) A s t u d y has been made of the interaction of Ni (II) and Co (II) ions with t h e following polypeptides: poly-(L-lysine), poly-(L-ornithine), poly-(L-diaminobutyrie acid) and poly-(L-histidine). I t is shown t h a t complexes involving amino nitrogens or deprotonated peptide nitrogens as ligands are formed a t several p H values. Deprotonated peptide nitrogens participate in complex formation even in the case of p h y siological p H values, when a stable chelate system m a y be formed. When deprotonated ' peptido nitrogens in m a t r i x backbones are involved in complex formation, no ordered structures is preserved in the complexes regions of a polypeptide. One m a y conclude from the results obtained t h a t conformational properties of the polypeptides are strictly correlated to complex formation.

A MAJORrole is played by ferment-metal complexes in biological systems. However, investigation of the properties of systems of this type is difficult both from a theoretical and from an experimental standpoint. It therefore seemed appropriate that a study should be made of corresponding systems that could simulate chemical and stereochemical structures of the active acentres of natural ferments. Recent investigations relating to interaction between transition metal ions and low molecular peptides have shown that Cu(II), Ni(II) and Co(II) ions facilitate * Vysokomol. soyed. A21: No. 12, 2754-2757, 1979.

Interaction of polypeptides with metal ions

304I,,"

ionization of amide nitrogens and strong chelate systems may be formed e v e n under physiological pH [1]. Since most of the latter investigations concern low molecular eompo~mds, we took as the binding matrices high molecular poly-a-amine acids, which a r e able to reproduce the conformational properties of polypeptide chains in albumins. In particular, we investigated polypeptides such as poly-L-lysine, poly-Lornithine, poly-L-diaminobutyric acid and poly-L-histidine, since it is known that basic amino acid residues are largely responsible for the binding of metals. to albumins. Earlier investigations of the interaction between copper ions and t h e above-mentioned amino acids [2-7] have shown that 1) poly-L-lysine and poly-Lornithine form complexes of two types at 7 < p H < 8 a~d p H > 8 respectively. Only amino nitrogens are included in the coordination sphere in complexes formed under a low pH, while in the Case of complexes formed under high p i t values deprotonated peptide nitrogens are likewise linked to copper ions; 2). poly-L-diaminobutyric acid forms two different complexes (at low and high pit values) in the pH range from 6 to 13, and in each of these 4eprotonated peptide ~ nitrogens are linked to Cu (II); 3) poly-L-histidene forms three complexes: a complex which incorporates imidazole and peptide nitrogens (at pH 5), a complex in which only imidazole nitrogens are linked to Cu(II) (at 5 < p H < 8 ) , and classical biuret type complex (alkaline pH values). Conformational states of the polypeptide matrices depend on the nature of t h e ligands in the metal ion co-ordination spere. Further investigations were carried out in which ions of other metals of biological importance, namely Ni(II) and Co(II) were used. The results of potentiometric and spectroscopic investigations of poly-Llysine and poly-L-ornithine in the presence of Ni(II) show that on increasing. the pH from 7 to 13 two complexes are formed. The absorption spectra and t h e absence of bands in the visible portions of the circular dichxoism (CD) spectra show that only amino groups of side chains are linked to Ni(II) in the complexes formed under low pH. In complexes formed under high pH deprotonated peptide groups are als0 included in the coordination sphere, as evidence by the C ] ) spectrum having a maximum in the negative region at 470 nm. The low intensity of the latter band even under very high pH, as well as the absorption spectra, show, however, that binding of amide ligands takes place only in N terminal segments of the macromolecnle, whereas the binding of ligands with nitrogens o f side amino groups predominates for units within the chain. In contradistinction to Cu (II) the addition of increasing amounts of Ni (II)to solutions of poly-L-lysine and poly-L-ornithine ir~ the completely or partially spiralized form does not. entail any major disruption of ordered structure. It was found that two optically active complexes were formed for poly-L-diaminobutyric acid in the pH range > 7. The absorption spectra and the CD data, point to the incorporation of amide nitrogens as ligands in both cases (Fig. 2)~ :~o ordered structure can be assigned to either of these adducts, as is clear from~

: 3042

M. PALUMBOe~ a[.

measurements carried out in mixed solvents, where the polypeptide is in spiralized form. In the case of poly-L-histidine a soluble complex is formed only under high p H in the presence of 1~i (II). The latter complex has optical properties similar to those of the corresponding complex of poly-L-diaminobutyric acid formed u n d e r high pH.

0 8~

z

-I

D

-2

-I -3

-2 I

350

I

I

~

FzG. 1

I

I

550 .,1,nm

I

l/O0

I

I

500

I

I

600 ,1~nm

FIG. 9.

IFIG. 1. CD spectra of the system poly-L-lysine-Ni(II) in aqueous solution. Metal : poly. peptide=0.16; pH 7-84 (1); 8.54 (2} and 13 (3). ~ o . 2. CD spectra of the system poly-L-diaminobutyrie acid-Ni(II) in aqueous solution. Metal : polypeptide=0.lS; pH 7.35 (1}; 7.67 (2); 7.86 (3); 8.11 (4) and 8.58 (5). All measurements carried out for Co (II) complexes were done in the absence o f oxygen so as prevent Co (II) oxidation to Co (III). However, our experiments ~how that the presence of oxygen is much less dangerous in the case in question t h a n in the corresponding low molecular peptide complexes. I n the light of the absorption spectra and the potentiometric data it appears t h a t poly-L-lysine and poly-L-ornithine each form two complexes with Co (II). ~The complex formed under low p H is optically inactive in the visible portion of the spectrum, from which one may surmice that only side chains are linked to *Co (II). The complex formed under high pH has a band in the negative region at ~30 nm, as do corresponding complexes in the low molecular peptides (Fig. 3). T h e degree of spirality of poly-L-lysine and poly-L-ornithine are dependent only to a slight extent, if at all, on the Co/peptide ratio if pH is constant. We would therefore surmise that as in the case of the corresponding complexes with Ni (II), deprotonated amide nitrogens participate in the formation of a complex solely in N terminal segments of the macromolecules.

Interaction of polypeptides with metal ions

3045

The OD pattern observed for poly-L-diaminobutyrie acid in the prosenc3 of Co (II) is considerably m~re complex, starting at p H 6. In view of the absorptiom spectra and CD spectra it may be assumed t h a t ligands in this case or preferentially deprotonated peptide nitrogens (Fig. 4). Similarly the interaction of poly-L-histidine with Co (II) leads to increased intensity of bands in the visible regions of the CD spectra, starting at pH 5. A comlalex precipitates at pH ~ 7, and dissolves once again in the alkaline range of pH. Bo,102

'f

0°~i0 z

!

-¢'0 -1"5

-1 500

550

600 FIO. 3

650 ,~, nm

-2 450

550

650 ~l,nm

FIG. 4

Fie. 3. CD spectra of system poly-L-lysino-Co(II) in aqueous solution. Metal :polylmptide--0.16; pH 9.39 (1); 10-36 (2), 10.90 (3) and 13 (4). l~io. 4. CD spectra of system poly-L-diaminobutyrie acid-Co(II) in aqueous solution. Metal : polypeptido=0.16; pH 6'45 (1); 6.83 (2);. 7.17 (3) and 7.40 (4). The most interesting feature of the Co-poly-L-histidiue complexes is the fact t h a t simple dissociation of the latter takes place on reducing p H below 5, either (luring dialysis in water, or even in a~trilen B solution. This points to rather hight chemical inertia on the part of the adduct question. The use of poly-L-histidine to extract Co (II) may be of interest from a practical standpoint. The results of a study of structure in relation to the nature of the ligand have been tabulated for the systems under study (see Table). The data presented a b o v e support the following conclusions. If deprotonated nitrogens of the polymer backbone participate in complex formation with Cu (II), Ni (II) or Co (II), no ordered structure can be preserved in the complexed regions of a polypeptide. Whereas complex formation in the case of Cu(II) involves the participation of amide groups of all the units even with low basic pH values for poly-L-lysine a n d poly-L-ornithine, it has been seen that Co(II) and Ni(II) are capable of inducing the ionization of peptide groups solely in N terminal fragments of the macromolecules. This might well be due to chelate ring formation involving a basic terminal unit.

M. P~_~UMBO e t a l .

3044

The properties of poly-L-diaminobutyric acid and poly-L-histidine complexea in the disordered form are practically identical. This shows that from a chemical a n d a structural standpoint the metal ions environments are similar for b o t h pelypeptides. CONFORMATIONAL

PROPERTIES

OF THE

Metal ion

OF POLYPEPTIDE--METAL

NATURE

OF THE

Matrix

Cu (II) • Poly-L-lysine Ditto Poly-L-ornithine Ditto Poly-L-diaminobutyric acid Poly.L-histidine Ditto

~ i (n)

Co (II)

LIGAND

pH

8

11 8

11 6-13 5

7 13

Poly-L-lysine Ditto Poly-L-ornithine Ditto Poly-L-diaminobutyr,~e acid Poly-L-hlstidino

11 6-13 13

Poly-L-lysine Ditto Poly-L-ornithine Ditto Poly-L-diaminobutyrie acid Poly-L:histidine Ditto

11 8 11 6-13 5 13

8

11 8

8,

COMPLEXES,

LINKED

TO THE

Conforma tion of the free m a t r i x

AND THEIR DEPENDENCE~ METAL

ION

I Conforma- q I tion of m a t r i x in complex

Ligand

Coil Helix Coil Helix* Coil Coil B-form Does not dissolve

Coil ~Coil Coil i Coil !Coil Coil Coil

Amino Amido Amino Amide Amide Amide Imidazole Amide

Coil Helix Coil Helix* Coil Does 'not dissolve

i Coil I Helix Coil i Helix* Coil C oil i

Amino Amino Amino Amino Amido Amido

Coil Helix Coil Helix* Coil Coil Does n o t dissolve

Coil Helix Coil Helix* Coil Coil Coil*

Amino Amine Amino. Amine Amide Amide Amido

! B-form

F

* Some ordered structure is formed.

Deprotonatcd peptide nitrogens certainly do particil~ate in complex formation even under physiological pH, when a stable chelate system is formed. In the light of the results of this investigation one m a y conclude that t h e conformational properties of the polypeptides are strictly corre]ated to complex formation. It follows that the use of polymeric ligands should provide more scope for model systems, and may serve as an effective aid towards a fuller underatanding of the interaction of metal ions with biological systems. Translated by R. J. A. HE~DBY

Kinetics of 3-methyl-, 3-chloromethyl-oxetane polymerization

304~i

REFERENCES l . R. B. MARTIN, In: Metal Ions in Biological Systems, p. 142, Ed. H. Siegel, N.Y., 1974 2. A. LEV1TZKI, I. PECHT and A. BERGER, J. Amor. Chem. See. 94: 6844, 1972 S. M. PALUMBO, A. COSANI, M. TERBOJEVICH and E. PEGGION, J. Amer. Chem. Soc. 99: 939, 1977 4. M. PALUMBO, A. COSANI, M. TERBOJEVICH and E. PEGGION, Maeromolecules 10: 813, 1977 ~i. E. PEGGION, A. COSANI, M. PALUlVIBO and M. TERBOJEVICH, Prec. Vth Amerioan Peptide Symposium, p. 368, Ed. M. Goodman and J. Meienhofer, N.Y., 1977 6. M. PALUMBO, A. COSANI, M. PERBOJEVICH and E. PEGGION, Biopolymers 17: 243, 1978 7. M. PALUMBO, A. COSANI, M. TERBOJEVICH and E. PEGGION, Maeromoleoules 11: 1978

laolymer Science U.S.S.R. Vol. 21, pp. 3045-3053. Pergamon Press Ltd. 1980. Printed in Poland

0032-3950/79/1201-3045507.5010

THE KINETICS OF 3-METHYL-, 3-CHLOROMETHYL-0XETANE POLYMERIZATION ON THE CATALYST SYSTEM AI(iso-C4H9)3-H20 AND THE MOLECULAR WEIGHT CHARACTERISTICS OF POLYMETHYLCHLOROMETHYLOXETANE* G.

P.

ALEKSYUK, L. V. ALFEROVA and V. A. KROPACHEV High Polymer Institute, U.S.S.R. Academy of Sciences

(Received 28 November 1978) The kinetics of 3-methyl-, 3-chloromethyl-oxetane polymerization have been investigated on the catalyst system Al(iso-C~H0)a-H20 I>2.0 as well as the molecu l a r weight characteristics of the resulting polymer. I t is shown that the molecular weight and polydispersity of the polymer do not depend on the degree of polymeri,zation or on the initial monomer and catalyst concentrations. The molecular weight reaches high values ( ~ 6-8 × l0 -5) in the earliest stages of polymerization, and does 'not vary up to the very end of the process. The molecular weight distribution is relatively narrow; the ]Vlw/ffln ratio is equal to.29-i-0.01, and likewise does not vary with ,conversion. Tim experimental values of 2~n exceed the calculated ones by a factor of 600-700, which points to a very low degree of catalyst utilization in initiation steps. "the polymer formed inhibits the process as a result of interaction with catalyst. The kinetic scheme of polymerization proposed in the light of the data obtained involves four elementary stages: slow initiation, rapid propagation, spontaneous monomolec•Jlar termination a n d the inhibiting stage. ~* Vysokomol. soyed. A21: No. 12, 2759-2765, 1979.