Solution properties of synthetic polypeptides. light scattering and viscosity of poly-γ-ethyl-l -glutamate in dichloroacetic acid and trifluoroethanol

European PolymerJou.,mal, 1967, VoL 3, pp. 681-689. PergamonPress Ltd. Printed in England.

SOLUTION LIGHT

PROPERTIES SCATTERING

ETHYL-L-GLUTAMATE AND

OF

SYNTHETIC

AND

VISCOSITY

POLYPEPTIDES. OF

IN DICHLOROACETIC

POLY-,/ACID

TRIFLUOROETHANOL

M. TERBOJEVICH,E. PEGGION,A. CosA~, G. D'ESTE and E. SCOFFONE Institute of Organic Chemistry, University of Padua, Italy and VIII Sez. Centro Nazionale di Chimica Macromolecolare, Italy

(Received 17 April 1967) Abstract--Poly-y-ethybL-glutamate (PELG) has been studied by viscometry and light scattering in trifluoroethanol CITE) and dichloroacetic acid (DCA) solutions. In D C A the following MarkHouwink equation was obtained: bl] = 2.02 x 10-4 x M 0"73; the exponent is typical of random coiled polymers. In TFE, the equation was [)}]=5.01 x 10-7 × M 1"30, the exponent in this case indicates a rod-like structure with some flexibility. From the experimental radius of gyration (Re) in TFE, determined by light scattering, the average length per monomeric residue h was calculated. This quantity was found to be strongly dependent on the molecular weight of the polymer. By extrapolation to zero molecular weight, h0 ~ 2.0 ,~.

INTRODUCTION IT IS weU known that many polypeptides can adopt helical conformations in solution, their existence depending on the nature of the solvent. Conformational studies on polypeptides in solution have been carried out by optical rotatory dispersion, light scattering and viscosity, flow birefringence, dielectric dispersion, rotatory diffusion and low angle X-ray scattering. Most studies have been carried out on poly-y-benzyl-Lglutamate (PBLG). (1-1°) For this polymer, the type of helical structure present in moderately polar solvents like dimethyfformamide (DMF) or chloroform-formamide mixtures has been the subject of controversy. Some workers (1°' 11) suggest an a-helix conformation; others, mainly on the basis of low angle X-ray scattering and hydrodynamic properties, proposed a 3:10 helical structure.(~, s. 9) Whatever is the type of helical structure postulated, it is generally agreed that in solvents like DMF no completely rigid conformations are present either for PBLG or for other polypeptides.(9' 12) In all the investigated polymers some flexibility has been detected, the extent being dependent on the natures of the polymer and solvent. This work deals with conformational studies on PELG in TFE and DCA solutions by light scattering and viscosity techniques. Since the first solvent is an helix-supporting solvent, we tried to get information about the type of helical structure present in solution and about its flexibility. A random coiled conformation should be present in the strongly interacting DCA. 681

682

M. TERBOJEVICH et aL EXPERIMENTAL

Solvents Dioxane (reagent grade) was refluxed several days over sodium metal; it was distilled from sodium immediately before use. Dichloroacetic acid (DCA) and trifluoroethanol fiFE) were of reagent grade and were used without further purification. Monomer and initiators ?,-Ethyl-/.-glutamate N-carboxy-anhydride (NCA) was prepared from v-ethyl-t-glutamate and phosgene according to the literature.¢I~) It was crystallized several times from ethyl acetate--carbon tetrachloride. Sodium methoxide solution was prepared by the method of Katchalski and Sela.¢I~) Reagent grade diisoprolylamine and di-n-butylaminewere dried over potassium metal and then fractionally distilled. Polymer preparation Poly-?,-ethyl-L-glutamate (PELG) samples were prepared by polymerization of ?,-ethyl-L-glutamate NCA in dioxane as summarized in Table 1. The polymers were isolated by pouring the reaction mixture into petroleum ether (b.p. 40°-70 °) with vigorous stirring. The precipitate so obtained was filtered, washed and dried to constant weight. Diisopropylamine, di-n-butylamine and sodium methoxide were used as initiators. These compounds in dioxane are known to induce the polymerization of NCA's by mechanisms of the strong base type, leading to polymers with narrow molecular weight distribution (~w/IFln ~ 1-3).¢1~) For this reason the polymer samples examined by light scattering and viscosity were not fractionated. Light scattering measurements Measurements were made with a Sofica L.S. photometer Mod. 42.000, using cylindrical cells plunged in a bath of pure benzene. The instrument was standardized using pure dust-free benzene as a reference liquid, taking the values 48'5 x 10-6 and 16.3 x 10-6 for the Rayleigh ratios Rg0 at wavelengths 4360 and 5460 A.¢16) The cells were cleaned with chromic mixture, water and steam, and then dried in a vacuum desiccator. Solvents and solutions were obtained dust-free by centdfugation at 14,000 rev/min in a Phywe "Pirouette" centrifuge. A special device on the instrument allows checking of the cleanliness of solutions before measurements are made. All measurements were made at 250 in TFE, using unpolarized light of wavelengths of 4360 and 5460 A. For each concentration scattered light was measured at angles between 37.5° and 150~, and the data were treated by the Zimm method.¢17) We thus determined the weight average molecular weight ~ , of the solute, the second virial coefficient B, and the radius of gyration Re of the polymer particles. Samples having l~lw lower than 100,000 do not show appreciable disymmetry and in these cases it was impossible to use the Zimm procedure. For this reason bTIwlower than 100,000 were determined by the standard plots KclRgo vs. c. The refractive index increments (dn/dc) were determined at wavelengths of 4360 and 5460 A using a Brice-Phoenixprecision differential refractometer. ForthesystemPELG-TFE, wefound(dnldc)436o= 0.168_+0-002 ml/g and (tin/de)s460=0-160_+0"003 ml/g. For the system PELG-DCA we found (dn/dc)4a~0=0'050_+0"003 ml/g. From these values it appears obviously convenient to make light scattering measurements in TFE. Mw, Re and B were determined using both 4360 and 5460 A wavelengths. For all samples, identical data were obtained using light of both wavelengths. Viscosities Viscosity measurements have been made at 25 + 0.01° in an Ubbelhode viscometer having a flow time for the solvent longer than I00 see. A multigradient viscometer was used for solutions of the highest molecular weight samples in TFE. The data were treated by the method of Holtzer et aL (tS) In these cases considerable gradient dependence was detected.

RESULTS AND DISCUSSION P o l y m e r samples having different molecular weights were prepared u n d e r selected conditions, as s h o w n in Table 1. The light scattering data are s u m m a r i z e d in Table 2. The molecular weights 1Viw a n d the second virial coefficients B of the first 5 samples

683

Solution Properties of Synthetic Polypeptides TABLE 1. CONDrnONS FOR PREPARATIONOF THE POLYMERSAMPLES Sample code

Solvent

Initiator

A/I*

I1 G1 M2 M5 G2 G3 L3 LI G4 G6 G5 15 G7 L2 C1

DMF Dioxane Dioxane Dioxane Dio×ane Dioxane Dioxane Dioxane Dioxane Dioxane Dioxane Dioxane Dioxane Dioxane Dioxane

Et hyldiisopropylamine di-n-butylamine sodium methoxide sodium methoxide di-n-butylamine di-n-butylamine di-n-butylamine sodium methoxide diisopropylamine diisopropylamine diisopropylamine diisopropylamine diisopropylamine sodium methoxide diisopropylamine

20 5 10 15 20 40 12 50 20 40 30 10 60 160 79

*A/I is the molar ratio of monomer to initiator.

TABLE 2. LXGH'rSCATTERINGDATAIN TFE AT 4360 A Sample code I1 G1 M2 M5 G2 G3 L3 L1 G4 G6 G5 I5 G7 L2 C1

lf4w 20,500 21,100 31,300 59,700 67,100 132,000 162,000 250,000 280,000 323,000 376,000 365,000 398,000 482,000 502,000

Re -----405 496 642 648 696 734 735 685 792 892

L = R~/ (A) -----1403 1718 2220 2245 2411 2543 2543 2374 2744 3095

(dn/dc)= 0-0168 + 0'002

h

B x 104

-----1"67 1"66 1"40 1"25 1' 17 1.06 1"09 0"94 0.90 0-96

8'8 8"3 7"5 22"9 11"2 17'4 13"5 9" 1 5" 1 6"9 8"6 10'0 7" 1 13"5 3"4

* L--Length of the macromolecules.

were determined by the usual plot Kc/R9ovs. c. The light scattering data on the remaining polymer samples were analyzed using the Zimm method. A typical Zimm plot is reported in Fig. 1. Corrections for depolarization of the scattered beam are negligible for molecular weights higher than 32,000 (Table 3). Such corrections where applied only for the first three samples of Table 2. Table 4 shows the viscosity data and Huggins' k' ~19) in DCA and TFE. From Tables 2 and 4 double logarithmic plots [7] vs. l~Iwwere obtained for both solvents. From slopes

684

M. TERBOJEVICH et aL TABLE 3. CABANNESFACTORS Sample code I1 GI M2

lf,lw =

1 (Kc/Rgo)c.o

23,850 22,800 32,000

G*

~f . . . . . .

1.165 1.080 1-022

20,500 21,100 31,300

* Fc is the Cabannes factor.

TABLE 4. Vmcosrrv DATA Sample code

I~7I.

(dllg)

I1 G1 M2 M5 G2 G3 L3 L1 G4 G6 G5 I5 G7 L2 C1

20,500 21,100 32,000 63,700 71,100 132 000 162 000 250 000 280 000 323 000 376 000 365 000 398 000 482 000 502 000

0"31 0"29 0"42 0'67 0-75 1'02 1"25 1"77 1"86 2"34 2"33 2"45 2"38 3"18 3'53

["rflDCA

["r'/]T F E

k'DC A

0-40 0"45 0"32 0"26 0"31 0"14 0-35 0-27 0"35 0"27 0"26 0"32 0"26 0"15 0"35

o

(dt/g)

kTrE

0.22 0-20 0-40 0"79 1'06 1"63 3"22 5.20 5.94 8.20 7"30 9.00 8.90 10"80 17"40

0.32 0"28 0"45 0"46 0.42 0"41 0"53 0-39 0"53 0"50 0"65 0-52 0"33 0"66 0"47

O

X

I I ¼c ~c

) ~c

I -~c

c

senz ~ + Kc

FIo. 1. Zimm plot of PELG in TFE: sample 15. The initial concentration c was 5-21 x 10 -4

g/ml.

Solution Properties of Synthetic Polypeptides

685

and intercepts of Fig. 2 the K and a coefficients of the Mark-Houwink equation have been determined. We obtained: [~7] = 2.022 x 10-4 x M °'73

in DCA

[~1] = 5.017 × 10-7 × M 1"30

in TFE.

Since the a coefficient of the Mark-Houwink equation in DCA is 0"73 we can conclude that in this soNent the conformation of P E L G is that of a random coil. In Fig. 2 we report for comparison the plot of the Doty relation (z) [7/]=2.78 x 10-s x M°'S7 valid for PBLG in DCA. It can be observed that there are considerable differences between PELG and PBLG in the same solvent. For example, a [~7]value of 0"3 leads to 1Vlw= 21,000 in the case of P E L G and to 1~I, = 44,000 in the case of PBLG. This observation shows that the molecular weights of a number of polypeptides reported in the literature and determined by intrinsic viscosity in DCA using the Dory relation, must be considered as very approximate or even erroneous. tOO

o

/

/

/ J . j /S /,,y.>';, ,

I0 '4

IO s

I0¢

Mw FIG. 2. Double logarithmic plot [~1]vs. l~Iw. o PELG in TFE. • PELG in DCA. Dotted line: PBLG in DCA. In TFE, the a coefficient of P E L G is quite high and about the same as for poly-~carbobenzoxy-L-lysineO2) (PCBL) in DMF. Since PCBL is in a helical conformation in DMF(9,12, 20) we can conclude that such a conformation occurs also for P E L G in TFE. This result is not new since Goodman et aL have already reported evidence for a rigid conformation of P E L G in T F E on the basis of N M R data. (21) Association and rigidity of the polypeptide in T F E must be considered. We obtained normal values of Huggin's k' constant in this solvent. No value exceeds 0-7 (Table 4) indicating that important association phenomena do not occur in TFE. Concerning the rigidity of the

686

M. TERBOJEVICH et al.

helices in solution, we present here some evidence for the existence of a considerable degree of flexibility of the molecular rods associated with the helices. In fact the experimental values of Ro (Table 2) plotted vs. l~Iwdo not give a linear plot (Fig. 3) as

X

I

2

3

4

5

MwXI0-~ FIG. 3. PELG in TFE. Experimental RG values plotted vs. bTIw. should be obtained for completely rigid macromolecules. (22) Surprisingly, we found a linear plot .Re vs. M~/2, as should be obtained for a random coil conformation (Fig. 4). However the possibility of a random-coiled conformation in TFE can be ruled out, because if this were the case no explanation could be found for the different viscosimetric behaviour in DCA and TFE.

5

0

1

2O0

I

400

I

600

t

803

FIG. 4. PELG in TFE. Experimental RG values plotted vs. I~'I~'2 Moreover, according to Spach e t al. ~9) the plot ffl~/[-~] vs. log ~ , should be linear in the case of completely rigid rods in solution. This plot for PELG in TFE exhibits a marked curvature (Fig. 5). According to this criterion of rigidity, we must conclude further that the polypeptide heLices in solution have considerable flexibility. From the data of Table 2 we calculated also the length per monomeric residue h; this quantity was found to be strongly dependent on the molecular weight of the samples, increasing with decreasing molecular weight. It can be observed from Fig. 6 that a value of about 2.0 for h0 is obtained by extrapolation to zero molecular weight. This value must be considered as an indicative one. In fact, an exact experimental determination of Ro in the low molecular weight region is very ditticult and subject to errors. Moreover, the lowest molecular weight sample for which it is stiU possible to determine Re is sample G3 having Mw = 132,000. Such a value is still too high for an unambiguous extrapolation in Fig. 6.

Solution Properties of Synthetic Polypeptides

687

2'~0

200 --

,o

c/

o/,

15¢ -

x

o I00

--

50

0

!

v 1 ) !I]I]

IO ~

)

T ) t

10 5

tltl I0 6

FIG. 5. Spach's plot Iv"I-~[[~/]vs. Iog~w for PELG in TFE.

!

!

I.

i

I

1

?.

3

4

5

MwX I0"s

FIG. 6. PELG in TFE. Average length per monomeric residue as a function of the molecular weight.

As reported in the Experimental section, we do not consider th~at polydispersity can affect appreciably our light scattering measurements. In fact, the experimental conditions of polymerization lead to polymers having I~iw/Mam 1"3, as demonstrated by fractionations carried out in our laboratory. (Is) It is interesting to compare the behaviour of PELG in TFE with that of PCBL in D M F and PBLG in DMF. It has been reported that, in DMF, PCBL exhibits a helical conformation with a degree of flexibility greater than that for PBLG in the same solvent.(9, 12) In fact the exponent of the Mark-Houwink equation is 1.27 for PCBL and 1.75 for PBLG (the theoretical value for completely rigid rods in solution is 1.8). (2) 44

688

M. TERBOJEVICH et aL

J I0

X

d 5

r 2

4

6

8

©

FIG. 7. PBLG in DMF. Experimental Ro values as a function of the molecular weight. o Our experiments. • Data of Fujita et al. • Data of Moha et aL [J. Chim. Phys. 1239 (1964)]. M o r e o v e r plots M~/[~7] vs. logMw are almost linear for P B L G in D M F and nonlinear for PCBL, indicating a greater rigidity o f P B L G . (9) Figure 7 shows the plot Ro vs. Mw for P B L G in D M F . The graph is linear, indicating the presence o f rigid rod like particles in solution.* In conclusion, f r o m our data we can say that the behaviour o f P E L G in T F E is qualitatively similar to that o f P C B L in D M F . Both polymers appear to be more flexible than P B L G in D M F . Unfortunately no comparison can be made between the conformations o f P E L G , P B L G and P C B L in the same solvent. In fact P E L G does not dissolve in D M F if 1Ulwexceeds 50,000 and P B L G or P C B L do not dissolve in TFE. Acknowledgement--We thank Dr. L. Strasorier for some measurements of light scattering.

REFERENCES (1) (2) (3) (4) (5) (6)

P. Doty, Rev. mod. Phys. 31, 107 (1959). P. Doty, H. J. Bradbury and A. M. Holtzer, J. Am. chem. Soc. 78, 947 0956). I. Tinoco, J. Am. chem. Soc. 79, 4336 (1957). J. T. Yang and P. Doty, J. Am. chem. Soc. 79, 761 (1957). J. T. Yang, d. Am. chem. Soc. 80, 1783 (1958). V. Ln~ati, M. Cesari, G. Spach, F. Masson and J. M. Vincent, in: Paly-=-aminoacids, Polypeptides and Proteins, p. 121, edited by Stahmann, Univ. Wimconsin Press (1962). (7) A. Wada, ibid., p. 131. (8) V. Luzzati, M. Cesari, G. Spach, F. Masson and J. M. Vincent, J. MoL Biol. 3, 566 0961). (9) G. Spach, L. Freund, M. Datme and H. Benoit, J. Mol. Biol. 7, 468 (1963). (10) H. Fujita, A. Teramoto, K. Okita, T. Yamashita and S. Ikeda, Biopolymers, 4, 769 (1966). (11) D. A. D. Parry and A. Elliott, Nature, Lond. 206, 616 (1965). (12) E. Daniel and E. Katchalski, in: Polyaminoacids, Polypeptides and Proteins, p. 183, edited by Stahmana, Univ. Winsconsin Press (1962). (13) E. R. Blout and R. H. Karlson, 3".Am. chem. Soc. 78, 941 0956). (14) A. Berger, M. S¢la and E. Katchalski, Analyt. Chem. 25, 1554 (1963). (15) E. Scoffone, E. Peggion, A. Cosani and M. Terbojevich, Biopolymers, 3, 535 (1965). (16) C. I. Can" and B. H. Zimm, J. chem. Phys. 18, 1616 (1950). * However, appreciable flexibility must be considered also for PBLG since the length per monomeric residue h appears to be molecular weight dependent in DMF.Cg) However h does not change so much with M~, as in the case of PELG in TFE.

Solution Properties of Synthetic Pol.~zocptides

689

K. A, Stacey, Light Scattering in Physical Chemistry, chap. 2, Butterworths, London (1956). A. M. Holtzer, H. Benoit and P. DoUr, J. Phys. Chem. 58, 624 (1954). M. L. Huggins, J. Am. chem. Soc. 64, 2716 (1942). J. Applequist and P. Dour, in: Polyaminoacids, Polypeptides and Proteins, p. 161, edited by Stahr~ann~ Univ. Winsconsin Press (1962). (21) M. Goodman and Y. Masuda, Biopolymer 2, 107 (1964). (22) C. H. Tanford, Physical Chemistry of Macromolecules, chap. 6, John Wiley, New York (1961).

(17) (18) (19) (20)

R4sum4--On a 4tudi4 le poly-y-4thyl-L-glutamate (PELG) par viscosim6trie et diffusion de la lumi~re en solution darts le trittuoro4thanol (TFE) et l'acide dichloracf:tique (ADC). Darts I'ADC on a trouv4 la relation de Mark-Houwink suivante: [,/] = 2.02 x 10-4 x M o"73; l'exposant obtenu est caract6ristique des polym~res formant des pelotes statistiques. Dans le TFE la relation obtenu: [7]= 5.01 x 10-7 x M r30 indique par son exposant 61ev6 une structure en b~.tonnets ayant une certaine flexibilit4. La longueur moyenne par r4sidu monom~rique h a 4t4 calcul4e tt partir du rayon de giration exp4rimental, d4duit de la diffusion de la lumi~re. Cette grandeur 4tait fonction de la masse mol4culaire du pol.vm4re. En extrapolant tt masse mol4culaire nuUe on a trouv~ h0 = 2.0 A. Sommari(~--Poli-y-etil-L-glutammato ~ stato studiato mediante le tecniche della viscosit/~ e della diffusione della luce usando come solventi acido dicloroacetico 03CA) e trifluoroetanolo (TFE). In acido dicloroacetico ~ stata ottenuta la seguente relazione: [-~] = 2.02 x 10-4 x M0" 73. L'esponente tipico di polimeri aventi forma di "random coil". In trifluoroetanolo 6 stata invece ottenuta la relazione: ['d = 5.01 x 10-7 x Mr30. L'esponente in tale caso indica una conformazione a bacchette aventi elevato grado di flessibilitt~. Dai valori sperimentali di raggio di girazione (Ro) in TFE, ~ stata calcolata la lunghezza media per residuo h. Tale grandezza ~ stata trovata fortementa dipendente dal peso molecolare. Per estrapolazione a peso molecolare hullo si ~ ottenuto h0= 2"0A. Zusammenfassung--Poly-y-tRhyl-L-glutamat (PELG) wurde dutch Viskosittit und Lichstreuung in Trifluor~ithanol (TFE) und Dichloressigsaure (DCA) als L6sungsmittel untersucht. In D C A wurde die folgende Mark-Houwink Gleichung erhalten: [-,7]= 2.02 x 10-4 x Mo'73; der Exponent ist typisch for statistisch gekntiuelte Polymere. In TFE lautet die Gleichung [-,7]= 5'01 x 10-7 x M r 3o; der Exponent weist in diesem Fall auf eine stabchenartige Struktur mit gewisser Flexibilit~.t hin. Aus dem experimentellen Wert ftir den Tragheitsradius (Ra) in TFE, dutch Lichtstreuung bestimmt, wurde die durchschnittliche Lange der Monomereinheit h berechnet, Es ~urde gefunden, dal3 diese GrSI3e stark vom Molekulargewicht des Polymeren abh~ingt. Durch Extrapolation auf Molekulargewicht Null erh~tlt man h0=2"0 A.