Nakajima, Akio

Tanaka, Seiji

Kyoto University Research Information Repository

Title Conformational Properties of Poly (γ-methyl-L-glutamate) inDilute SolutionAuthor(s)Tanaka, Seiji; Nakajima, AkioCitationBulletin of the Institute for Chemical Research, KyotoUniversity (1976), 54(4): 229-233Issue Date1976-10-20URL http://hdl.handle.net/2433/76675RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityBull. Inst. Chem. Res., Kyoto Univ., Vol. 54, No. 4, 1976  Conformational Properties of Poly (r-methyl-L-glutamate)                   in Dilute Solution                    Seiji TANAKA and Akio NAKAJIMA*                             Received April15,1976     From viscosity and osmotic pressure measurements, the characteristic ratio <R02)/n12 of poly  (y-methyl-L-glutamate) in random coil conformation was estimated. The numerical value obtained  supports the theoretical result reported previously.                          INTRODUCTION    Recently, theoretical calculations on unperturbed chain dimension have been carried out for random coil chain of polypeptides.1-6) Conformational properties of polypeptides in dilute solution have also been investigated experimentally.7-11)    In order to develop the method of conformational analysis toward more complicated molecules such as proteins, and to discuss precisely the thermodynamic properties on helix-coil transition, it is necessary to evaluate the validity of the conformational energy contour map and potential functions for polypeptides.1 612,13)The information on ordered structure of polypeptides in the solid state, obtained from the crystallographic study by X-ray diffraction, contributes only to the potential energy minimum in the contour map calculated theoretically. The characteristic ratio, on the other hand, depends not only upon the location of the potential minimum but also upon the form of potential functions or the shape of the overall surface of conformational energy map. Thus, the measurement of characteristic ratio <Ro2>/n12 affords rather exact experimental test on the validity of the theoretical investigation. In this paper, we report experimental results on conformational properties of poly(y-methyl-L-glutamate)(PMLG) in dilute solution.    PMLG is one of popular synthetic polypeptides at the present stage. However, on account of its relatively poor solubility in solvents and of corrosive nature of solvents in which the polymer is soluble, little has been known about average molecular weights and molecular chain dimensions of the polymer. Further, in m-cresol, a helicogenic solvent, for example, the concentration dependence of refractive index is too small to obtain appropriate data from light scattering measurements.    Unavoidably, the choice of the method to determine the chain dimension is restricted. Thus, in the present study, we estimate the characteristic ratio of PMLG by combining the limiting viscosity number in dichloroacetic acid (DCA) with the osmotic pressure data in m-cresol.  * pj[ ' i ,: Department of Polymer Chemistry, Faculty of Engineering, Kyoto University,     Kyoto. ( 229 )                                S. TANAKA  and A. NAKAJIMA                          EXPERIMENTALMaterials y-Methyl-L-glutamate N-carboxy anhydride (NCA) was prepared according to the method of Blout and Karlson.14) Poly(y-methyl-L-glutamate) samples are prepared by polymerizing y-methyl-L-glutamate NCA under conditions as summarized in Table I.    Solvents used in the preparation of polymer samples and for measurements of viscosity and osmotic pressure were purified in appropriate manners. Osmotic Pressure Measurement    Stock solutions were filtered and the concentrations were determined by dry weight analysis. Osmotic pressure of polymer solutions was measured by using a Knauer Membrane Osmometer. Gel cellophane No. 400 membranes, supplied by Tokyo        Table I. Summary of Preparative Information on Poly (y-methyl-L-glutamate) Samples    Sample CodeSolventInitiator[M]/[I]b     ML311NitrobenzeneDiethyl amine65 ML312Ethyl acetateDiethyl amine32     ML313Ethyl acetateTriethyl amine60 ML310NitrobenzeneTriethyl amine55 ML406Dioxane-MDCaTriethyl amine1620     ML306Dioxane-MDCTriethyl amine150    a; Methylene dichloride.    b; Monomer-initiator mole ratio. Cellophane Co., were used after appropriate conditioning. Measurements were made in m-cresol at 57.8°C. Number-average molecular weight Mn and the second virial coefficient A2 are obtained from the following equation. (77./c)1/2=(RT/Mn)1/2(1+A2 Mn c/2) (1) where zr is the osmotic pressure and c the polymer concentration in g/ml. Viscosity Measurement    Viscosity measurements were performed  in DCA at 25±0.01°C with an improved Ubbelohde viscometer having a flow time for solvent longer than 100 sec.                     RESULTS AND DISCUSSION    Experimental results from viscosity and osmotic pressure measurements are summarized in Table II. In Fig. 1, the limiting viscosity number [77] (dl/g) is plotted against the number-average molecular weight Mn on double logarithmic scale. A straight line is drawn as to fit in with experimental points. From the straight line in Fig. 1, the coefficients K and a of Mark-Houwink-Sakurada equation N]=KMa have been determined. The equation obtained is [n]=2.9 x 10-4Mn°•74, in DCA(2) ( 230 )                        Conformational Properties of PMLG  0A - 02 -(a),"""                                         ,~'V(b) S 0 - -Q2 - . -0.6-                     4.3 4.5 4.7 49 51 53                                       Log M            Fig. 1. Relations between the limiting viscosity number [n] (g/dl) in DCA                at 25°C and the molecular weight of poly(y-methyl-L-glutamate) on                double logarithmic scale. Open circles represent the experimental               points on [n] vs. M. The broken lines (a) and (b) represent the [ii]                vs. Mu relations obtained from full line by assuming that M/M„=1.5                 and 2.0, respectively.               Table II. Results of Viscosity and Osmotic Pressure Measurements    Sample Code[n](dllg)DCAM„(cmmoleg-)                          32     ML3110.512.96 x 1044.0 x 10-4     ML3120.864.64 x 1043.5 x 10-4    ML3131.095.40x1046.2x10-4 ML3101.317.62x1045.5x10-4     ML4061.6711.2 x 1048.1 x 10-4     ML3062.5820.5 x 1044.8 x 10-4 The experimental result that a=0.74 means that PMLG exists in random coil confor-mation in DCA. m-Cresol used in the osmotic pressure measurements is known as a helicogenic solvent for PMLG. The second virial coefficient shown in Table II, therefore, is for the molecules in helical conformation.    It was found that polypeptide samples obtained from the polymerization initiated by strong bases had rather narrow molecular weight distribution. For example, it was reported that Mto/M„=1.2,1.4 for poly(y-benzyl-L-glutamate)15) initiated by either diethyl-, di-n-butyl-, or diisopropyl amine, or by sodium methoxide, and M,u/M„=1.15,1.5 for poly(y-benzyl-L-aspartate)16) initiated by triehtyl amine (TEA).    Unfortunately, with respect to PMLG, trials to fractionate the samples or to measure the weight-average molecular weight have not been succeeded in. It may not be so absurdity to assume that the molecular weight distribution of the present PMLG is similar to those of polypeptides15,16) cited above. In this respect, we assumed that M„,/M„=1.5 and that Mu,/M„=2.0; the latter corresponds to the "most probable distribution” for the present samples. The broken lines (a) and (b) in Fig. 1 represent the relation between h] and derived from the relation given by Eq. (2) with M,u/M„=1.5 and 2.0, (231)                             S. TANAKA and A. NAKAJIMA   5 ----------------------------------------------------------  -  0                     ;,3                   - 3E 2 1- 0 1 2 3 4 5 6 7 8 ^,'2 (x102)             Fig. 2. [71]/M,01/2 plotted againstM.'/2. Curves (a') and (b') are recast                from curves (a) and (b) in Fig. 1, respectively. respectively.    The straight line relationships given by curves (a) and (b) in Fig. 1, respectively, are recast to the H/MW1/2 vs. MM1/2 plots, i.e., curves (a') and (b') in Fig. 2, according to Stockmayer-Fixman's relation.17) 1/2=K+(3/2.7r)3/2CCoBMw1/2(3)    From curves (a') and (b'), the values of K are evaluated, and the characteristic ratio <Ro2>/n12 is calculated by using the well-known relation, <Ro2>/n12 =(K/0o) 2/ 3(Mo/l2)(4) in which <Ro2> is the mean square unperturbed end-to-end length, n is the number of amino acid residues in a chain, 1 is the length (3.8 A) of a virtual bond, Mo is the weight of an amino acid residue, and 0o=2.5 X 1021.    The characteristic ratios obtained were 8.5 and 7.7, respectively, with M,,/M„=1.5 and 2.0. Taking into consideration of deviation of experimental points from the curve represented by Eq. (2), <R02>/nl2 is estimated as 8.5±0.7 and 7.7+0.8 for 114/M„=1.5                     Table III. Characteristic Ratios of Polypeptides          Polymer[<1'o2>/n12]cale. [<h'o2)/n12]0bs. Ref. Poly(y-methyl-t-glutamate)*7.7±0.8 this work   Poly(L-alanine)8.385    Poly(y-benzyl-L-glutamate)8.88    Poly(y-benzyl-L-glutamate)7.5±1.2**    Poly(y-benzyl-L-aspartate)9.68    Poly(y-ethyl-L-glutamate)8.2±0.310   Na-poly(L-glutamic acid)8.88   HBr-poly(L-lysine)8.68  Polyglycine2.175    Poly(N-methyl glycine)2.971.8±0.211 Poly(N-methyl-L-alanine)0.585     * The observed characteristic ratio is for M„,IM„=2.0. ** The characteristic ratio was estimated by the authors from the data in ref. 9. (232)                         Conformational Properties of PMLG and 2.0, respectively. Both values are not different significantly. In Table III, the characteristic ratio of PMLG obtained above is listed together with the values for various polypeptides observed by other  authors8-11) and the values calculated theoretically in our previous paper. 5) The characteristic ratio obtained in this work is in agreement with the observed values for other a-L-polypeptides having -CH2R side chains and with the calculated value for poly(L-alanine), though some discrepancies are observed among experimental values of characteristic ratio for a-L-polypeptides having different side chains. However, it is difficult to find any systematic trend depending on the kind of side chains.    The experimental evidence presented here appears to support the validity of theoretical treatments on the conformational analysisi,2,5,6,12,13) for the polypeptide chain, in particular, for poly(L-alanine) chain. Further studies are expected to clarify in detail the effect of side chains on the characteristic ratio for a-L-polypeptides having -CH2R side chains.                           REFERENCES (1) D. A. Brant and P. J. Flory, J. Amer. Chem. Soc., 87, 2791 (1965). (2) D. A. Brant, W. G. Miller, and P. J. Flory, J. Mol. Biol., 23, 47, 67 (1967). (3) P. R. Schimmel and P. J. Flory, ibid., 34, 105 (1968). (4) P. J. Flory, "Statistical Mechanics of Chain Molecules," Interscience Publishers, New York,       N. Y., 1969, Chapter VII. (5) S. Tanaka and A. Nakajima, Polymer J., 2, 717 (1971). (6) S. Tanaka and A. Nakajima, ibid., 2, 725 (1971). (7) P. Doty, J. H. Bradbury, and H. A. Holtzer, J. Amer. Chem. Soc., 78, 947 (1956). (8) D. A. Brant and P. J. Flory, ibid., 87, 2788 (1965). (9) H. Fujita, A. Teramoto, T. Yamashita, K. Okita, and S. Ikeda, Biopolymers, 4, 781 (1966).    (10) M. Terbojevich, E. Peggion, A. Cossani, G. D'Este, and E Scoffone,Europ. Polym. J., 3, 681      (1967).    (11) J. H. Fessler and A. G. Ogston, Trans. Faraday Soc., 47, 667 (1951).   (12) R. A. Scott and H. A. Scheraga, J. Chem. Phys., 45, 2091 (1966).    (13) T. Ooi, R. A. Scott, G. Vanderkooi, and H. A. Scheraga,T Chem. Phys., 46, 4410 (1967).    (14) E. R. Blout and R. H. Karlson, J. Amer. Chem. Soc., 78, 941 (1956).    (15) E. Scoffone, E. Peggion, A. Cossani, and M. Tervojevich, Biopolymers, 3, 535 (1965).    (16) Y. Hayashi, A. Teramoto, K. Kawahara, and H. Fujita, ibid., 8, 403 (1969).    (17) W. H. Stockmayer and M. Fixman, J Polym. Sci., Part C, 1, 137 (1963). (233)

Conformational Properties of Poly (γ-methyl-L-glutamate) in Dilute Solution

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Conformational Properties of Poly (γ-methyl-L-glutamate) in Dilute Solution

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