Polymer Journal, Vol. 17, No. 4, pp 621-631 (1985) Random-Coiled Conformation of Polypeptide Chains II. Experimentally Evaluated Characteristic Ratio of Poly(N5-2-hydroxyethyl­ L-glutamine) and Theoretical Conformational Analysis of Poly(L-glutamine) and Poly(L-glutamic acid) Masahito 0KA, Toshio HAYASHI*, and Akio NAKAJIMA Department of Polymer Chemistry and *Research Center for Medical Polymers and Biomaterials, Kyoto University, Kyoto 606, Japan (Received September 12, 1984) ABSTRACT: Random-coiled conformations of fractionated po1y(N 5 -2-hydroxyethyl-L-glu­ tamine) were experimentally investigated and the characteristic ratio was obtained by the Stockmayer-Fixman equation with the experimental results for intrinsic viscosity and weight­ averaged molecular weight. Moreover, random-coiled conformations of poly(L-glutamine) and poly(L-glutamic acid) were theoretically analyzed by conformational energy calculations based on intra-residue interactions, and the calculated characteristic ratio was in good agreement with the experimental results. KEY WORDS Random-Coiled Conformation I Characteristic Ratio I Intrinsic Viscosity I Ultracentrifugation I ECEPP 1 Conformational Energy Calculation I Poly(N 5-2-hydroxyethyl-L-glutamine) I Poly(L-glutamine) I Poly(L-glutamic acid) I The characteristic ratio, a basic property of for normal synthetic polymers. 2) A random­ random-coiled polymer chains in dilute solu­ coiled conformation can exist only in a tions, provides information on local confor­ limited solvent system16·17 in which intra- and mations of polymer chains. The values of side­ inter-molecular hydrogen bonds of polypep­ chain derivatives of poly(L-glutamic acid) and tides are broken. 3) The EJ-state14 of synthetic poly(L-lysine), treated as alanine-type poly­ polypeptides has not been found yet because peptide chains, 1 •2 were experimentally investi­ of helical conformations due to favorable gated. 3 - 13 The experimental values of 6 to I 0 short-range interactions such as backbone/ distribute over a somewhat wide range, due to backbone hydrogen bond; i.e., short-range experimental difficulties, polydispersity of the interactions are favorable compared to long­ samples and uncertainty of the universal con­ range interactions and polypeptidejsolvent in­ stant 4J0 14·15 to calculate the characteristic teractions in the e-state. ratio from viscosity data. Experimental diffi­ Such relations as the Stockmayer-Fixman, 18 culties are concerned with the following Orofino-Flory, 19·20 and Flory-Fox,21 used for points. 1) Synthetic polypeptides have the evaluation of the characteristic ratio from tendency to take helical conformations sta­ viscosity data are derived for the mono­ bilized by favorable intra-molecular hydrogen disperse polymer systems. However, many bonds and to cause molecular aggregations characteristics ratio3 ·7 ·9 · 10· 13 have been with intramolecular hydrogen bonds in evaluated from data on unfractionated sam­ common solvents which are good solvents ples. Moreover, several values [(2.1-2.6) x 621 M. OKA, T. HAYASHI, and A. NAKAJIMA 1021 ] have been used as the universal constant introducing phosgene gas into suspensions of cf>0 14 in the Stockmayer-Fixman, Orofino­ BLG in tetrahydrofuran (THF). BLG-NCA Flory, and Flory-Fox equations. This ambigui­ was purified by repeated recrystallization from ty causes uncertainty in the characteristic ratio ethyl acetate solution with the addition of by as much as 5% to 20%. petroleum benzene. BLG-NCA was dissolved Using the fj-methylene group approxima­ in the mixture of dry methylene dichloride and tion, the characteristic ratio of polypeptide dioxane (volume ratio I: 2), and polymerized chains composed of alanine-type residues were at room temperature in the presence of tri­ theoretically investigated, 1.2.22 and 9.272 and ethylamine as the initiator ([M]/[I] =50). After 8.3822 were obtained. These values are polymerization for 3 days at room tempera­ slightly higher than those experimentally ob­ ture, poly(y-benzyl-L-glutamate) (PBLG) was tained.4-11 As shown in the previous paper,23 precipitated in excess cold methyl alcohol, a more precise analysis in which all intra­ filtered and dried under reduced pressure. residue interactions including side-chain/ PBLG was aminolyzed through a reaction backbone interactions and freedom of internal with 2-amino-1-ethanol for 24 h at 55°C, 26 and rotations of the side chain are considered, dialyzed against five times the volume water of gives 11.24 and 12.33 for poly(L-phenyl­ the reactant solution using a CU-8000 tube alanine) and poly(L-tyrosine), respectively, and with a Imp pore. After dialysis, poly(N5-2- the latter value was very close to that 12.324 of hydroxyethyl-L-glutamine) (PHEG) was pre­ poly( 0-benzyloxycarbonyl-L-tyrosine ). These cipitated from an aqueous solution into ace­ results indicate that side-chain/backbone inter­ tone. The precipitated PHEG sample was actions are very important to stabilize local separated into 6 fractions with the water­ informations of polypeptide chains even dioxane system, and each was freeze-dried. though they are not composed of fj-branched Completion of aminolysis was confirmed by residues. It should be of interest to make a the disappearence of absorption spectra of precise analysis of alanine-type polypeptide the benzyl-group at 257 nm, using a Hitachi chains such as poly(L-glutamine) and poly(L­ Model ESP-3T Spectrometer. glutamic acid), considering the dependence of side-chain/backbone interactions on backbone Measurements conformations. Viscosity measurements were performed in In this paper, poly(N5-2-hydroxyethyl-L­ 0.05 M sodium phosphate buffer at pH 7.4 and glutamine), which takes on a random-coil con­ with an imposed Ubbelohde-type vis­ formation12 in water at room temperature, was cometer having a flow time for the solvent synthesized as a model polypeptide of poly(L­ longer than I 00 s. glutamine), and its characteristic ratio was The molecular weight of the samples was evaluated. Moreover, theoretical analyses of determined from equilibrium runs on polymer poly(L-glutamic acid) and poly(L-glutamine) solutions by a MOM Type-3170 B ultracen­ were made, considering the side-chain/back­ trifuge. As the solvent, 0.05 M sodium phos­ bone interactions and freedom of i, i, and x3 phate buffer was used. Solution concentra­ in the side-chain conformations. tions from 0.05 to 0.20 (g dl-1) were used in the sedimentation equilibrium runs. EXPERIMENTAL Optical rotatory dispersion (ORD) mea­ surements were carried out with a JASCO J-20 Materials Recording Spectropolarimeter at 25oC. The The N-carboxy anhydrides (NCA) of y­ concentration of the polymer solution was benzyl-L-glutamate (BLG) were prepared25 by 1.0 g dl-1 throughout the measurements to 622 Polymer J., Vol. 17, No.4, 1985 Random-Coiled Conformation of Polypeptides II. cancel out the effects of polymer concentration Glu-NHMe has 60 energy minima with AE < 3 on optical properties. The Moffit-Yang pa­ kcal mol- 1 and all their x4 ·2 are almost 180° ± rameter -b0 was determined by the Moffit­ 2o except that only two energy minima have Yang procedure.27 x4 ·2 = 24° (AE = 1.82 kcal mol- 1) and -26° (AE=2.14kcal mol- 1). Their results suggest THEORETICAL that fixing x4 of Gln and x4 ·2 of Glu at 180° has minor effects on the stability of backbone The nomenclature and conventions adopted conformations with intra-residue interactions, were those recommended by an IUPAC-IUB since the contributions of these conformations nomenclature commission.28 Assumptions and to the partition function (eq 1-1 33) are negli­ definitions used in this work corresponded to gible. All other dihedral angles of backbone those in the previous work. 23 Conformational were fixed at 180°. energy E;(c/J;, t/1;, x;) of residue i was calculated for a model single residue peptide with two RESULTS blocking end groups, acetyl- and N-methyl­ amide- (i.e., Ac-X-NHMe). All interactions in Experimental Evaluation of the Characteristic this model peptide are referred to as intra­ Ratio residue interactions. The validity of this The weight-average molecular weight Mw, treatment is discussed in the paper 1. 23 degree of polydispersity Mz/ M w' intrinsic Conformational energy calculations were viscosity [ry], and Moffit-Yang parameter - b0 carried out for Ac-L-Gln-NHMe and Ac-L­ are summarized in Table I. The degree of Glu-NHMe with ECEPP29 using the standard polydispersity Mz/ M w is relatively small, ex­ geometry for bond lengths and bond angles, cept for the last two fractions, PHEG5 and except that the ca-H bond length was in­ PHEG6. The value -b0 =0 indicates that PHEG takes on a random-coil conformation creased from 1.00 to 1.09 A. 30·31 The backbone conformations ¢ and t/J, and two side-chain in 0.05 M sodium-phosphate buffer at pH 7.4 and 25°C. Figure 1 shows a double logarithmic dihedral angles x1 and x2 were changed at !5° intervals. l was changed at 30° intervals, and plot of [ry] versus Mw for PHEG in 0.05 M sodium-phosphate buffer at pH 7.4 and 25°C. x4 of Gin and x4 ·2 of Glu were fixed at 180°. As shown by Zimmerman et al., 32 Ac-Gln-NHMe has 69 energy minima with AE < 3 kcal mol- 1 1-0 and all their l are nearly 180° ± 4°; also, Ac-L- Table I. Molecular characterization of fractionated PHEG samples [I]] Sample Mwx J0-4a M.fMw' code dl g-1 b PHEGl 0.86 1.24 0.093 PHEG2 1.36 1.16 0.125 0-1 PHEG3 2.53 1.15 0.184 0 PHEG4 4.47 1.18 0.264 PHEG5 6.69 1.45 0.355 PHEG6 14.3 1.80 0.572 0 Figure I. Double-logarithmic plot of [17] vs.