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COXXUNICATIONS TO THE EDITOR
VOl. 79
OPTICAL ROTATION AND HELICAL POLYPEPTIDE CHAIN CONFIGURATION IN a-PROTEINS
ably consist of cables of a a-helices in a supercoiled c o n f i g ~ r a t i o n it ~ ~ would ~ ~ ~ appear that this asSir: sociation in aqueous solution, as .well as superiinA theory of optical rotatory dispersion for helical posed backbone dissymmetry, do not markedly macromolecules has recently been developed by affect rotatory behavior. Moffitt.' This has been applied successfully to In the denatured state one expects absence of the rotatory behavior of synthetic polypeptides in helical backbone contribution to the rotation.'O the a - c o n f i g u r a t i ~ n , ~ ,and ~,~,~ of "globular" pro- Indeed, we have found that bo is approximately teins6 Here we present the result of an examina- zero for all the proteins except paramyosin whose tion of a series of "fibrous" proteins which yield in bo is of the order of -190". Moffitt's equation the condensed state the a-type wide-angle X-ray then reduces to a one-term Drude equation, aqd diffraction diagram. the Xc's found from the latter are close to 2100A. In the native state the proteins examined have a (see Table I). This fact supports our use of bo as rotatory behavior in aqueous solution similar to a measure of helical content. There is independthat of synthetic polypeptides in helix-inducing sol- ent evidence that paramyosin is not completely vents. With the exception of fibrinogen, none of denatured by the urea treatment." The values of these proteins shows "simple" dispersion, ;.e., fol- the specific rotations listed in Table I are in agreelows a one-term Drude equation. The data fit ment with those found for other denatured proRIoffitt's equation for helical systems teinsln and synthetic polypeptides in the random coil configuration.? The rotation of myosin is close to the proportional sum of the rotations of light and heavy where M is the molecular weight per residue and n meromyosin; hence it would appear that there is is the refractive index of the solvent. Within the no large-scale unfolding of helical domains in the experimental error of about i loOK., Xo equals myosin molecule when split by trypsin. We note that although there is a variation of 2100K., in agreement with the XO reported for poly-y-benzyl-L-glutamate and poly-L-glutamic helical content in "fibrous" proteins, the values are acid.3 A value of near -600" for the constant bo generally higher than those characterizing "globucharacterizes a fully-coiled, right-handed a-helix lar" proteins. Asymmetry is thus an expression of synthetic polypeptide^.^ Assuming that Xo of high helical content. The relationship of proline content to helical for the helical and for the non-helical configurations are close, one can use bo as a measure of helical con- configuration in these a-proteins will be described tent. Table 1 lists the bo's obtained on ;his as- in another communication. We thank Professors Paul Doty and William sumption, using a value of XO equal to 2100A. The Noffitt, and Dr. J. T. Yang, for discussions, and for allowing us to read their manuscripts before TABLE I publication. lye thank Professor Richard S. Natives Denaturedb /almoc bud Ial~iso Xo ( h , ) e Bear for interest and advice, and Professor David Waugh for the fibrinogen sample. Light meromyosin fraction I' Tropomyosin Paramyosin Light meromyosin Myosin Heavy meromyosin Fibrinogen
-13.0" -16.0" -11.1" -20.4" -28.7' -34.5' -58.2'
-660" -620' -600' -490' -370" -300" -210'
-118" -118' -63' -107'
-108" -103' -110'
2120 2130
... 2130 2180 2150 2130
I n 0.6X KCl, PH 7.0; fibrinogen in 0.3M KaCI, pH 6.2. * I n 9.5M urea. Measurements were made with a Rudolph High Precision Polarimeter with photoe1ect;ic attachment at four wave lengths: 3650, 4360, 5160, 5780A., isolated by glass and interference filters from a mercury Obtained from plots of arc; room temperature, 20 f 3'. [alx($!/lOO) (3/(n2 2) ( A 2 - X o 2 ) us. l / ( X Q - XO*), Xo = 2100A. e Obtained from plots of X 2 [ a ] x us. [ a l ~ .f ~P a r t of light meromyosin resistant to ethanol treatment representing about half of light meromyosin.
+
negative sign indicates that the helices in the "fibrous" proteins have the same sense of twist as those in synthetic polypeptides and other proteins. Since the macromolecules of these a-proteins prob(1) W. Moffitt, J . Chem. Phys., 25, 467 (1986). (2) P. Doty and J . T. Yang, THIS JOURNAL, 78,498(1956). (3) W. Moffitt and J. T. Yang, Proc. Wall. A c n d . Sci. ( W a s h . ) , 42, 597 (1956). (4) W. Moffitt, ibid.,42, 736 (1956). ( 8 ) P. Doty, A. Wada. J. T. Yang and E. R . Blout, J . Polymer S c i . , in press. ( 6 ) P. Doty ann J. T. Yang, TEISJOURNAL, in press.
(7) F. C. Crick, Acta Crysl., 6, 689 (1953). (8) I>.Pauling and R . J. Corey, Natirre, 171, 59 (1953). (9) R. S. Bear and C. C. SeIby, J . Biophy?. R i o c h ~ m .Cytol.. 2, 55 (1956). (IO) C. Cohen, S a l t w e . 176, 129 (1985). (11) A . G. Szent-Gyorgyi, unpublished d a t a .
(12) The work in this laboratory was aided by a grant from t h e National Foundation for Muscular Dystrophy. (13) Aided in part by Research Grant A-901 from the National Institutes of Arthritis and hfetabolic Diseases of the National I n stitutes of Health, United States Public Health Service. (14) This work done during the tenure of an Established Investigatorship of the American Heart Association. C H I L D R E N ' S C A N C E R RESEARCH F O U N D A T I O N , AND THE DEPARTMENT OF PATHOLOGY OF THE CHILERET'S MEDICALCENTER,^^ BOSTOS,MASSACH'CSETTS DEPARTMEST O F BIOLOGY13 CAROLYN COHEN
MASSACHUSETTS ISSTITUTE OF TECHNOLOGY CAMBRIDGE, MASSACHUSETTS
ISSTITUTEFOR MUSCLERESEARCH^^ MARISEBIOLOGICAL LABORATORY A'OODS
HOLE,k f A S S A C H U S E T T S h N D R E W G. SZENT-GYORGYI RECEIVED SOVEYBER 28, 1956
THE STRUCTURE OF T H E TRANSITION STATE FOR ELECTROPHILIC AROMATIC SUBSTITUTION
Sirs: Present theories of aromatic electrophilic substitution allow only an indefinite formulation of
Jan. 5, 1957
COMMUNICATIONS TO
THE
EDITOR
249
the transition state structure, somewhere between the extremes A and B. Most frequently the transition state is approximated by B,' although there are indications that in some systems B is a discrete intermediate.2,3s4 We present herein kinetic and
I)6fail to show significant aryl participation, except to a small degree for R equal to 2,4-dimethoxyphenyl, and contrast with the kinetic results indicating important Participation for the 4-aryl-1-butyl brosylate series,b in which for aryl equal to p-methoxyphenyl and 2,4-dimethoxyphenyl, k A r / k c s H s is 1.8 and 10, resp.5 Since the rate constant for direct formolysis should be about the same for I and 11 series, it follows that kiar/k;ir must be considerably larger than unity. A B The difference in free energy change for the procthermodynamic data which indicate that structure esses -(CHZ)~--+ cyclopentane (AF5) and -(CH2)6B is not a good representation of the transition + cyclohexane (A F 6 ) , calculated accurately from state, a t least for certain cases. thermodynamic data on ~yclopentane,~cycloUnder suitable conditions the ionization of aryl- hexane,' -(CH2)5--8 and -(CH2)6-,* ;.e., AFr sulfonate from 4-aryl-1-butyl and 5-aryl-1-pentyl AF6, is +4.3 kcal.!mole (25') and from this the arylsulfonates (I and 11, resp.) may proceed with difference in free energy, AF(I+. IB) - AF(II+ IIB) aryl participation (intramolecular electrophilic is obtainedQas f3.6 kcal./mole. On the basis of I1 substitution) via IA, IB and IIA, IIB, resp. Com- transition state B, therefore, kiar/kpar 2.5parison of the rate constants for these participation For a transition state of type A the rereactions (kiar and k&) can be used to decide verse is expected since the incipient ring in IA is between A and B type transition states, since, as is strainless, with all hydrogens staggered and an opderived below, for extreme A, kiar/kFar > 1 and timum (linear) arrangement of C A ~C0Ts , and OTS, for extreme B,
