NOTES
2820
Vol. 70
ignated as above according to the system employed in the “Ring Index.”2 It has not been studied further since its preparation from 5aminotetrazole and acetylacetone in the presence of piperidine, the method which was also employed in this work.3
shifts cannot be developed. However, if one admits ionic structure^,^ this behavior can apparently be explained satisfactorily. The dipolar ions (“Zwitterions”) shown in (11) to (V) contribute to the arrangement of the compound in acid, and, to a somewhat lesser extent, in neutral solution. These considerations would lead one to expect that the resulting spectrum, with respect to the position of the maximum, should be similar to that of a benzenoid ring with a conjugated double bond. Indeed, a measure of similarity to the spectra of styrene, i n d ~ l e ,benzotriazole8 ~ and even benzoxazole9 is observable. In alkaline medium, however, this polarization tendency, due to the addition of protons in position 1 or 3, disappears and the structure (I) is the only one remaining. Such a spectrum should more closely resemble that of a polyene and shift the absorpCH3 tion maximurn toward the visible range. This is I borne out by the similarity of the spectrum of 5,7From an examination of the spectra of 5 , i - dimethyltetrazolo [alpyrimidine in alkali with dimethyltetrazolo [alpyrimidine shown in Fig. 1, 4 that of 1,3,5,7-octatetraene which was studied by a tremendous batho- and hyperchromic shift is to KovnerlO and Hausser.” Both of the combe noted in comparing the spectrum at pH 13 pounds have four conjugated double bonds and a with that in neutral and acidic solutions. As the “maximum extinction coefficient” beyond 300 mp.
CHa I1
p e113 IV
X ‘y
( 5 ) The authors are grateful to Dr. Elmer J. Lawson for his helpful discussion on the formation of dipolar ions. (6) Elliott and Cook, I n d . Eng. Chem., Anal. E d . , 16, 20 (1944); Rodebush and Feldman, THIS JOURNAL, 68, 896 (1946). ( 5 ) Johnson, Bruce and Dutcher, ibid., 66, 2005 (1943). ( 8 ) Specker and Gawrosch, Bcr., 75B,1338 (1942). (9) Ramart-Lucas and Vantu, Bull. soc. chim., [ 5 ] 146, 1165 (1936). (I(]) Kovner, Acta Physicochim. ( U . R . S . S . ) ,19, 385 (1944). Cf. also Ferguson and Branch, THISJOURNAL, 66, 1467 (1944). (11) Hawser, 2. fcchn. Physik, 16, 10 (1934).
220
260
300
340
X in mr.
of 5,7-dimethyltetrazolo[a]ppirnidine Fig. 1.-Spectra in - ethanol (gayo), ---- 0.01 N “21, - - 0.01 N NaOH.
nitrogen atoms do not bear any hydrogen [atoms], a classical picture on the basis of tautomeric (2) Patterson and Capell, “The Ring Index,” Reinhold Publishing Corp., New York, N. Y., 1940. (3) The authors wish to express their appreciation to Miss R . Pauline Brundage for her preparation of the compound. (4) All spectral determinations were made with a Beckman quartz spectrophotometer, Model DU, Serial No. D-377, as in earlier work [cf. Ewing and Steck, THISJOURNAL, 68, 2181 (1046)].
THESTERLING-WINTRHOP RESEARCHIXSTITUTE RECEIVED APRIL20, 1948 RENSSELAER, N. Y.
Remarks on the Physico-Chemical Mechanism of Muscular Contraction and Relaxation B Y JACOB
RISEMAN AND
JOHN
G. KIRKWOOD
The physico-chemical processes underlying the contraction and relaxation of muscle have been the subject of much speculation. Recently significant between the behavior Of and that of rubber and synthetic elastomers have
Aug., 1948
NOTES
282 1
been investigated by Bull’ and others. That the work of St. Gyorgi suggests may play a r0le.5 structure of striated muscle, and possibly smooth Nevertheless, both of these charging mechanisms muscle, is much more ordered than those of elasto- leave obscure the manner in which the chain of meric cross-linked high polymers is clearly demon- carbohydrate oxidation reactions supplies free strated by the studies of Schmitt and his collabo- energy for the muscular processes. We are inclined to the view that phosphorylaratom2 Nevertheless, important aspects of structural similarity exist. The basic structural unit of tion of the hydroxy amino acid residues of the the muscle fibril is considered to be the linear poly- myosin or actomyosin molecule by adenosine peptide chain of the myosin or actomyosin mole- triphosphate provides a charging mechanism cule. Like the segments of a polymer network, in best accord with known facts. From its the long polypeptide chain possesses many inter- amino acid analysis, myosin is known to contain nal rotational degrees of freedom which allow i t to a large proportion of hydroxy amino acid residues; gain configurational entropy on contraction. serine, 3.9% ; and threonine, 4.95%.6 Myosin Therefore i t is probable that the elastic modulus is also considered to be one of the enzymes inof a structure composed of such elements is in part volved in the dephosphorylation of ATP to ADP determined by the dependence of their configura- and inorganic phosphate. Assuming that the first step in the dephosphorylation of ATP contional entropy upon elongation. The contraction or relaxation of a muscle seg- sists in the phosphorylation of the -OH groups ment under constant stress is the consequence of the hydroxy amino acid residues of the myosin of a change in the elastic modulus arising from al- molecule, we conclude that a t a PH of 7, the teration of its structural units. Following cer- approximate PH of the sarcoplasm, each - H2P04 will be approximately singly ionized to tain ideas suggested by the work of K. H. M e ~ e r , group ~ we wish to examine the hypothesis that the essen- - HP04-. Thus the phosphorylation process tial alteration of the structural unit, leading to a would impart to the neutral sites originally occhange in.modulus, is a change in its net electric cupied by -OH, approximately one unit of charge. A structural unit consisting of a long negative charge. The extension of the myosin polypeptide chain can gain or lose electric charge chain and that of the structure of which it is the in several ways in response to changes in its phys- unit, resulting from electrostatic repulsion beico-chemical environment.. If the absolute mag- tween the negatively charged - HP04- groups, nitude of the charge, whatever the sign, is in- stores up the free energy, released in the degcreased, electrostatic repulsion between the ele- radation of the high energy phosphate bond of mentary charges comprising the increment will ATP, in the form of negative configurational decrease the elastic modulus by destroying the bal- entropy of extension. Subsequent dephosphorylance between the external stress and the contrac- ation of the myosin molecule with release of intile force arising from configurational entropy. organic phosphate ion to the sarcoplasm would Conversely, if the magnitude of the charge is de- remove negative charge from the molecule and creased, the modulus will increase. According to release the stored free energy as mechanical work this view, the relaxed state of muscle is an elec- in contraction of the structure. The coupling trically charged state and the contracted state between the carbohydrate oxidation process and one in which the polypeptide chains are in an the mechanical processes of muscle activity is uncharged or “ i s o e l e c ~ c ”condition. We shall thus clarified by the proposed mechanism. ATP, presently make some rough estimates of the regenerated in the chain of oxidation reactions, change in elastic modulus produced by electro- serves as a carrier of free energy released in these static repulsion between charges distributed a t reactions to the myosin units of the muscle strucintervals along a polypeptide chain, after dis- ture. cussing possible mechanisms by which it might In order to examine the quantitative implicagain or lose charge. tions of our hypothesis, we will make some rough Since a polypeptide chain is an ampholyte, estimates of the change in elastic modulus E of a it can gain or lose charge in response to a change flexible linear molecule produced by attaching in the PH of its environment. This mechanism electric charges of equal magnitude at equal inwas proposed by K. H. Meyer3 as a basis for the tervals along its length. If the terminal groups analysis of the energetics of muscular relaxation of the molecule are separated by a distance L and contraction. Meyer’s proposal was re- and n charges of the same sign and magnitude jected by Weber4 on grounds which still must be e are attached to the chain a t points separated regarded as inconclusive. Adsorption of cations, by equal numbers of bonds, the random coil for example potassium, by actomyosin segments model of a flexible linear molecule leads to the provides a second method of charging, which the following approximate estimate of the increment in elastic modulus AE, produced by the charge (1) H. €3. Bull, THISJOURNAL, 67, 2047 (1945). increment ne (2) F. 0. Schmitt, M. A. Jakus and C. E. Hall, Biol. Bull., BO, 32 (1946); M. A. Jakus and C . E. Hall, J . B i d . Cheni., 167, 705 (1947). (3) K. H. hleyer. Biochefn. Z.,214, 1 (1929). (4) H. H . Weber. i b i d . , 217, 430 (1930).
( 5 ) A. St. Gyorgi, “Chemistry of Muscular Contraction,” Acndemic Press, Inc., N. Y . ,1947, p. 30. (6) M . L. Anson and J. T. Edsall, “Advances in Protein Chemistry,” Vol. I, Academic Press, Inc., New York, N. Y . , 1944, p. 310.
NOTES
2822
Vol. 70
methylphthalimide. This compound was first prepared by Sachs' by the hydrolysis of N-browhere De is an ef€ective dielectric constant, N momethylphthalimide. It was later prepared by is Avogadro's number, M the molecular weight Sachs2 from formaldehyde and phthalimide in of the linear molecular unit of the structure and sealed tubes and by Buca from the same reactants a t atmospheric pressure. The melting. points rep is the density of the structure, regarded as a three-dimensional network with L the average ported by these authors were 141-142 , 139-140' distance between net points. Since electrostatic and 137-141°, respectively. Buc also reported interactions between the charges of neighboring that the melting point is not improved by crystalchain segments are neglected and other gross lization from ethanol. We attempted to eliminate the impurities by approximations have been employed in its deriadding about 1 g. of fuller's earth per 100 ml. of vation, Eq. (1) is intended to provide no more than an estimate of the order of magnitude of AE. solution in BUC'Sprocedure. This yielded a prodThe elastic modulus of muscle1 is of the order uct melting a t 14e145' whicb was, however, still of magnitude 10" dynes/sq. cm. In order to not pure. A product of high purity melting a t 149.5' was produce a change of this order of magnitude by 'ng the structural units, we estimate from finally obtained through an unstable, previously Eq. 1) a magnitude of n of the order of 100, with unreported complex formed from N-hydroxythe rough assignments of value P = 1, M = lo6, methylphthalimide and one mole of pyridine. Chloro-, bromo- and iodomethylphthalimides L = 104A, De = 100 to the other parameters. The values of L and M are those determined for were prepared from the pure compound and the the myosin or actomyosin molecule in solution, appropriate halogen acid. I n all cases the prodand De is assumed to be of the order of magnitude ucts had higher melting points than those previof the dielectric constant of water. The esti- ously reported. Procedure.-A solution obtained by warming 17.7 g. mated number of charges would require phosN-hydroxymethylphthalimidea in 30 ml. of pure phorylation sites situated a t intervals of 100 A. of pyridine was filtered, if necessary, and left to crystallize. along the myosin or actomyosin molecule. This If crystallization did not occur, seed crystals were obtained value is not inconsistent with the hydroxy amino by placing a few drops of the solution in a desiccator over sulfuric acid. As soon as the first crystals appeared, they acid content of myosin. added to the solution. The new compound crystalThe observation of Needham' that the flow were lized in long, bright needles which after cooling in an birefringence of myosin solutions is diminished ice-bath were filtered with suction. by the addition of ATP seems a t first to be in To determine the pyridine the crystals were dried briefly contradiction with our hypothesis. However, on a porous plate over calcium chloride. A weighed was then dried in vacuum over sulfuric acid. it seems that the effect was observed under condi- sample The crystals gradually lost their brightness and came to tions leading to the dissociation of actomyosin constant weight after twenty-four hours. into actin and myosin, according to St. Gyorgi.6 Anal. Calcd. for CeHtOaNCbHsN: pyridine, 30.58. We have deliberately avoided placing undue Found: pyridine, 30.68. The residue melts at 148.5-149 '. One crystallization emphasis on hypothetical structural details of acetone brings the m. p. to 149.5'. muscle and on the detailed analogy between the from Anal. Calcd. for C&03N: N,7.91. Found: N, 7.88. elastic properties of muscle and elastomers. The halogenomethylphthalimides were prepared esThe essential qualitative aspects of our sugges- sentially according to Gabriel,' heating at 50" for one hour tions, (a) change in the elastic modulus of the after the crystals separated. The crystals were filtered, structure due to alteration of the electric charge of washed with the appropriate acid and then dried over acid and potassium hydroxide. The results obthe structural unit, considered to be a polypeptide sulfuric tained are summarized in the table. chain rich in hydroxy amino acid residues; (b) TABLEI charging of the structural unit through phosphorylN-HALOMETHY LPHTHALIMIDES ation of the hydroxyl groups by ATP, are to a large Yield extent independent of assumed structural details. Comcrude, 7-M. p.. '(2.-
7
(7) J. Needham, Shih-Chang Shen, D. Needham and A. S. C. Lawrence, Natura, 147, 766 (1941).
INSTITUTE FOR HIGHPOLYMER RESEARCH BROOKLYN POLYTECHNIC INSTITUTE RECEIVED APRIL3, 1948 BROOKLYN, NEWYORK GATESAND CRELLINLABORATORIES OF CHEMISTRY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA
Purification of N-Hydroxymethylphthalimide through a Molecular Compound with Pyridine BY EUCLID J. SAKELLARIOS
In connection with the synthesis of N-alkylated phthalimides, we have prepared N-hydroxy-
pound
%
Crystn. solvent
Obs.
Prev.5
Ethyl acetate 136.5 132-1334 Chloro- 93 Bromo- 91.1 Ethyl acetate 151.5 149-1W 92.3 Benzene-ethyl acet. 155.5 153' Iodo5 Best previously reported. RESEARCH LABORATORY THEPIRAEUSDYEWORKS (FORMERLY s. A. OECONOMIDES AND CO.) NEWPHALERON, NEAR PIRAEUS RECEIVED FEBRUARY 24, 1948 ATHENS. GREECE (1) Sachs, Bey., 81, 1231 (1898). (2) Sacha, ibid., 81, 3230 (1898). (3) Buc, THISJ O U R N A L , 69, 254 (1947). (4) Gabriel, Bcr., 41, 242 (1908). ( 5 ) Gabriel, Ber., 81, 1229 (1898).
