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polyacetylenes. Except in the case of Id, only exploded on attempted combustion and gave low small amounts of the linear coupling products carbon values. I n all the four series studied, cyclic result. polyacetylenes of higher mol. wt. than those deFor example 1 part of nona-l&diyne (Ia) in 100 scribed were also obtained; their structures are now parts of pyridine was heated and stirred with 15 under investigation. parts of neutral cupric acetate a t 55' for 3 hours. The present method makes available in one step The product on chromatography on 200 parts of highly unsaturated large ring alicyclic hydrocarbons alumina into ca. 300 fractions gave successively : (a) (and by hydrogenation the corresponding saturated the Cis-tetrayne IIa (loyo), m.p. 210-212' (identi- ones) over a wide range of size, including considercal with that made previously1-2b); (b) the CZi- ably larger ones than the thirty-four membered hexayne IIIa (130/0), m.p. 125-126' (hydrogenated cycle which is the biggest alicyclic ring prepared to m.p. 47-48', mol. wt., 3745; calcd. 378) ; previously. (c) the Cas-octayne IVa (11%)) m.p. 135-136' DANIELSrEFF RESEAKCH FKANZ SOXDHEIMER INSTITUTE (hydrogenated t o C ~ B H ,m.p. ~ , ~ 70-71', mol. wt., INSTITUTE OF SCIENCE YAACOV AYIEI, 517; calcd., 504); (d) the C%-decayne Va (4y0), WEIZMANN ISRAEL REUVEN WOLOVSKY m.p. 144-145' (hydrogenated to C46H0~,4 m.p. 78.5- REHOVOTH, RECEIVED JUNE 17, 1957 79') mol. wt., 618; calcd., 630); (e) probably the C ~ d o d e c a y n eVIa (4%), m.p. 144-145' (hydrogenated to m.p. 90-91', mol. wt. not THE CHANGE OF OPTICAL ACTIVITY OF POLY-)BENZYL-L-GLUTAMATE I N AN ELECTRIC FIELD determined due t o insolubility). A similar coupling of octa-1,7-diyne (Ibj gave: Sir: (a) the Cia-tetrayne I I b (9%), m.p. 162-163' The optical activity of a molecule is different (identical with that made previously2"); (b) the along different directions. Therefore we expect C ~ h e x a y n eI I I b (14%)) m.p. 173-174' (hydro- the optical activity of a solution of these molecules genated to C24H@,m.p. 46.547', mol. wt., 330; to change if the molecules are oriented. However, reported: m.p. 46-17') mol. wt., 336); (c) the this effect has not been described. We wish to reC ~ o c t a y n eIVb (8%), m.p. 154-155' (hydrogen- port the change of optical activity of a solution of ated to C32H64, m.p. 58-59', mol. wt., 429; re- poly-7-benzyl-L-glutamate (PBLG) caused by oriported: map. 59-60') mol. wt., 448); (d) the GO-entation of the molecules in an electric field. decayne Vb (9%), m.p. 155-157' (hydrogenated to An equation relating the change in optical acC40H80,4 m.p., 74-75', mol. wt., 568; calcd. 560). tivity to molecular parameters a t infinite dilution Coupling of hepta-1,6-diyne (IC)did not give the has been derived.' For a helical molecule the (214-tetrayne ITc (if formed, it probably decom2 ____ but the C21-hexayne IIIc (3%), m.p. = -- ( [ a n ) - ia,tl)[P*2 (P3J - 4n)I E2 43 174-17.5' (hydrogenated to C21H42,4 m.p. 63-64', mol. wt., 289; calcd. 294) and the Cz8-octayne IVc change in the specific optical activity in an electric (4%), m.p. 213-214' (hydrogenated t o CrsH56, field2 E is proportional to the difference in optical activity for light incident parallel to the helical m.p. 47-48', mol. wt., 389; reported: m.p. 47-45', axis [a331 and perpendicular to the helical axis mol. wt., 398). Coupling of hexa-1,5-diyne (Id) and chromatog- [a!~].The proportionality factor is the electrical raphy into 450 fractions gave: (a) the linear dimer orientation term which involves the permanent (9%), m.p. 98-99' (identical with that made pre- and induced dipole moments of the molecule. viously1,2b); (b) the &-hexayne I I I d (6%), de- The specific optical activity in the absence of a composes on heating (hydrogenated t o C18H36, field [a10is just (2[a11] [(~33]),/3. I n order to measure the change of optical acm.p. 72-73', identical to that obtained from IIa) ; (c) the Cz4-octayneIVd (67,), decomposes on heat- tivity in an electric field, the light must be incident ing (hydrogenated to C24H4.3, n1.p. 46-17', identi- parallel to the field. Only along this direction is cal t o that obtained froin IIIb); (d) the C30- the refractive index and optical absorption the decayne Vd (6%)) decomposes on heating (hydro- same for all directions of the plane of p ~ l a r i z a t i o n . ~ A one-em. cell with transparent conducting glass6 genated to C30Hso,m.p. 57-58"; mol. wt., 416; reported m.p. 57-58O, mol. mt., 420); (e) probably electrodes was used. The optical rotation with the cyclic Cd*-tetradecayne, decomposes on heat- the electric field off, then on, was measured from ing (hydrogenated to C4*HBl14 m.p. 75-78', mol. X = 330 to 550 mp in a Rudolph automatic spectropolarimeter6 for two concentrations of AVw wt., 598; calcd., 588). All the above cyclic polyacetylenes were highly = 64,000 PBLG' in ethylene dichloride. The crystalline. They were shown to differ from each (1) I. Tinoco, Jr., and W.G. Harnrnerle, J . Phys. Chcm., 60, 1619 other since they gave mutual depressions in m.p. (1956). (2) The units of E are e.s.u./cm. (1 e.s.u. 300 volts). Their cyclic nature mas shown b y the absence of (3) The change of refractive index in an electric field is electrical acetylenic hydrogen (no band a t ca. 3300 cm.-' in birefringence The change of optical absorption is or Kerr effect.' the infrared, no precipitate with silver nitrate) and electrical dichroism; it has not been reported. (4) C. G. LeFevre and R. J. W. LeFCvre, Revs. P u r e A p p l . Chcm., by the absence of terminal methyl groups (no band 261 (1955). at ca. 1380 cm.-l) in the corresponding saturated 6, (5) wlsh to thank Dr. E. M. Grcist of Corning Glass Worka for compounds. The latter gave satisfactory an- klndly We furnishing this glass (E-C 67720). alytical results, but most of the polyacetylenes (6) We wish to thank Professors J. B. Nielanda and H.K. Schachman, and Mr. B. Burnham for t h e use of thls Instrument. (4) Thfa Is a previously unknown cpcloalkane.
+
+
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(5) All molecular weights were determlncd by the Rast method in camphene.
(7) We wlsh to thank Dr. E.R.Blout for klndly supplylug a sample of PBLG ({ES-508) for these measurements.
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rotatory dispersion in the absence of the field was in good agreement with the measurements of Doty and Yang.8.9 I n the presence of the electric field an increase in optical rotation was observed at all wave lengths. The change was found to be proportional t o the square of the electric field strength as predicted; an easily measurable change of about 0.1" occurred for a field of about 2,000 volts/cm. As the electrical orientation term is not known, quantitative values of [cy331 and [all] cannot be obtained. However, an estimate of this term from electrical birefringence studieslO allows us to make the following conclusions. Both [a331 and [a111 are very large in absolute magnitude, but [a331is positive and [a111 is negative. Both values increase in absolute magnitude as the wave length decreases; however [all]increases faster, thus leading to the change in sign of the average optical activity [a10a t X = 425 mp. These conclusions are in good agreement with Moffitt's prediction."
phosphate, C14-amino acid, ATPB and GTPs for one hour in the Dubnoff shaker. They were then inactivated by the addition of TCAI6and the proteins were isolated and counted a t infinite thickness in a GM gas-flow counter. The results are given in Table I.
(8) P. Doty and J. T. Yang, TRISJOURNAL, 78, 498 (1956). (9) J. T. Yang and P. Doty, ibid., 79, 761 (1957). (10) I. Tinoco, Jr., ibid., in press. (11) W. E. Moffitt, Proc. Null. Acad. Sci., U .S., 42, 736 (1956).
I n another series of experiments, the enzyme preparations were supplemented with vitamin Bl2. The results are given in Table 11.
DEPARTMEKT OF CHEMISTRY OF CALIFORXIA UNIVERSITY IGNACIO TINOCO,JR. BERKELEY, CALIFORNIA RECEIVED MAY15, 1957
VITAMIN Biz AND PROTEIN BIOSYNTHESIS. 11. EFFECT OF VITAMIN Biz ON AMINO ACID INCORPORATION IN MICROSOMAL PREPARATIONS
Sir: It has been reported from this laboratory that, while vitamin B12 has no effect on nucleic acid biosynthesis, there is a decreased incorporation of CI4-1abeled amino acids into liver protein in vitamin B12-deficient ani mal^,^.^ indicating that vitamin B12 is involved in the incorporation of amino acids into protein. Following these findings with the whole animal, the incorporation of C14-labeled amino acids into protein has been studied in vitro using the microsomal fraction of liver and of spleen from both B12deficient and normal rats. The rats were killed by decapitation and the livers removed and microsomes prepared by the procedure of Zamecnik and Keller.4 This microsome fraction was freed only of mitochondria and nuclei since, as Keller and Zamecniks have reported, the supernatant fraction is essential along with microsomes for the incorporation of amino acids into protein. These preparations containing microsomes and supernatant were then incubated under oxygen (95% 0 2 , 570 CO2) with fructose di(1) S. R. Wagle and B. C. Johnson, Fcdcrafion Proc., 16, 401 (1957). (2) S. R. Wagle and B. C. Johnson, Arch. Biochcm. Biophys.. in press. (3) Presented in part before annual meeting, National Vitamin Foundation, March 6, 1957, New York; B. C. Johnson, A m . J . Clin. Nulrition, in press. (4) P. C. Zamecnik and E. B. Keller, J . B i d . Chcm., 209, 337 (1954). ( 5 ) E. B.Keller and P. C. Zamecnik, ibid., 221, 45 (1956).
TABLE I INCORPORATION OF C i 4 - A m ~ oACIDS INTO PROTEIN BY MICROSOME PREPARATIONS' FROM THE LIVER AND THE SPLEEN OF VITAMINB 1 2 - D AND ~ ~NORMAL ~ ~ ~ R A~T S~~ ~ Liver microsome preparation, c.p.m./mg. protein
Spleen microsome preparation, c.p.m./mg. protein
Biz status
-Biz -Bi2 +Bip +Bit Ci4H8-Methionine 16 76 26 94 2-Ci4-Alanine 12 57 21 69 Complete system contained 0.1 p M . F D P (6), 0.5 p M . ATP, 0.25 pM. GTP, 0.5 ml. of microsome preparation and labeled amino acid, made t o 1 ml. with 0.15 molar KCl. * Each figure is the mean for three rats and run in duplicate. Excellent agreement was obtained between replicate animals.
TABLE I1 THE EFFECTOF ADDITION OF VITAMINBIZTO LIVERAND SPLEEN MICROSOME PREPARATIONS" ON THE INCORPORATION O F C"-AMINO ACIDSINTO PROTEINb BIBadded t o microsome prep. Bln status of animals
Liver microsome preparation, c.p.m./mg. protein -Bir +Bir
Spleen microsome preparation, c.p.m./mg. protein -Bit +Bin
Cl4-Methionine 19 64 14 81 None 51 88 50 mpg 53 73 CI4-Methionine 2-Ci4-Alanine None 12 44 21 67 50 mpg 40 66 48 90 2-Ci4-Alanine See Table I. Each figure is the mean for two rats, run in duplicate. Agreement was excellent between animals.
Table I clearly shows that there is much less incorporation of labeled amino acids into protein in the case of microsome preparations prepared from the livers and spleens of vitamin BIZ-deficient rats than in those from normal animals. These results agree with our previous data on intact animals.2 In an additional experiment, in which the effect of level of substrate (labeled amino acid) concentration on incorporation was studied, it was found that an increase in the incorporation of amino acids was obtained with microsome preparations (both liver and spleen) from Bl2-normal animals, upon incubation with increasing levels of amino acid, but that these substrate increments had no effect in preparations from deficient animals. This indicates an enzymatic block in protein synthesis in deficient animals. Again, when the microsome'preparations, particularly from the deficient animals, were supplemented with crystalline vitamin BIZ increased incorporation of the amino acids occurred, bringing the incorporation almost t o a normal level (the stepwise increase obtained with graded levels of vitamin Blz and also amino acid increment data will be reported in full
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(6) A T P adenosine triphosphate; G T P = guanosine triphosphate; T C A = trichloroacetic acid; FDP = fructose diphosphate.
