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m.p. 199-20lo, [ u ] ~ ~-82O, D kindly furnished by dilute aqueous solutions is due primarily to an inDr. Sannik, was converted to the A',4-dien-3-one duced polarization, and that the permanent dipole in 20% yield of crystalline material. An infrared moment contribution is neg1igible.l One indication spectrum of the mother liquors indicated that they of this was the fact that reversal of a square polarizcontained approximately another 30% of this ing pulse after a steady-state orientation was material. I n contrast to the results obtained by achieved did not produce a transient in the bireBurn, et al.,3 our substance proved to be identical fringence. In further studies of various strains, with the dienone obtained, by selenium dioxide de- similar behavior has been observed with the hydrogenation,' from diosgenone.' We believe Masked and Green Aucuba strains, but a large the yield of dienone obtained in our sequence was transient was obtained with the Holmes Rib high enough to preclude its chance origin from an Grass strain (HR). All experiments were carried impurity. out in solutions sufficiently dilute to eliminate comOxidative degradation of the side chain of rusco- plications arising from macromolecular interacgenin diacetate,' followed by saponification, fur- tions. Figure 1 contains oscillograms illustrating nished 1~,38-dihydro~y-A~~'~-pregnadiene-2O-one, the wave form of the polarizing field and the m.p. 228-235O, tEF1 9,100; AN"?' a t 2.93, transient electric birefringence signal of HR. 6.06 and 6.28 p. Upon hydrogenation in pyridine over palladium-charcoal, one mole of hydrogen was absorbed and there was obtained 1{,3@-dihydroxyA5-pregnene-20-one, m.p. 1S5-19So, which showed no selective absorption in the ultraviolet between 220 and 300 mp; A::?' a t 2.96, 3.03 and 5.83 p, [ a ] " D +22.4" (17, in CHClJ. When this substance was subjected to the qction of Flaerobacterium dehydrogenrcn~~ there was obtained the hydroxylated progesterone, l{-hydroxy-A4-pregnene-3,20dione, m.p. 155-157", AN:?' a t 2.98, 5.84, 6.01, 6.19 p ; C , 76.58; H, 9.05; $FH 15,800. In methanolic alkali a t 6Oo,l0 a shift from 241 t o 244.5 mu (characteristic of a dienone) was observed. The absence of any selective absorption near 370 mpl0 under these conditions establishes the formation of a dienone which should result from the dehydration of the proposed structure. Further 1-hydroxylated hormone analogs are being synthesized, and we are investigating the stereochemistry of this hydroxyl group as well as the relationship of these substances to other l-hydroxylated steroids.",'* (81 T.Miki and Y.Hare, Phorm. Buii. (Jnpnn), 4, 421 (1958). (71 F. Sondheimer, C. Amendalla and C. Rosenkranz, T ~ I Joua. S NAL, 75, 5932 (1953). (8)Inlcr d i n R. E. Marker and E. Rohrmann, Tam J o u a r r ~61, ~, 3592 (19391; D. H. Could, H. Staeudle and E. B. Hershberg, ;bid., 1 4 , 3685 (1952). (9) C . Aroaudi, Z e n i ~Paroriimk.. . lOS, 352 ( 1 9 4 2 ) . (10) A. S. Meyer. J. Org. Chem., 90. 1240 (1955). (11) W. Schlezel and C. Tamm. Hcio. Chim. Acin, 40, 1GO (1957). (12) G. Creen3pao. C . P. Sehaffoer, W. Charney, H. L. Herrug and E. B. Hershberg, T H ~ S JoueNAL, 19, 3922 (1957).
A. L. NUSSRAUM F. E. CARLON versal of the field for the Holmes Rib Grass strain of D. GOULD tobacco mosaic virus: (a) oscillogram of the polarizing field; E. P.OLIVETO (b) transient response. The c e n t n l minimum does not Is. R . HERSHBERC occur with the common strain.' M. L. G I L M ~ R E
CHEMICAL RESEARC~I DEPARTMENT INDUSTRIAL MICROMOLOGY DEPARTMENT SCHERINC CORPORATION BLOOMFIELD, Narv JERSEY RECEIVED JULY 22, 1957
W. CHARNEY
ELECTRIC PROPERTIES OF MACROMOLECULES. 11. EVIDENCE FOR A PERMANENT DIPOLE MOMENT IN THE RIB GRASS STRAIN OF TOBACCO MOSAIC VIRUS
Sir: A previous investigation of the common strain of the tobacco mosaic virus by means of electric birefringence showed that the large Kerr effect in
The transient upon field reversal was observed with three separate preparations of the Rib Grass straimz Analysis of the field-free decay of the birefringence revealed that each preparation had two relaxation times, corresponding to rotational diffusion constants of 4.S X lo2and 2 X lo2 set?. The larger value was assigned to the HR monomer. I t is 1.4 times the corresponding value for the common strain, and corresponds to a rod length (11 C . T. OKonrki and A . I. Halfner (Paper 11, THISl o u a l r ~ ~ , 19, In press (1967). ( 2 ) Kindly furnished by C. A. Knilrht of t h e Virus 1,ahoratnry. Prepared by the general method af W. M. Stanley, (bid.. 64, 1804 (191".
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around 10% less than that reported for the monomer of the common strain.3 The Kerr constant increased a factor of about 20 as the pH was increased from 5.6 to 8.3. The variation resembled a titration curve with an inflection around pH 7. Experiments with the square reversing pulse as a function of pH indicated that the permanent moment contribution also increased with pH. The amino acid analyses, which have been reported4 for all four strains mentioned above, show that histidine is present only in the Rib Grass strain. Titration of the imidazole group of histidine occurs around pH 7, and there is no other known constituent which is titrated in this reg i ~ n . ~This . ~ leads to the suggestion that an asymmetric arrangement of histidine residues in the macromolecular structure might be the origin of the large changes of dipole moment and Kerr constant around pH 7. The lower rotation diffusion constant cannot be explained by rigid end-to-end dimers, which were suggested for the common train.^ It is the value expected for a staggered side-by-side dimer in which the monomer units overlap about one-half of their lengths, but it may be due to other types of aggregates. Analysis of the transient behavior of the birefringence, which is complicated by the presence of two components, leads to the tentative conclusions that both components have permanent dipole moments, and that they are ca. lo4 to lo5 debye units. A more complete account of this work is available elsewhere.6 Further studies are in progress on the macromolecular properties as a function of preparation procedure. Ih?e are happy to acknowledge financial support from the National Science Foundation and the California Research Corporation.
TABLE I
pH
HILL REACTION AS REPORTED BY DIFFEREST~ ~ ‘ O R K E R S
OPTIMA FOR THE
Holt, Smith and French1 Warburg and Liittgensz \Vessels3 Arnon and Whatley4 Spikes, Lumrp, Eyring and K’nyryneii5 Punnett and FabiyiO Clenderining and Gorhain7 Holt and French* Punnett$ Hill and Scarisbricklo Davenport, Hill and \Tliatleyll
5.9 6.5 6.5 6.9 6.0
G.9 7,I.i
7.6-7.9 7.9 8.(1 8 . :3
chloroplasts. The leaves were ground with a mortar and pestle in the presence of 0.03 -11phosphate buffer, pH 7.0, containing 0.01 AT KCI. The suspension was filtered through glass wool, then centrifuged for 90 seconds at 100 g to remove whole cells and other debris. It was then decanted and recentrifuged for five minutes at 900 g to bring down “whole chloroplasts.” The supernatant was discarded, the centrifuge tube and the surface of the chloroplast pellet were rinsed with fresh, cold buffer, and the inside of the tube was wiped dry. The chloroplasts were resuspended by gentle stirring during slow addition of fresh buffer of the same composition as the grinding medium. All operations were carried out as close to 0” as possible, and no effort was made to avoid exposing the preparation to either light or air. Reaction rates were measured by following the rate of reduction of purified 2,G-dichlorophenolindophenol (molar extinction coefficient 22,400) The reaction vessel contained 0.03 X phosphate or Tris buffer, 0.01 ST KCI, 1.5 X JI dye, and chloroplasts giving a final concentration of chloro(3) C. T. O’Konski and A. J. Haltner, THISJ O U R N A L , 78, 3604 phylls a plus b of 3.5 pAI./l (3.2 kg.,,’nil.). In the (1956). dark the rate of reduction of the dye by the chloro(4) C . A. Knight, J . B i d . Chem., 171, 297 (1947). (5) R. A. Alberty in H . Neurath and K. Bailey, “ T h e Proteins,” plasts corresponded to a Q;” of 30, which is Vol. 1, P a r t A, Chapter 6, Academic Press, Inc., New York, N. Y., roughly the same as the error in the determination 1953. of the rate in high intensity red light. (6) Details are presented in t h e P h . D . Thesis of R. 11. Pytkowicz, When freshly prepared chloroplasts were tested June, 1957, University of California, Berkeley, California. for p H response within one to two hours after DEPARTMENT O F CHEMISTRY USIVERSITVOF CALIFORNIA C. T. O’KOSSKI grinding the leaves, the optimum was found t o be R. hl. PYTKOKICZequal to or greater than pH 8.7. Following storage BERKELEY, CALIFORNIA RECEIVED JUSE 21, 19j7 of the preparation at 0”, the pH optiiiium decreased half a pH unit during the first nine hours of storage, and another unit and a half during the next 43 hours (Table 11). T H E p H OPTIMUM OF THE HILL REACTION
Sir: One of the contradictions in the Hill reaction literature is that concerning the pH optimum at high light intensity. The ‘‘optimal” values previously cited range from pH 5.9 to 8.3 (‘Table I). As will be shown in this paper, this discrepancy is largely due to the fact that the pH optimum is dependent on the activity of the chloroplast material. A high pH optimum is characteristic of high activity preparations, and the optimum decreases as the chloroplasts lose activity. In the experiment from which the following data were taken, washed, turgid leaves of field grown chard (Beta azdgaris) were used as the source of
(1) A. S. Holt, R . F. Smith and C . S . French, P l a n t Physiol., 26, 164 (1951).
( 2 ) 0. Warburg and W, Liittgens, Biokhim., 11, 303 (1940). (3) J . S. C. Wessels, “Investigations into Some Aspects of the IIill Reaction,” Thesis, University of Leyden, 1954. (4) D. I. Arnon and F. R. Whatley, Arch Biochem., 23, 141 (1049). ( 5 ) J. D . Spikes, R. \\‘. Lumry, H. Eyring a n d R. \Vayryn?n, ibid., 28, 48 (1950). (6) T. P u n n e t t and A. Fahiyi, S a t i w e , 172, 947 (1953). (7) R. A . Clendenning and P. R . Gorham, Can. J. Res., C28, 75 (1 950). (8) A. S. Holt and C . S . French, Avch. Biochem., 9, 2 5 (1920). (9) T. Punnett, Revues des Fermentations, 19,X; paper presented a t 3rd I n t . Cong. Biochem., Brussels, 1955. (10) R . Hill and R . Scarisbrick, P Y O CR. o y . Soc., Bl29, 238 (1940) (11) H . E. Davenport, R. Hill and F. R. X’hatley, ibid, B139, 346 (1952) (12) N. Savage and T. Punnett, uupublislitd reiu!t;.
