Helix formation by isoguanosine

PRELIMINARY NOTES

603

These values are likely to be directly useful for the study of covalent binding although extrapolation to biologically active estrogens which are not brominated is not possible presently and must await comparable X-ray data on natural estrogens and reliable :information on the biological activity of brominated derivatives. However, one would recall that HECKER AND MUELLER5 have stated that the substitution of fluorine at C-4 of steroids does not affect the rate of covalent binding to protein. One direction in which we are continuing theoretical research in our Department is to locate the possible site of amide linkage and to see how such a link would modify the electronic structure along lines parallel to the investigations of PULLMAN AND PULLMAN 13 o n the carcinogenic hydrocarbons. One of the authors (K.S.) thanks the Indian Council of Medical Research for a fellowship grant for doing this work.

Department of Biophysics, All India Institute of Medical Sciences, New Delhi (India) I 2 3 4 5 6 7 8 9 io II 12 13

K . SUNDARAM-

R. K. MISHRA

U. WESTPttAL, Mechanism of Action of Steroid Hormones, P e r g a m o n Press, L o n d o n , 1961, p. 33~ B. F. ERL2kNGER, V. BOREK, S. M. BEISER AND S. LIEBERMAN, f . Biol. Chem., 228 (1957) 713 I. L. RIEGEL AND G. C. MUELLER, J. Biol. Chem., 21o (1954) 249. W. ZILLIG AND G. C. MUELLER, Federation Proc., 15 (1956) 503 . E. HECKER AND G. C. MUELLER, J. Biol. Chem., 233 (1958) 991. C. HEIDELBERGER AND M. G. MOLDt~NHAUER, Cancer Res., 16 (1956) 442. D. A. NORTON, G. N. KARTHA AND C. T. LU, Acta Cryst., 16 (1963) 89. D. A. NORTON, G. N. KARTHA AND C. T. LU, Acta Cryst., 17 (1964) 77. R. PARISER AND I~. G. PARR, J. Chem. Phys., 21 (1953) 466. B. GRABE, Arkiv Fysik, 17 (196o) 97M. GODFRF.Y AND J. N. MURRI~LL, Proc. Roy. Soc. London., Set. A, 278 (I964) 64. C. A. COOLSON, Valence, Oxford U n i v e r s i t y Press, London, 1961, p. 259. A. PULLM~,N AND B. PULLMAN, Advan. Cancer Res., 3 (1955) 117.

Received November I9th, 1964 Biochim. Biophys. Acta, 94 (1965) 6Ol-6O3.

PN 41027

Helix formation by isoguanosine Dilute aqueous solutions of isoguanosine [(erotonoside; 2-keto-6-amino-9-fl-Dribofuranosylpurine; structure shown in Fig. I) the nucleoside was first isolated from croton beans; by CHERBALIEZAND BERNHARD1. It was synthesized by DAVOLL 2, whose preparative procedure we have followed, omitting the lead salt step but using large amounts of charcoal for purification. Our material had the same ultraviolet spectra in acid, basic, and neutral solution as those reported by DAVOLLand the same RF value in the solvent system he reported. The compound was homogeneous when examined b y paper chromatography in four other solvent systems. Further confirmation of structure was provided by deamination to xanthosine, identified by ultraviolet and infrared spectroscopy. DAVOLL noted during the deamination of 2,6-diaminopurine riboside the formation of a "gelatinous mass" but did not further characterize t h e Biochim. Biophys. Acta, 94 (1965) 603-606-

604

PRELIMINARY NOTES

aggregated state of the nucleoside] become viscous on cooling and at higher concentrations form thick opalescent gels. We present here evidence that this phenomenon reflects the formation of an asymmetric, regular, ordered structure, presumably helical.

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Fig. 2. Dependence of relative viscosity of isoguanosine upon concentration. Measured with O s t w a l d viscometer at 6 °, p H 5.5.

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Fig. 3- Ultraviolet spectra of isoguanosine in non-aggregated (upper curve, 3 o°, ;tmax 293 mff ( 8 = I I 3 0 0 ) a n d 248 m p (8 = 9090)) a n d in helical (lower curve, 3 °, Zmax 293 mff (e = 793 o) a n d 248 m # (8 = 5700)) form measured w i t h Zeiss P M Q II spectrophotometer. Concentration 4.05 m g / m l ; p H 5.5 ; p a t h length, o. IO m m . L o w e s t curve : difference spectrum. Fig. 4. Infrared spectra of isoguanosine (solution 2HzO; pZH 5.6; concentration 4.05 mg/ml). The lowest t e m p e r a t u r e represents the helical form (Vmsx 1652 cm-1; 1581 cm -1) the highest, fully dissociated solute (Vmax 1652 cm-1; 1587 cm-Z). Measured w i t h B e c k m a n I R - 7 s p e c t r o p h o t o m e t e r using m a t c h e d CaF~ cells of 55-~u p a t h length. The scale expansion w a s 4.1 fold, a n d o r d i n a t e index m a r k s are o.i units apart, uncorrected for this expansion.

Biochim. Biophys. Acta, 94 (1965) 603-606

605

PRELIMINARY NOTES

In addfition to its intrinsic interest, the observation of aggregate formation by a nucleoside should help to provide further insight into the forces stabilizing such gels. The method of structural variation of the base may be conveniently applied to a simple monomer to see which changes decrease or enhance the tendency to helix formation. The formation of the aggregate depends upon the concentration of nucleoside at a constant temperature, as illustrated by the viscosity-concentration curve (the dependence of viscosity upon aggregate formation has been discussed by PETICOLAS ~, who has reported the dependence for the 5'-GMP gel as well as for other specific examples) in Fig. 2. The dependence of aggregate formation upon temperature at constant concentration is seen in Fig. 5. The ultraviolet spectra of the unassociated and helical forms of isoguanosine are shown in Fig. 3. The peak at 248 m~ shows a reduction in intensity of 37.9 % ([e~0°-e~o/~8o °] X IO~), and that at 293 m p a reduction of 29.8 %, upon helix formation, both without shift of wavelength. The difference spectrum shows two distinct maxima and hyperchromism at longer wavelengths, resembling in these characteristics the difference spectrum reported for the 5'-GMP gel4.

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Fig. 5. Temperature profile of a.bsorbance of ring vibration of isoguanosine (concentration 4.0 5 mg/ml) ; conditions of Fig. 4. Fig. 6. Optical rotation of isoguanosine (concentration 5.0 mg/ml; p H 5.6; 4.o-mm path length) ordered structure (I. 2 o, upper curve) and non-aggl egated monomer (42. 5°, lower curve). Measured with Rudolph recording spectropolarimeter. The upper curve is the average of two runs which varied from the average by ± 5-8 %.

We observe in the infrared spectra (Fig. 4, spectra measured in *H,O solution) a decrease in intensity and some increase in frequency of the ring vibration at ca. 159 ° cm -1 upon gel formation, but, in contrast to the spectra of the 5'- and 3'-GMP Biochim. Biophys. Acta,. 94 (1965) 6o3~5o6

606

PRELIMINARY NOTES

gelsS,6, no change in frequency and little change in intensity of the carbonyl vibration at 1652 cm -1. We conclude that the carbonyl group does not undergo inter-base hydrogen bonding in the ordered structure but that the purine ring is probably hydrogen-bonded by the amino group of another molecule. Comparison of the spectra with those of the isoguanosine cation (bands at 1697, 1652, and 1613 cm -1) and anion (broad band with Vmax at 1631 cm -1) demonstrates that the nucleoside is uncharged in the ordered structure. The temperature dependence of infrared intensity (Fig. 5) reflects the cooperative melting of an ordered structure. The transition is much broader than those observed with the homopolynucleotide helices, but similar to those of the guanylic acid gelsS,6. Rotatory dispersion curves of the aggregate and of the unassociated monomer are given in Fig. 6. The gel has a positive specific rotation about six times that of unassociated monomer, suggesting that the structure is highly asymmetric and probably helical. We shall later report demonstrations of the tautomeric forms of the neutral and charged molecules and propose a structure for the helix based upon these studies. Syntheses of the 5'-phosphate and pyrophosphate of the nucleoside are in progress. National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.) i 2 3 4 5 6

-

R. V. RAVINDRANATHAN H. TODD MILES

E. CHERBALIEZAND I{. BERNHARD,Helv. Chim. Acta, 15 (1932) 464, 978. J. DAVOLL,J. Am. Chem. Soc., 73 (1951) 3174. W. PETICOLAS,J. Chem. Phys., 4° (1964) 1463. M. GELLERT,M. N. LIPSETTANDD. R. DAVIES,Proc. Natl. Acad. Sci. U.S., 48 (1962) 2Ol3. H.T. MILESAND J. FRAZlER,Bioehim. Biophys. Acta, 79 (1964) 216o. B. HOWARDANDH. T. MILES,J. Biol. Chem., 24o (1965) 8Ol.

Received January I2th, 1965 Biochim. Biophys. Acta, 94 (1965) 6o3-6o6

PN 41028

Fluorescence decline in purple bacteria resulting from carotenoid absorption An initial decline in bacteriochlorophyll fluorescence as a result of excitation in the region of carotenoid absorption was measured with chromatophores of purple bacteria. This decline occurs only at high intensities of exciting light, exceeding 1.5" lO4 erg. cm -2- sec-1 with Rhodospirillum molischianum and 4.5" 104 erg- cm -2. sec-1 with Rhodospirillum rubrum. In Table I the intensity of fluorescence of chromatophore suspensions of R. molischianum and R. rubrum measured after 15 sec of illumination, is given. The intensity of exciting light at different wavelengths is adjusted such that the fluorescence intensity amounts to ioo units at the onset of illumination. The chromatophores were adsorbed on filter paper. Fluorescence was measured at 900 nm. Exciting light was obtained from a Ioo-W, I2-V incandescent lamp (Philips P35S, type 13 II6C/C4). Monochromatic light of indicated wavelengths was isolated with interference filters. Fluorescence was analysed with a Bausch and Lomb grating monochromator (transBiochim. Biophys. Acta, 94 (1965) 606-609