July 5 , 1955
COMMIJNICATIONS TO THE EDITOR
methosulfate, n 2 9 ~1.5023, a thick viscous liquid, could not be induced to crystallize and was purified by repeated precipitations by dissolving iL in chloroform and adding to a 10-fold excess of ether; calculated for C10H2607PS3; P, 8.1; found, P, 8.3. The infrared spectrum4 in chloroform solution showed strong absorptions for the P=O and P-0-C moieties a t 1250 and 1015 cm.-', respectively, and a weak shoulder for the P-O-CH2CH3 moiety a t 1160 cm.-'. The formation of the sulfonium salt increased the anticholinesterase activity by a factor of about one hundred fold: from a 50% fly-brain cholinesterase inhibition concentration of 3 X M for the 0,O-diethyl S-2-mercaptoethyl phosphorothiolate to a 3.3 x lo-* M for the methosulfate. I t is of interest to note the similarity in structure of the sulfonium ion (I) to the natural cholinesterase enzyme substrate acetylcholine (11).
307 I
FRACTIONATION OF DEOXYRIBONUCLEIC ACID (DNA) BY ION EXCHANGE'
Sir: Recent evidence2-6 clearly indicates that the total DNA of the cell is heterogeneous. Further exploration of this finding has been aided by the development of new fractionation procedures which afford greater resolution and are applicable t o small quantities of material. Highly polymerized calf thymus DNA7 (ca. 1 mg./ml., pH 7) was applied to columns of ion-exchangers. The adsorbed DNA could not be eluted with concentrated NaCl solution a t neutral PH from the strong-base anion-exchanger Dowex-1 Cl-. It was eluted from the weak-base anion-exchanger Amberlite IR-4B OH- with stepwise increase in NaCl concentration (0.1-3.0 M ) yielding some 16-20 fractions, but this resin had several disadvantages. Of the substituted-cellulose derivaCH3 tive$ tested, the weak-acid cation-exchanger CMCH3CHn cellulose had no affinity for DNA, but the weakbase anion-exchangers DEAE-celluloses and ECCH3SO4TEOLA-celluloseg adsorbed it quantitatively from I, Methosulfate solution. 0 CHs Because of its favorable capacity, low shedding C H 3 d *I \O-CH~CH~?JCH~ blank, the essential reproducibility of elution patI tern and high recovery of DNA obtained with 6% neutral eluting solutions, ECTEOLA-cellulose apxpears to be the most promising chromatographic 11, Acetylcholine medium. Most of the calf thymus DNA adsorbed The product is extremely soluble in water and on i t was eluted by either discontinuous or continushowed excellent systemic activity in the cotton ous change of concentration of sodium chloride s o h plant. For the determination of the systemic ac- tion (Figs. 1 and 2 ) . DNA from pneumococcus postion, the bases of young cotton plants (6-leaf stage) sessing transforming activities10 gave patterns simiwere treated with 5X of the methosulfate. After lar to Fig. 2. The fractionation appears to depend, various intervals leaves were picked, and contact toxicity in Munger cells and house fly-head cholin- in part, on the molecular size or state of aggregation of the polynucleotide components of the nucleic esterase inhibition by the leaf homogenates were acids. A mixture of mono-deoxyribonucleotides determir~ed.~The results are shown in Table I. was completely eluted with 0.01 M phosphate, PH TABLE I 7, devoid of sodium chloride, whereas a deoxyriboSYSTEMIC ACTIVITY O F O,O-DIETHYLS-8-ETHYLMERCAPTO- nuclease digest of calf thymus DNA containing a ETHYL PHOSPHOROTHIOLATE METHOSULFATE large proportion of oligonucleotides required inPer cent. mortality in creases in sodium chloride concentration up to 0.22 Munger cells after 48 hours Time House fly-head HelioTetranyM for quantitative elution (cf. Fig. 2 ) . h highly after cholinestearase thrips chus polymeric ribonucleic acid (RNA) from pneumococtreat% inhibition harmor- bimacula- A p h i s , . ment 1 0.1' O.OIQ rkoidalis Ius ~ O S S Y ~ P C cus exhibited the same heterogeneous type of be24 hr. 71 51 0 90 100 havior as did DNA. After treatment with ribonu48 hr. 96 93 90 clease, the remaining RNA fragments were also 1 week 100 100 93 95 2 weeks 100 100 100 80 3 weeks 100 100 100 20 6 weeks 97 97 48 5 a Leaf homogenate diluted 1 t o 10 and 1to
90 100 70 100 90 100 80 100 100 with water.
(1) This investigation was supported by grants from the United States Public Health Service, and from the Aromic Energy Commission, Contract No. AT(30-1)-910. (2) A. Bendich, E x p . Cell. Res., 3, SUPPI. 2. 181 (1952). (3) A. Bendicb, P. J. Russell, J r . , and G. B. Brown, J. Biol. Chcm. 208,305 (1953). Evaluation of contact toxicity of the methosul(4) E. Chargaff, C. F. Crampton and R. Lipshitz, Natuue, 172, 289 fate to the house fly, thrips, and mosquito larvae, (1953). ( 5 ) G.L. Brown and M. Watson, Natuve, 172,339 (1953). gave L G o values of 0.4%) 0.01%) 0.001%, respec(6) J. A. Lucy and J. A. V. Butler, ibid., 174,32 (1954). tively. The intraperitoneal L D ~to o the mouse was ( 7 ) H. Schwander and R. Signer, Helv. Chim. Acta, 33, 1521 (1950). between 1 and 5 mg. per kg. (8) H. A. Sober and E. A. Peterson, THISJ O U R N A L , 76, 1711 DEPARTMENT O F ENTOMOLOGY T. R. FUKUTO(1954). UNIVERSITY OF CALIFORNIA R. L. METCALF (9) Peterson and Sober have prepared ECTEOLA-cellulose by CITRUSEXPERIMENT STATION R. B. MARCH treating alkaline cellulose with epichlorohydrin and triethanolamine RIVERSIDE,CALIFORXIA M. MAXON (unpublished). The batch of exchanger used in these studies had a capacity of 7.2 mg. DNA per g. We are indebted t o Drs. Peterson and RECEIVED MAY9, 1955 Sober for their generous gifts of these exchangers, and to Dr. G. B. (4) A Perkin-Elmer model 21 self-recording infrareds pectrophoBrown for bringing their previously unpublished observations t o our tometer was used in this work. attention. (5) R. L. Metcalf, R. B. March, T. R. Fukuto and Marion Maxon, (10) R. D.Hotchkiss and J. Marmur, PYOC. N u l . Acad. Sci., 40,55 J . Ecun. Enl., 47, 1045 (1954). (1954).
46
1
3 6 mq calf thymus DNA (Schwonder-Signer) on 0 5 9 ECTEOLA-CELLULOSE column I O 8 x 5 5 im i Flow rote CU 5 ml /hr
DISCONTINUOUS CONCENTRATION CHANGE
J I
j I
# C J
20
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2 I mg. calf thymus DNA (Schwonder-Signer) on 0.59.EGTEOLA-CELLULOSE coliimn
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290
,'I
1'
47
12.7
1-1.20
( 0 . 8 x 5.5 cm.)
l
5
a0 100 FRACTION NUMBER
60
40
I1
Flow rate :tu.7 m l . / h r
081 I
Solutions made up in
d 07 :: 2 0.6 1
0.01M phosphate, pH 7
0
0.5 >
+
Z 0.4
z
0" 0.3 _1
