1769
V O L U M E 2 4 , NO. 11, NOVEMBER 1 9 5 2 Each pair of ~inglc-turnpotentiometers in the wave-length changer has been replaced by ten-turn Helipots. All of the wiring of the wave-length changer has been shielded from stray currents. The network of resistors involved in st.andardizations has been replaced, bringing the recorder print wheel into position on the chart a t 100% transmittance. The standardization compensator has been overhauled to eliminate defective standardizations which occurred occasionally. The relays which carry the low-vol tage currents have been hermetically sealed to lessen noise. All relays niid sensitive parts have been protect,ed more effectively from corrosion, the system of electrical grounds has been rearranged to eliminate ground loops, and t,he solenoid-operated solvent valves have been overhauled to eliminate occasional internal nntl external leakage. ACKNOWLEDGMEST
The authors are indebted to Edgerton Caldwell, Ross II-.Farmer, Edn-in 11.Griffiths, Frank Sehuster, John Tanaka, and Ed-
\tin L. \\'heeler of the C.C.L.A. technical staff for assistance in constructing the apparatus and the engineers of Beckman Instruments, Inc , for much valuable advice. LITERATURE CITED
(1) Cohn, W ,E., Abstracts of Papers, Division of Biological Chemistry, 115th Meeting, A M . CHEM.Soc., p. 26C, 1949. (2) Cohn, W. E., J. Am. Chem. Soc., 71, 2275 (1949). (3) Cohn, W.E., Science, 109, 377 (1949). (4)
Deutsch, A., Zuckerman, R., and Dunn, 31. S., ANAL.CHEM.,24, 1769 (1952).
RECEIVED for review January 4, 1952. Accepted July 28, 1952. Paper 88. Presented before Section 3, Biological Chemistry, X I I t h International Congress of Pure and Applied Chemistry, New York, N. y . , September 10 to 13, 1951. Work aided b y grants from the Kesearch Corp., Swift and Co., and the University of California. The material in this paper was taken from the thesis of Alfred Deutsch, presented in February 1952 to the University of California in partial fulfillment of the requirements for the degree of doctor of philosophy.
Nucleic Acid Derivatives Separation and Quantitative Analysis by Ultraviolet Spectrophotometry 4LFRED DEUTSCHI, RICHARD ZUCKER>IhN,
AYD
MAX S. DUSN
Chemical Laboratory, University of California, Los Angeles, Calif.
The ribose nucleic acid nucleotides, adenylic acid, cytidylic acid, uridylic acid, and guanylic acid, were separated in pure form and determined in yeast nucleic acid by the authors' chromatographic and autonlatic continuous flow-recording ultraviolet spectrophotometric methods. The purity of the nucleotides was judged by the criteria of chromatography, spectrophotometry, and elementary analysis. The nucleotides were determined w-ith reasonable accuracy, as indicated by the recovery in the individual nucleotides of 10270 of the nitrogen in the nucleic acid sample. These results indicate that the authors' procedures may be employed satisfactorily for the separation, isolation, and quantitative determination of nucleotides in nucleic acids.
T"'-
apparatus designed by the authors (4)has been applied to the quantitative determination of the nucleotide compo-ition of yeast nucleic acid. The prerequisites for this analysis included the purification of a sample of nucleic acid and its hycli olysis to nucleotides, the development of a quantitative separation of the nucleotides from each other, and the purification of samples of the individual nucleotides for use as spectrophotometric standards. PURlFlCATION OF RIBOSE NUCLEOTIDES
Solvents and conditions for the complete separation of the four nucleotides were determined in preliminary experiments (data iiot shown) and applied to purification of commercial samples of the nucleotides using the described apparatus. Each product was separated into fractions collected automatically under control of thr optical density of the eluate. Each fraction was initiated when the optical density of the eluate approached infinity and was cut when it dropped. The material was elut,ed as slowly as practicable t o minimize the possibility that each fraction would cont:tin inore than one component. The chromatographic column was packed with Dowex-2 anion cwhange resin which had been washed t,oremove the fine particles aud admixed with 25y0 Celite to improve the flow characteristics. The packed resin bed was 15 mm. in diameter and 130 mm. high. Purification of adenylic acid, cytidylic acid, and uridylic acid was readily effected utilizing optical densities a t the single 1 Present address, California Foundation lor Biochemical Research, Loa hngeles, Calif.
.
ave length of 270 mp, but optical densities a t four wave lengths (260, 265, 270, and 280 mr) were employed in the purification. of guanrlic acid because of the many components in the starting material. M
bj
The per cent transmittance records of the nucleotides prepared the procedures described below are shown in Figures 1 to 4.
Adenylic Acid. -4 solution, formed by suspending 0.591 gram of commercial (Schwartz) adenylic acid in 8 ml. of distilled water and adding 6 drops of concentrated ammonium hydroxide, was placed on the resin column and eluted n i t h 0.005 S hydrochloric acid at the rate of about 1.1 ml. per minute. The main fraction (Figure 1 ) was distilled rapidly under reduced pressure belox 35' C. to a few drops of yellow sirup. The latter was taken up in 5 nil. of water, the mixture was warmed, and acetone v a s added to slight turbidity. The flask was cooled for 1 hour, acetone was added to bring the volume to 45 ml., and the flask was placed i n the refrigerator overnight. The suspension Mas filtered, and the R hite precipitate was washed n i t h acetone and ether and dried overnight a t 90" C. in a vacuum oven over phosphorus pentoxide. The yield was 0.12 gram. h n additional 30 mg. was recovered from the mother liquor and Tvashings. Cytidylic Acid. A solution, formed by dissolving 1.00 gram of commercial (Xutritional Biochemicals) cytidylic acid in 10 ml. of 1 ammonium hydroxide, was placed on the resin column arid eluted u ith 0.007 S hydrochloric acid a t the rate of about 6 nil. per minute. The main fraction (Figure 2) was distilled as described before to about 20 ml. of light yellow liquid which crystallized. -in equal volume of ethyl alcohol was added and the miature was distilled under reduced pressure. The suspension was filtered. the rn hite precipitate was suspended in boiling absolute ethyl alcohol, 10% of water was added, and the mixture was cooled overnight in the refrigerator, The product was filtered and the precipitate was washed with ethyl alcohol and ether and dried ovei night as described. The yield was 0.57 gram.
1770
ANALYTICAL CHEMISTRY
Uridvlic Acid. -1 solution. formed-by dissolving 1.00 gran; Table I. Elementary Composition of Purified Nucleotidesn of commercial (Nutritional Nucleotide Nitrogen, % Phosphorus, % Hydrogen, 70 Carbon, % Biochemicals) uridylic acid in Xame Formula Calcd. Found Calcd. Found Calcd. Found Calcd. Found 5 ml. of distilled water was Cytidylic acid CpHlrOaNaP 13.1 13.0 9.59 9.59 4.37 4.66 33.5 34.0 placed on the resin column and Ammonium urieluted with 0.005 S hydrodylate CeHnOgNzP. NHI 12.3 12.9 9.09 9.10 4.73 5.12 31.7 30.2 chloric acid followed by 0.03 S .4denylic acid CloHlrO7XiP. l/zH20 18,8 19,l 8.34 8.39 4.35 4,80 32,3 32.0 Guanylic acid CmH1rOaNsP.2H20 17.6 18.3 7.76 7.63 4.54 4.40 30.1 30.3 hydrochloric acid. The main fraction (Figure 3) was distilled 0 Analyses performed by Adelbert Elek, who employed special methods for the determination of carbon and hydrogen as required in the analysis of phosphorus-containing organic compound4 under reduced pressure almost to dryness. The residual material was taken up in 25 ml. of concentrated ammonium hydroxide, the solution was filTable 11. Molar Extinction Coefficients of tered and warmed, and acetone was added to a slight turbidity (final Ribosenucleotides volume, 100 ml.), On standing in the refrigerator for several days Piesent Work Literature the precipitate coalesced into a white oil which was dissolved in 5 Sucleotide EM X 10-4 X max. E M X 10-4 h max. Ref. ml. of water. The solution was placed in a carbon dioxide-acetone Uridylic acid 0.963 261 0.989 262 (f.8, IS) bath, 15 ml. of acetone were added slowly with shaking, the misCytidylic acid Guanylic acid Adenylic acid
1.32 1.24 1.44
279 257 257
1.272 1.22 1.5
278 250 262
(Id, I S ) (7) (7)
E M , Molar extinction coefficient. Wave length of maximum,
X max.
I
The elementary analysis of the purified nucleotides is s h o w in Table I. The ultraviolet absorption spectra, determined nith the aid of a Cary recording spectrophotometer between the wave lengths of 220 and 310 mp, are shown in Figure 5 as optical density curves. The concentrations per 100 ml. of the components, each dissolved in the same solvent used for its purification, were
Figure 1. Per Cent Transmittance of Fractions Obtained in Purification of Adenylic Acid Adenylic acid was isolated from fraction indicated b y arrows and solvent was changed from 0.005 .V to 3 N HC1 a t dotted line.
ture was allowed to stand 1 hour, and the yellow supernatant liquid was decanted. Upon standing for several hours, a small amount of white crystals formed. .hetone was added slowly until crystallization was complete, the suspension was filtered, and the precipitate was washed with acetone and ether and dried as described. The yield was 0.14 gram. Guanylic Acid. The following purification procedure was found satisfactory. A 0.656-gram portion of commercial guanylic acid (Schwara), a light tan powder which gave an amber-colored solution, was suspended in 7 ml. of 0.25 N sodium hydroxide, 20 ml. of water were added, and the relatively large precipitate was separated by centrifugation. The supernatant liquid was placed on the column and elution was begun with 0.005 .V hydrochloric acid. As may be noted from Figure 4, a relatively large component appearing virtually with the solvent front was eluted. There followed closely a second, small component and a third, slowmoving component. The column was washed with water and the solvent was changed to 0.02 M sodium chloride which eluted a large quantity of a fourth, slow-moving component. When most of this component had been eluted, small amounts of 0.03 -V hydrochloric acid were mixed with the solvent, causing the response curve to dip sharply and producing the fifth peak (guanylic acid). After about 20 ml. of eluate with infinite optical density had collected, the solvent was changed to 0.03 S hydrochloric acid and the eluate was collected separately until the transmittance rose to about 2 %. Subsequently, guanylic acid was recovered from this 150-ml. fraction. The sixth component, which was eluted slowly by 0.03 S hydrochloric acid, and a seventh component were eluted by 3:O S hydrochloric acid. The guanylic acid solution was distilled rapidly to dryness under reduced pressure below Z O O , yielding a small amount of white crystals. The latter were suspended in warm ethyl alcohol, water was added almost to complete solution, and the mixture was filtered and cooled. Ether was added to the cold solution dropwise until precipitation was complete. The mixture was allowed to stand overnight in the refrigerator, the sus ension was filtered, and the small crystals were washed with etlyl alcohol and ether. The product was dried for several days in a vacuum desiccator over phosphorus pentoxide. The yield as 0.05 gram.
l.E
1.0
0.6
2.2
2.5
C.5
Figure 2. Per Cent Transmittance of Fractions Obtained in Purification of Cytidylic Acid Cytidylic acid was isolated from fraction indicated by arrows and solvent
was changed from 0.007 .l to ’ 3 .V HC1 a t dotted line.
Old
I
0.4
0.8
C.P
Figure 3. Per cent Transmittance of Fractions Obtained in Purification of Uridylic Acid Uridylic acid was isolated from fraction indicated by arrows and solvent was rhanged from 0.005 .V to 0.03 N HC1 a t dotted lines.
V O L U M E 2 4 , N O . 11, N O V E M B E R 1 9 5 2
1771 was repeated once, yielding a light-tan powder containing 13.60% nitrogen and 7.88% phosphorus. A portion (0.3797 gram) of the purified nucleic acid was hydrolyzed by heating it wit! 0.3 N sodiuni hydroxide for 19 hours a t 37 The hydrolyzate n-as calculated to contain 0.516 mg. of nitrogen per ml. A standard solution, containing a known amount of each of the four purified nucleotides, and the described nucleic acid hydrolyzate were analyzed for nucleotides by the following procedure. Two- to 5-ml. aliquots of the standard solution or the hydrolyzate were pipetted onto the top of the chromatographic column. This solution was permitted to flow by gravity until the level of the liquid in the column dropped to the top of the resin bed. Then the column was washed x-ith three 5-1111. portions of distilled
.
J
Guanylic acid was isolated from fifth fraction. Eluting solvent -as 0.005 .V HCI for first three components, 0.02,If S a C l for fourth component, 0.03S HC1 for fifth and sixth components, and 3 N HCl for seventh component. Per cent transmittances are silotin a t four wave lengths (260, 265,270,and 280 mp).
Table 111. Cytidylic .kcid Standard
x
Run 37 39 49
Z
1\f g.
I‘SClW
3.64 13.86 11.03
0.791 1,92 1.54
0 140 0 . 139
77 79
10.57 10.80
263
37 39 49
7.581 17.96 14.3.5
270
37 39 49
9.252 22.21
270
77 79
18.34 18.28
280
37 39 49
26 32 20 47
260
260
17.66
10 89
0.140 Mean 0.140 1.64 0.155 1 64 0.152 Mean 0.154 0.791 0.104 1,92 0.107 1.54 0.107 Mean 0.106 0.791 0.0855 0 0865 1.92 1.54 0 0872 Alean 0 0864 1.64 0.0894 1,64 0.0897 Mean 0 0896 0 791 0 0726 1 92 0 0729 1.54 0 0752
r-r\
O L
250
Run 77 79 81
1Val.e length. S u m of optical densities multiplied by volume increment (see tevt).
260
37
guanylic acid dihydrate 2.25 nig., monoammonium uridylate 1.96 nig., adenylic acid hemihydratr 2.50 mg., and cytidylic acid 1.91 mg. l\Iolar extinction coefficients (E.w) calculated from these data and obtained from the literature are tabulated in Table 11. The authors’ values are in satisfactory agreement Kith those found by other uorkers. The literature values listed for guanylic acid and adenylic acid were derived by applying corrections of typographical and sector errors in Holiday’s ( 7 ) published data which were communicated by him to Heyroth and Loofbourow (6).
280
A.
Z.
77 70
22.11 21.68
Mean 0 0736 1,64 0.0742 1.64 0.0756 Mean 0.0749
4 N A L Y S I S OF RIBOSE NUCLEIC ACID
.4sample of commercial yeast nucleic acid was purified by dissolving it in sodium acetate solution and precipitating it with ethyl alcohol containing 10% hydrochloric acid. This procedure
-
Table IV.
x
..__
\
Adenylic Acid Standard P
14,iO 14.92 18.60
49
9.37 23.93 18.95
260
77 70 81
17.42 17.36 21.50
26.5
37 39 49
8 68 20.89 16 90
270
37 39 49
6.38 17.26 12 80
270
77 79 El
12.66 12.66
39
-
LCD
\\
G I
15 48
Ale.
Factor 0.114 0.113 0.112 hlean 0.113 0.829 0.0884 2.01 0.0839 0.0849 1.61 Mean 0 0857 1.68 0.0864 1.68 0.0867 2.09 0.0972 Mean 0.0968 0 829 0 095.5 2 01 0.0986 1 61 0,0952 Mean 0.09.54 0.130 0 829 2.01 0.127 0.126 1.61 Mean 0 . 1 2 8 0.133 1.68 1.68 0.133 2.09 0.138 Mean 0.134
1.68 1.68 2.09
See footnotes t o Table 111 for explanation of symbols.
ANALYTICAL CHEMISTRY
1772
Table V.
Uridylic Acid S t a n d a r d I:
b1R
77 79 81 83
8.83 9.06 10.97 11.01
1.65 I .65 2.06 2.06
26" . ._
77 .. 79 81 83
11.34 11.37 14.67 14.47
1.65 1.65 2.06 2.06
270
77 79 81 83
9.09 9.02 11.02 11.18
1.65
250
raptor
Mean
0.187 0,182 0.188 0.187 0.186
nIehn
0.146 0.145 0.140 0.142 0.143
Mean
0.182 0.183 0.187 0.184 0.184
1.65 2.06 2.06
See iootnotw to Table I11 for owlanation of symbols.
water, t,he column was stoppered, and the flaw of eluting solvent was started. Circuit connections were made on the control panel (4)which would provide automatically the following sequence and volumes of eluting agents found previously to effect compleie separation of the nucleotides: 500 ml. of 0.005 N hydrochloric acid, 5000 nil. of 0.03 M sodium chloride, 260 ml. of 0.03 N hydrochloric acid, 160 ml. of 3 A' hydrochloric acid, and 400 ml. of d i 5 tilled water. The last two liquids were employed to remove any uneluted material and excess hydrochloric acid. The wave lengths 260, 265, 270, and 280 mp first employed were changed bo 250, 260, 270, and 280 mw, which yielded more useful information on the composition of the sample stream. The dump cup was not. calibrated precisely to memure 10 ml. but this did not result in significltnt error, as the conditions of the analysis were identical for the standards and the unknowns.
speotra of the purified nucleotide. The unsymmetrical shapes of the peaks result, i t is believed, from incomplete resolution of the nucleotide isomers, first reported by Carter ( 8 ) to he present in yeast nucleic acid hydrolynates. As the output of the Beckman photoelectric cell is linear with respect to incident light, the absorption is recorded in terms of per cent transmittance, a logarithmic function of the concentration. It was necessary, therefore, to convert the observed readings to optical densities t,a obtain values which are a linear function of the concentration. This was accomplished by constructing a logarithmic ruler of proper length and calibration suitahle to indicate optical density directly when placed on the recorder tape. Optical densities were determined in this manner a t equal volume increments along the curves. The sum of such optical density values for each curve multiplied by the volume (ml.) increment gave a value, 2, which is the area under the logarithm of the curve. The area, 2, was calculated for each peak a t each wave length except for three peaks under which the area8 were too small to be determined with the degree of accuracy possible in the other cases. Three or four replicate values were obtainable, therefore, for each ~
Table VII.
Nucleotide R u n Cybidylioaoid 31 33 47 51 67 73 Adenylic noid
Table VI. Guanylic Acid S t a n d a r d R""
z
Mg.
Facto,
230
77 79 81 83
16.53 16.42 19.85 20.75
1.68 1.68 2.10 2.10
Mean
0.102 0.102 0.106 0.101 0.103
77
16.71 16.16 20.33 20.28
1.68 1.68 2.10 2.10
Mean
0.101 0.104 0.103 0.104 0.103
Mean
0.131 0.130 0.131 0.132 0.131
79 81 83
.
270
77 79 81 83
12.83 12.92 15.97 1s.94
1.68 1.68 2.10 2.10
280
77 79 81 83
11.25 11.23 14.03 13.86
1.68 1.68 2.10 2.10
250 nip
.. ,.
.. .. ..
ME Found 260 265 mp mp 1.25 1.28 1.96 1.95 2.5C2.56 1 . 9 U 1.92
.. ..
at h 270
280 mu 1.29 1.29 1 . 9 5 1.92 mil
l:Q7 1168 2.81 2.60 1.88 1.82
..
2.55
31 33 47
..
67 73
3'78 2.89
1 . 8 6 1.87 1 . 8 6 2 . 7 8 2.78 2.74 3 . 7 2 3 75 3 . 8 9 2.782.782.79 3.88 .. 3 . 5 1 2.82 .. 2.70
.. ..
51
h
260
Nucleotide C o n t e n t of Yeast Nucleic Acid
.. ..
1.93
.. .. .. ..
Uridylie acid
65 2.51 2 48 67 3 . 2 4 3.51 73 2.52 2.49 85 2.47 2 . 5 3
.. .. .. ..
2.45 3.25 2.46 2.44
.. .. .. ..
Guanylio acid
65 67 73
.. .. ..
2.72 3.84 2.88
2.87 3.92 2.73
2 . 8 5 2.90 3.79 3.99 2.88 2.93
h.fe&n,
Mc.
Mg. N/ML 0.0833 0.0842 0.0824 0.0842 0.0861 0.0816 M e a n 0.0836 1.86 0.188 2.77 0.186 3.79 0.191 2.78 0.187 3.72 0.188 2.80 0.183 M e a m 0.188 2.48 0.0716 3 33 0 0719 2.49 0.0717 2 48 0.0715 Mean 0.0716 2.83 0.182 3.88 0.187 2.85 0.183 Mean 0.184 1.28 1.94 2.53 1.94 2.65 1.88
0.149 0.150 0.150
Mean
0.132 0 130
See footnote%t o Table 111 ior exDlanation of symbols.
A reproduction of the record obtained from a typical run (No. 67) made in the analysis of a nucleic acid hydrolyzate is shown in Figures 6 and 7. The hydrolyzate contained four major companents correspondingtofour major peak8,designated as A (cytidylic acid), B (adenylic acid), C (uridylie acid), and D (guanylic acid), as well as several minor peaks representing relatively small amounts of extraneous material. The four curves, indicated by K , L, M , and N , represent per cent transmittance a t the four wave lengths 250, 260, 270, and 280 mg, respectively. The curves are identified by the numbers 1 to 4 printed by the recorder beside each paint. The 10-ml. increments of volume of eluates, recorded by the solenoid-operated pen, are indicated by marks of the type shorn a t H . Standardization is indicated by marks of the type shown a t I and both standssdization and solvent change are indioated by marks of the type shown a t J . The identity of each component (peak) was determined by comparison with the
Figure 6 . Per C e n t Transmittance of Fractions Obtained fmm a Yeast Nocleie Acid Hydrolyzate
V O L U M E 24, NO. 11, N O V E M B E R 1 9 5 2
1773
individually determined nuclectides. Accordingly, t h e r e was 102% recovery of the 0.516 mg. present per ml. in the nucleic acid hydrolyzate. Values are not recorded in the tables for all four nucleotides in every run. Numerous difficulties YiTere encountered, psrticulady in the early runs, in the electrical, plumbing, and chromatographic systems, resulting in loss of many experimental data. I n general, faulty operations were immediately obvious and data obtained during defective runs were discarded. In no case were data discarded after complete runs. Unobserved temporary aberrations of the apparatus account for some v&Iucs whirh deviate rather widely from the mean' Figure 7. Per Cent Transmittance of Fractions Obtainr:d from a Yeast Nucleic Acid Values far nucleotides in p a s t Hydrolyzate nucleic acid obtained by the authors and reported in the lib ectture are summarized in Table VIII. Tahln V .l_ l l... C ~s i~n nof Nazrl, - nm ~~ - i t~ ~.Yeast .. ~ _. . . ..~.~eio . ~Acid ~ ~ . The authors' values fall near the mean Moles per Mole of Adenylic Acid of the literature values. It has been Guanylic Cytidylic Uridylio Recovery, suggested that yeast nueleicacid variesin aoid acid aoid % Method" Worker Ref. composition, depending upon the method 0.WR n 740 0,952 102 IX Present work 0. '5 0.67 85 PP'. Chargaff (3) of preparation (S) and the conditions 1. 11 0.73 PPr. .Marshank (lo) under which the yeast u m cultured (5). 2. z 1.1 ZIBA, grav.. ~~
y5
1
0.85 1.17
1.21 1.17 0.6-
1
?
0.48 0.79 0.769 0.81
1.06 0.89 0.903 0.94
?
~-~
-~,.
(3) Chargaff,E., Msgasanik, B., Visoher. E., Green. C., Doniger. R., and Eleon. D.. J . Bioi. Cfia.. 186.51 (1950).
(10) (11) (12) (13) (14) (15)
Marshank, A,, and Vogel, H.. Ibid., 9, 1, 85 (1950). Piroio. A,, and Cerecedo, L. R., Ibid., 9, 214 (19501. Ploeser, J. M., andLoring, H. S., J . Bid. Cfim., 178,431 (1949). Seraidarian. K.. and Warpon. M.. Ibid., 181,761 (1949). Smith. J. D., and Markham, R., Biocfim.J.. 46,509 (1950). Wyatt, G. R., Ibid., 48,584 (1951).
RECEIYED for review S a n u ~ r y4, 1952. Accepted July 28, 1952. P S P ~ 89. For the preceding paper in this series (No. 88) see Deutsch el (11. (4). This work was aided by grants from the Researoh Corp., Swift a n d Co.. and the University of Cslifornia. The material in this p & m i vas taken from the thesis of Alfred Deutmh which was presented in February 1952, in partid fvlfillment of the rcquirements for the degree of dootor of philoswhy.
