Simultaneous Separation of Purines, Pyrimidines, Amino Acids, and Other Nitrogenous Compounds By Ion Exchange Chromatography JOSEPH S . WALL’ Department of Biochemistry, College of Agriculture, iiniversity of Wisconsin, Madison, Wis.
To study the pathway of utilization of N’Lenriched
PROCEDURE
compounds by certain bacteria, the isolation and determination of the numerous nitrogenous compounds of the bacterial cell were necessary. .Mixtures of purines, pyrimidines, amino acids, and other nitrogenous substances were found to be simultaneously separated by chromatography on a single column of the cation exchange resin, Dowex 50. The compounds were eluted with hydrochloric acid and determined by either their reaction with ninhydrin or their ultraviolet absorption. By this method approximately 97% of the nitrogen contained in bacterial hydrolyzates were accounted for in 25 known isolated compounds. The procedure is applicable to the study of the nitrogenous compounds Contained in a wide varietyof biologicalfluids, extracts, and hydrolyzates. It is precise, rapid, and eliminates difficult chemical separations. The relative order of elution of the compounds gives additional information as to the factors affecting ion exchange chromatography.
Dowex-50 resin (Dow Chemical Corp., Midland, LMich., or Microchemical Specialties, Berkeley, Calif.), 200 to 400 mesh and 12% cross linked, was initially prepared by means of successive batchwise washings in a beaker with 8 N hydrochloric acid, water, 2 N sodium hydroxide, and water. It then was mixed with 1.5 N hydrochloric acid and poured as a slurry to form a column above a sintered-glass disk in a glass c linder. After settling, the height of the resin bed was 55.0 cm. T l e resm was washed further by passing 1.5 N hydrochloric acid through the column for 48 hours. The capacity of the resin is such that hydrolysates containing 2.4 to 4.8 mg. of nitrogen can be applied per square centimeter cross section of the column. After the hydrolysate was added and allowed to move into the column of resin, it was washed in further with three small aliquots of 1.5 N hydrochloric acid. The components of the sample were eluted from the resin by hydrochloric acid, the concentration of which was increased from 1.5 to 2.5 N and then to 4.0 N a t volumes of eluate indicated by Stein and Moore (6). However, after lysine was eluted, the concentration of the hydrochloric acid was increased from 4 to 6 N , as this resulted in better resolution and more rapid elution of the remaining compounds. A model D U Beckman spectrophotometer was employed to measure the ultraviolet absorption of portions of the fractions transferred to matched quartz cells. It was essential to use as a blank solutions obtained from fractions that exhibited minimum ultraviolet absorption but were of the same acid strength as the sample being analyzed. This corrected for a slight ultraviolet absorption contributed by hydrochloric acid-soluble decomposition products of the resin. Authentic purines and pyrimidines were dissolved in the same concentrations of hydrochloric acid as were used for their elution from Dowex-50, and their optical densities a t 260 mp were measured. The optical densities were proportional to the concentrations of the purines or pyrimidines and were employed for the quantitative determinations of these substances in the eluate from the column. For the amino acid determinations, 0.25- or 0.10-ml. aliquots were taken from the fractions and transferred to matched colorimeter tubes. Fifty of these were placed in a rack in a vacuum desiccator which then was evacuated continuously with a water pump while the desiccator was partially immersed in a hot water bath. This dried the samples in about an hour, and then they were analyzed by the ninhydrin method of Moore and Stein ( 5 ) which detects amino acids, ammonia, and certain amines. The identities of the purines and pyrimidines eluted from the column were established by comparing their absorption spectra and their R / s on paper to those of authentic compounds. The absorption spectra were determined directly with a fraction of eluate from the center portion of an ultraviolet absorption peak. The combined fractions of a peak were concentrated for paper chromatography; the solvent systems of Vischer and Chargaff ( 7 ) were employed. The positions of the substances after migration on the paper were determined by their ultraviolet absorption. The bacterial cells to be analyzed were hydrolyzed by refluxing in 6 N hydrochloric acid for 24 hours. The filtered hydrolyzate was concentrated under vacuum to remove hydrochloric acid. The hydrolyzate was then taken up in a small volume of 1.5 N hydrochloric acid (ca. 4 mg. of nitrogen per nd.). Total nitrogen was determined by the Kjeldahl method of Hiller, Plazin, and Van Slyke ( 2 ) ; however, the digestion was prolonged for 3 hours to insure quantitative rerovery of nitrogen contained in aromatic rings.
M
ETHODS have been developed for the separation and analysis of mixtures of certain closely related nitrogenous compounds of biological importance by ion exchange chromatography. Stein and Moore (6) employed long columns of Dowex50, a sulfonated polystyrene cation exchange resin, in the acid form, to resolve a large number of amino acids. The normality of their hydrochloric acid-eluent was increased in steps to facilitate the separation. Wall et al. (8), with this method, were able to account for as much as 92% of the nitrogen in hydrolyzates of photosynthetic bacteria as nitrogen in amino acids or other substances reacting with ninhydrin. Cohn ( 1 ) has separated purines and pyrimidines by chromatography on short columns of Dou ex50 in the acid form with 2 N hydrochloric acid as the eluent. The purines and pyrimidines were detected in the eluate by their absorption in the ultraviolet. By Cohn’s technique it was established that much of the remaining nitrogen in our bacterial hydrolysates vias contained in purines and pyrimidines. However, this procedure did not resolve all of the ultraviolet absorbing components of the bacterial hydrolyzate, and it did not separate the purines and pyrimidines from the amino acids clearly enough to permit accurate determinations of their isotope concentrations. Experiments were performed with resin columns of greatei length than those employed by Cohn, and the normality of the acid eluent was increased in steps to determine if it were possible to resolve simultaneously on a single column of Dowex-50 mixtures of purines, pyrimidines, amino acids, and other cationic nitrogenous substances. The chromatographic system of Stein and Moore (6) with slight modification was found to achieve this goal and has been applied to the analysis of the complex mixtures of nitrogenous substances found in bacterial hydrolyzates. 1 Present address, Department of Pharmacology, New York University, New York, N. Y.
RESULTS
A solution containing a mixture of commercially obtained purines, pyrimidines, and growth substances that absorb in the ultraviolet wa8 chromatographed to determine the effectiveness of the
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V O L U M E 2 5 , NO. 6, J U N E 1 9 5 3
OPTICAL DENSITY
951
thine, guanine, and adenine; were found in the hydroly---Azates. These substances were -o-o--o-2QO mp all well separated from one URACIL another and the amino acids except xanthine which was 1.5 THYMINE eluted close to valine and proHYPOXANTHINE line. An ultraviolet a b s o r p tion peak a t 260 mp was ACID XANTHINE found in fractions in ~ h i c h ammonia waa eluted. This absorption arose from ferric ions which had been contained in the hydrolyzate solution and had been eluted a t that point. Other smaller ML. ELUATE ultraviolet peaks remain to -1.5 N HCl 2 . 5 N HCI be identified, including one that waa eluted together ith phenylalanine. NICFINIC ACID ADENINE The unknown ninhydrin- 1 . 0 CYTOSINE positive substance in the Chr o m a t i u m h y d r ol y z a t e eluted between alanine and ammonia appears to be ethanolamine on the basis of its Rj’s in three solvent systems 300 340 380 420 460 500 540 employed for paper chromaELUATE tography. I n an earlier re port it was tentatively iden-4.0 N HCI6.0 N HC1 tified as p-alanine (8). AnFigure 1. Separation of Purines, Pyrimidines, and Growth Substances on Dowex-50 other small ninhydrin-posiColumn tive peak lying between Zero volume of eluate indicates point of application of solution of nitrogenous substances to wlumn uracil and thymine remains to be identified. The quantities of purines and pyrimidines found in the hgdrolycolumn in resolving these compounds and to determine their relazates of Chromatiurn and Chlorobackrium cells are given in Table tive positions of elution. Approximately 0.166 mg. each of orotic 11. The concentration of puri,nes and pyrimidines found in these acid, uracil, thymine, cytosine, xanthine, hypoxanthine, guanine, hydrolyzates may bear little relationship to the amounta of the adenine, nicotinic acid, pyridoxine, and p-aminobenzoic acid (PAB) were applied in 2 ml. of 1.5 N hydrochloric acid. The bases occurring bound and free in the living cells. It is well known that both purines and pyrimidines are deaminated by Dowex colunin employed was 55 cm. in length and 1.0 cm. in diprolonged heating in strong acid solutions. Markham and ameter. The flow rate was 4 ml. per hour, and 4-m]. fractions were collected. The plot of the chromatographic separation achieved is shown in Figure 1. Orotic acid, uracil, and thymine are well separated and eluted early by the 1.5 K hydrochloric Table I. Recovery of -4uthentic Purines and Pyrimidines acid. Xanthine and hypoxanthine are eluted in the 2.5 A- hydroafter Chromatography on Dowex-50 Column as Determined chloric acid. Cytosine, nicotinic acid, pyridoxine, and p-aminoby Absorption at 260 rnw benzoic acid are eluted after changing to 4 N hydrochloric acid. Amount Amount Substance Applied, y Found, y Recovery, % ’ Guanine and adenine come off in the 6 N hydrochloric acid eluate. Uracil 173 156 90.2 Most of these substances are readily detected by their absorption Thymine 171 170 99.5 a t 260 mp. However, orotic acid and p-aminobenzoic acid were Cytosine 173 176 101.0 Guanine 167 164 98.5 better observed at 230 mp and pyridoxine a t 290 mp. .4denine 185 186 100.5 The strong adsorption of the purines and pyrimidines to the long column of aromatic resin and the high acid concentration required for their elution caused concern regarding their recoveries. In Table I are listed the recoveries for some of the components apSmith ( 3 ) have demonstrated that the purine rings are degraded plied to the column and separated as illustrated in Figure 1. The by heating a t 100’ C. in strong mineral acid solution for 24 hours data indicate that satisfactory recovery of these compounds is obto yield glycine, ammonia, formic acid, and carbon dioxide. This tained from the column. decomposition likely accounts for the small amounts of adenine Figure 2 shows a plot of the separation obtained when a hydrolyand guanine found in the hydrolyzates and contributes to their zate of the purple sulfur bacterium, Chromatium, containing 50 contents of ammonia and glycine. The concentration of santhine is much higher than that previously reported in bacterial mg. of nitrogen in 15 ml. of 1.5 AThydrochloric acid was applied to cells. It is probable that most of this Xanthine results from the a column of Dowex-50, 55 cm. in height and 5.5 cm. in diameter. The flow rate of the column was 100 ml. per hour, and 15-ml. fracdeamination of guanine. Purine and pyrimidine nitrogen actions of the 1.5 N , 30-m1. fractions of the 2.5 N , and 60-ml. fraccounted for 5.2% of the nitrogen in the Chromatiurn cell hydrolytions of the 4 and 6 AT hydrochloric acid eluates were collected. zate and 5.8% of that in the Chlorobackrium hydrolyzate. The amounts of the amino acids, ammonia, and ethanolamine found Similar separations were obtained with hydrolyzates of the cells of the green sulfur bacterium, Chlorobacterium. Three pyrimiin the hydrolyzates were similar to those reported earlier (8). dines: uracil, thymine, and cytosine; and three purines: xanThe total of the nitrogen contained in these is given in Table 11.
260 mp 230 m p
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-
-L A
r I
1
I
M4
_I
952
ANALYTICAL CHEMISTRY
Thus, 96.8% of the total nitrogen contained in the hvdrolyzate of Chromatiurn cells and 96.6% of the nitrogen of the Chlorobacterzurn (.ell hydrolyzate are accounted for in known isolated compounds DISCUSSION
w-as changed in the usual manner. It is. therefore, important that the proper resin be employed to achieve satisfactory resolution of all of the compounds. The variations of positions with
,
The technique described here for the ion exchange chromatogTable 11. Nitrogenous Composition of Hydrolj zates of Cells raphy of cationic substances contained in hydrolyzates of bacof Photosynthetic Bacteria as Determined b? terial cells offers a means for the efficient separation in a single Chromatographic -4nalysis operation and the precise determination of most of the nitrogenous Organism Chromatzum Chloroba le,aum substances in the preparation. The nature of the substances % % of Total of Total containing the remaining small amount of unaccounted for nitroN of h- of MicroN. hydrolg- MicroN hydrolygen was not determined. The method has proved equally adaptmg. zate moles mg zate Component moles able to the analysis of hydrolyzates, extracts, and fluids from Total other biological sources. It is especially applicable to surveys of hydrolyzate 95.0 58.0 Uracil 0,930 0.98 15.60 0.437 0.75 * 33.10 nitrogenous compounds in biological tracer studies involving the 18.25 0.504 0.87 Thymine 10.60 0,298 0.31 15.60 0.900 1.55 Xanthine 32.75 1,830 1.92 transfer of isotopic nitrogen. As some unaccounted for material 0.730 1.26 0.90 17.40 Cytosine 20.76 0.855 may be eluted together Tith the identified compounds, tests for 3 . 8 5 0.600 1.03 0.79 Guanine 13.0,5 0 , 7 3 0 0 . 2 0 0 0 .34 Adenine 4 . 0 7 0 . 2 7 3 . 5 3 0 . 2 5 6 purity should be made, and, when indicated, further purification Total purines should be effected before analyzing for isotopic concentration. and The hydrolysis of bacterial cells or other tissues by refluxing in pyrimidines 4.899 5.17 3.371 5.80 Total amino acids, 6 S hydrochloric acid for 24 hours results in the degradation of ammonia and other ninhydrid-positive purines and pyrimidines and analysis of this hydrolyzate yields an substances" 91.61 90.76 inaccurate picture of the concentration of these substances in the Total S recovered 96.78 96.56 intact cells. Marshak and Vogel ( 4 ) and Wyatt (9) employed a a Reported earlier in detail, Wall, et ol. ( 8 ) . milder condition of hydrolysis of nucleic acids and lipid-free trichloroacetic acid extracts of bacterial cells, 7270 perchloric acid a t 100" C. for 1 hour, to release the purines and pyrimidines without their destruction. The ion evchange chromatographic acid strength and resin may often br emplo>-ed to advmtage t o method described here and those of Cohn ( 1 ) are applicable to the achieve desired separations. separation of the products of this hydrolytic procedure and thus permit the determination of the bases as they occur in vivo. ACKNOWLEDGMENT It was observed that the position of guanine relative to phenylThe counsel of Rohert H. Burris in whose laboratory this work alanine can be shifted depending on h o x early the change from 1 iws prri'ormetl is .*iric,ei,elyapprrc+~te.tl. to 6 hydrochloric acid eluent is made. If the change is made just after histidine is eluted, guanine and phenylalanine -NINHYDRIN DETERM. OPTICAL ,mAAlMINO emerge from the resin tcACID/ML. * , DENSITY gether. If the change is ALANINE -o-o--U. V. ABSORPTION made after lysine is eluted, 260 mp THREONINE> guanine precedes phenylalanine Arginine and phenylalanine are eluted together from the re in by 4 N hydrochloric acid, but 6 A' hydrochloric acid causes arginine t3 2.0 precede phenylalanine. As has been emphaqi7ed by Stein and Moore (6\, two principles govern the affinity of a component for the Do~%ee\-50 1.0 resin: ( a ) ionic attraction and ( b ) adsorption on the aromatic resin. The less polar the substknee the greater this latter effect. An increase in 1200 . a& . 3600 4800 6000 7200 8400 acid normalityaffects the rate 1.5 N HC1 ;' ML. ELUATE 2'5 HC1 of elution of a substance of ARGININE low polarity the least. The relationship of the fractions in 1.0 LYSINE which the purines and pyrimidines appear relative t o the amino acids is also governed by the particle size and cross .o I 8400 9800 10800 ' 12000 ' 13200 14kO linkage of the resin. With c -4.ONHC1 -6.ONHCl another batch of resin it was found that vanthine came off Figure 2. Separation of Nitrogenolls Components of Chromutitrm Hjdrolj zate on with valine, cytosine with Dowex-50 leucine, and guanine mith arZero volume of eluate indicates point of application of hjdro1)zate t o column ginine. n hen the acid strength *Based on leucine *tandard, uncorrected for differences in color yield with ninh3drin
t
4
n
V O L U M E 2 5 , N O . 6, J U N E 1 9 5 3
953
LITERATURE CITED
W ,E., Science, 109,377-8 (1949). ( 2 ) Hiller, -2..Plazin, J., and Van Slyke, D. D., J . Biol. Cheni., 176, ( 1 ) ('ohn,
1401-20 (1948). ( 3 ) Markham, R., and
Smith, J. D., Nature, 164, 1052 (1949). ( 4 ) lIarshak, A , , and Togel, H. J., Federation Proc.. 9,85-6 (1950). ( 5 ) IIoore, S.,and Stein, W.H., J . Bid. Chem., 176, 367-88 (1948). (6) Stein, W.H., and JIoore, S., Cold Spring Harbor Sy??iposiu C)i((i?it. Riol., 14,179-90 (1949).
( 7 ) Vischer, E., and Chargaff, E., J . Biol. Chem., 176,703-14 (1948). (8) Wall, J. S., Wagenknecht, -4.C., Kewton, J. W., and Burris, R. H., J . Bacterid., 63, 563-73 (1952). (9) Wyatt, G. R., Biochem, J , , 48, 584-90 (1951).
RECEIVED for review January 13, 1953. Accepted March 23, 1953. Presented before the Federation of -4merican Societies of Experimental Biology, I f a r c h 1952. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part b y a grant from the Atomic Energy Commission.
Spectrophotometric Micromethod for Determining Polyunsaturated Fatty Acids S. F. HERB
AND
R . W. RIEMENSCHNEIDER
Eastern Regional Research Laboratory, Philadelphia, Pa.
'There is considerable need for a micromethod of analysis of polyunsaturated fatty acids in fats or lipides, because often more than a few milligrams of sample niay be difficult or impractical to obtain. A spectrophotometric method is described which requires 1 to 10 mg. of fat for determination of acids containing from two to five double bonds. Results of analyses by the micromethod were in agreement with those obtained by macromethods. The micromethod should find greatest application in studies of the changes in lipide composition of 5uids and tissues of living animals and plants owing to the small quantity of sample required for analysis.
I
1IPIIOVEIII:STS in spectrophotometric methods for determining polyunsaturated acids in fats and oils have been discauwd in recent papers ( 6 , 7 ) . Although the methods currently in upc require only 100 m g . of sample, there is a great deal of iiitweut in a niicromet~hodthat would require only a fen- milligrams. Sunicrous inquiiiea concerning such an adaptation of thr method have been rweived from persons engaged in biological :inti mediral research, herause often moi'e than a few milligrams of lipide may be difficult or iinprnctical to obtain-for example, i i i :inalysis of hody fluids Liken from living animals. A search of the literature. has furniqhed further evidence of keen illtorest iii spcctrophotornetric micromethods. Considerable \r.ork in this direction has been published (4,9, 10, 1 2 , 12). l-iifortun:ztcl~-, these invcstig:ttors did not have pure, natural, uris:itur:itcd acsiilr :ivailahle as reference standards from which they cmultl determine the correct spectrophotometric con.tants f o r thtir .spc&l ronditions of alkali isomerization. This is particdarly true for witis of greater unsaturation than linolenic acid. Coiiscquently, thrsv woi~lrersdid not recommend their published ronst:iiits, :inti :idvisetl othew to determine their on-n independcntlJ.. 111spit(, of t h r fact th:it no satisfactory micromethod resulted fi~onithis earlier work. the greater amount of conjugation protluwtl hy isomerizing in higher concentrations of potassium hyrlroxitle iii glycol intlicated possibilities for a more sensitive mc.thoti. For examplr. Holman and Burr (9) reported an extincGY tion cocfirient, E:,,",. = (i22, fur tetraene (3000 A , ) from Lie1~t~omiriatiori-ar:ic~liidonic :rcicl isomerized in glycol solution cont,:iiriing 22 to 23 gi':ims of potassium hydroxide per 100 nil. for 8 iuinutes at 178" C. This value is more than twice as great as the value 258, obtained under the conditions used by Beadle and I
