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A spectrophotometric procedure for the analysis of nucleic acid purines and pyrimidines, and its application to fish sperm nucleic acids
55 °
BIOCttIMICA ET BIOPHYSIC& ACTA
A SPECTROPHOTOMETRIC
PROCEDURE
OF NUCLEIC ACID PURINES
VOL. 2 8
(I958)
FOR THE ANALYSIS
AND PYRIMIDINES,
AND ITS APPLICATION TO FISH SPERM NUCLEIC ACIDS* C. F. EMANUEL AND I. L. CHAIKOFF Department o] Physiology o/ the University o! Cali[ornia School o[ Medicine, Berkeley, Cali/. (U,S.A.)
I. INTRODUCTION The quantitative analysis of nucleic acid purines and pyrimidines has been accomplished in several ways. In the most widely used procedure, the purine and pyrimidine components of the hydrolysed nucleic acid are first separated b y paper chromatography, then eluted and measured quantitatively in a spectrophotometer 1. In our hands, this method was found to be of low precision, necessitating m a n y replicate analyses in order to obtain acceptable values. The poor precision resulted from impurities in the filter paper and from extensive manipulations. Furthermore, the recoveries of the heterocyclic bases rarely exceeded 80%, with losses the result of inadequately controlled hydrolysis of the nucleic acid. A few reports have appeared on the use of starch-column chromatography 2 and ion-exchange resins for separating purines and pyrimidines 3 as well as nucleotides 4, ~.6. Although the recoveries are excellent, the methods are tedious and time-consuming, and require at least 5o h for the analysis of a single sample. KERR et al. 7 and, later, LORING et al. s devised a procedure for analysis of 5-1o mg of ribonucleic acid (RNA) based upon differential spectrophotometry. In this method, the RNA was digested for I h, at Ioo °, with N H2SO 4. The p H of the mixture was then adjusted to ~ and the purines were precipitated with silver nitrate solution. The silver-purine precipitate was filtered, washed, and decomposed to the free purines with hot hydrochloric acid. The purine-containing filtrate was then subjected to a twocomponent, differential spectrophotometric analysis by which the exact content of guanine and adenine was obtained. The pyrimidine nucleotide fraction of the hydrolysate (not precipitable with acidic silver solutions) was dephosphorylated with acid phosphatase, purified on an ion-exchange column, and also subjected to a twocomponent spectrophotometric analysis for cytidine and uridine content. Since 99% of the heteroeyclic content of RNA was accounted for in terms of total nitrogen, this procedure has a decided advantage over paper-chromatographic methods in accuracy and probably in time. In its present form, the LORIN6 procedure for RNA is not applicable to deoxyribonucleic acid (DNA) analyses. In order to apply the principles of LORING'S * This work was supported by a contract from the United States Atomic Energy Commission. Re/erences p. 56x.
VOL.
28 (i958)
SPECTROPHOTOMETRY OF PURINES AND PYRIMIDINES
55I
procedure to DNA, it was necessary to determine the optimum conditions for the perchloric acid hydrolysis of DNA. In addition, we determined the molar absorbancy coefficients of the pure purines and pyrimidines under the same experimental conditions as were used in our analyses. A method is described here for the hydrolysis and quantitative analysis of DNA which is precise and accurate. All quantitative manipulations, cooling, etc. of the silver-purine precipitates, and the enzymic hydrolysis and ion-exchange treatment used in the LORING procedure are eliminated. A complete duplicate analysis of at least a dozen samples can be carried out in one working day. II. MATERIALS AND EQUIPMENT The nucleic acid s a m p l e s were isolated f r o m s a l m o n s p e r m * or h e p a t o c a r c i n o m a tissue** b y the a u t h o r s ' procedures 9,1°. I n a f u r t h e r effort to eliminate final traces of protein, we dissolved the highly purified nucleic acid s a m p l e s in physiological saline (in which nucleoprotein is insoluble), and centrifuged t h e solutions at IOO,OOO × g for 6o rain at 7 °. The nucleic acid in the clear s u p e r n a t a n t s w a s precipitated w i t h e t h a n o l b y s t a n d a r d procedure. T h e p u r i n e s and p y r i m i d i n e s were isolated f r o m s a l m o n s p e r m D N A b y the m e t h o d devised for R N A b y HUNTER AND HLYNKA11. Deoxyribonucleotides were purchased from the Biochemical R e s e a r c h F o u n d a t i o n , a n d recrystallized f r o m w a r m w a t e r until suitable p u r i t y was obtained. P h o s p h o r u s and n i t r o g e n analyses were done as previously described 1°. The analytical properties of t h e crystalline s u b s t a n c e s are given in Table I. The nitrogen and p h o s p h o r u s c o n t e n t s of the nucleic acids (dried, i n vacuo, at r o o m t e m p e r a t u r e over Mg(C104) 2) are given in Tables I I and V I I . TABLE I ELEMENI'ARY COMPOSITION OF NUCLEIC ACID DERIVATIVES Compound
Adenine sulfate § (C~H~Ns)2" H~SO4 G u a n i n e hydrochloride CsHaNsO. HC1 T h y m i n e CsH6NzO 2 Cytosine C4HsNsO D e o x y r i b o c y t i d y l i c acid C9H14NzO7P. H 2 0 Calcium t h y m i d y l a t e CIoH14N2OsP- Ca. 2 H 2 0 Diammonium deoxyriboguanylate CIoH1,N~OvP- 2 N H 4. 2H~O Diammonium deoxyriboadenylate C10H14NsO6P. 2 N H 4
Calculated
C
H
29.7 °
3.99
31.99 47.61 43.27
3.20 4.79 4.54
N
Found
P
22.21 37 .82 12.91
9.52
C
H
29.98
3.79
31.89 47.63 43.28
3.34 4.93 4.72
N
P
21.82 37.74 13.37
7 .80
9.22, 9.3 ° 7.72
23.39
7.38
22.95
7.65
26.82
8.44
27.21
8.45
§ P u r i n e s a n d p y r i m i d i n e s were dried over Mg(CIO4) 2 and N a O H at i io ° for 18 11, in vacuo. Nucleotides were dried at r o o m t e m p e r a t u r e , i n vacuo, over Mg(C104) 2 and N a O H . Silver p e r c h l o r a t e w a s p r e p a r e d as follows: silver n i t r a t e was converted to silver hydroxide with a stoichiometric a m o u n t of s o d i u m hydroxide. After the silver hydroxide was t h o r o u g h l y w a s h e d w i t h water, it was mixed with a slight excess of 60 % perchloric acid. The colorless solution w a s filtered t h r o u g h a sintered disk and t h e n slowly c o n c e n t r a t e d over sulfuric acid, in the dark, until c r y s t a l s formed. The silver perchlorate was recrystallized t w o or three times from a small * O b t a i n e d from the I s s a q u a h Fish H a t c h e r y t h r o u g h the c o u r t e s y of the \ V a s h i n g t o n State I ) e p a r t m e n t of Fish and Game. ** C9.~a h e p a t o c a r c i n o m a (obtained f r o m Roscoe B. J a c k s o n Memorial L a b o r a t o r y , Bar H a r b o r , Maine) w a s carried b y serial t r a n s p l a n t a t i o n in C57 leaden mice. Re/erences p. 5 6 L
552
C . F . EMANUEL, I. L. CHAIKOEE
VOL. 2 5
(1958)
TABLE II ANALYTICAL
PROPERTIES
NaDNA source Pink ( H u m p b a c k ) s a l m o n s p e r m (Oncorhynchus gorbuscha) King (Chinook) s a l m o n s p e r m (0. lshawytscha) ( ' h u m (Dog) s a l m o n s p e r m (0. keta) Sockeye (Red) s a l m o n s p e r m (0. nerka) Silver (Coho) s a l m o n s p e r m (0. kisutch)
OF
DNA
SAMPLES
N (%)
P (%)
NIP
15.61
8.55
1.83
i4.78
8.15
1.81
15.19
8.25
1.84
15.1o
8.25
1.83
15.42
--
--
a m o u n t of hot w a t e r until a 20 % solution of it h a d an a b s o r b a n c y at 250 millimicrons of 0.25 or less. The B e c k m a n D U s p e c t r o p h o t o m e t e r was tested b y s t a n d a r d procedures 1~ for w a v e l e n g t h position and a b s o r b a n c y intensity. Deviations in a b s o r b a n c y (.4 s) f r o m National B u r e a u of Standards valnes were 1.2 % (low) at 250 m p , 0.6 % (low) at 260 m#, a n d 1.8 % (low) at 280 m/*. The a b s o r b a n c y values (Table I I I ) used in this w o r k were not corrected for these errors. Reproducibility of a b s o r b a n c y readings a n d w a v e l e n g t h settings required the elimination of p a r a l l a x errors in the m a n n e r described b y GIBSON AND BALCOM13. This is p a r t i c u l a r l y i m p o r t a n t for t h e procedure described here since the w a v e l e n g t h settings often fall on very steep p o r t i o n s of the a b s o r p t i o n curves where a small w a v e l e n g t h error will result in a large a b s o r b a n c y error. The w a v e l e n g t h settings finally selected here are n o t the theoretically o p t i m u m ones. A c o m p r o m i s e was necessary to avoid the s h o r t e r w a v e l e n g t h where interference from u n k n o w n foreign materials in nucleic acid h y d r o l y s a t e s was more likely to occur. The molar a b s o r b a n c y indexes (aM) o b t a i n e d from the analytically p u r e p u r i n e s and pyrimidines are given in Table I I I . The p u r i n e values deviate s o m e w h a t f r o m those r e p o r t e d by LORING et al. The r e a s o n s for this are n o t known. However, e q u a t i o n s derived from our a b s o r b a n c y values gave calculated p u r i n e concentrations, m u c h closer to k n o w n values for a s t a n d a r d m i x t u r e of p u r i n e s t h a n did the e q u a t i o n s given by LORING et al. TABLE III MOLAR ABSORBANCY
Compound
Thymine Cytosine Guanine Adenine
INDEXES
(aM)
FOR
DNA
PURINES
AND PYRIMIDINES
Wavelength 250 ml~
262 ml~
280 ml~
51 oo 2976 ---
~ -7842 1294 °
4030 9600 6866 4608
I I I . GENERAL EQUATIONS FOR TWO-COMPONENT DIFFERENTIAL SPECTROPHOTOMETRY I t was our original i n t e n t i o n to c a r r y out a f o u r - c o m p o n e n t differential analysis of D N A h y d r o lysates, and e q u a t i o n s for such an analysis were developed. H o w e v e r , because of the close overlapping of the a b s o r p t i o n curves for adenine, guanine, cytosine, and t h y m i n e , the precision obtained with such e q u a t i o n s w a s p o o r for several selected w a v e l e n g t h sets tested. F o r this reason we resorted to a t w o - c o m p o n e n t analysis of the t y p e described b y LORING et al. The success of such an analysis dcpeuds upon a q u a n t i t a t i v e separation of the m i x t u r e into t w o pairs of substances, tile purines aml tile pyrinlidines. Solutions to the required s i m u l t a n e o u s e q u a t i o n s yielded the fl)llowing general e q u a t i o n s for a n y t w o - c o m p o n e n t m i x t u r e . Re/erences p. 561.
VOL. 2 8
(I958)
SPECTROPHOTOMETRY OF PURINES AND PYRIMIDINES
553
General e q u a t i o n s / o r determining the concentration o / a n y p a i r o / c o m p o u n d s , x and y
1)., 2
(euw2)(Dwl)
C~ ~
euwl
(i)
(e.wl) (e~2)
exzt,2
eyw 1
(ea:w2) (-DwI)
D w2
ex.wl
Cv =
(2)
(ezw2) (eyw1) exwl
eyw2
D is a b s o r b a n c y (As) w I is w a v e l e n g t h one w 2 is w a v e l e n g t h two x is c o m p o u n d one y is c o m p o u n d two ex is m o l a r a b s o r b a n c y (aM) of c o m p o u n d x eu is m o l a r a b s o r b a n e y (aM) of c o m p o u n d y C~ conc. of c o m p o u n d x in m o l e s p e r liter C v conc. of c o m p o u n d y in m o l e s p e r liter ' S u b s t i t u t i o n of t h e m o l a r a b s o r b a n c y v a l u e s g i v e n in T a b l e I I I into e q n s . (I) a n d (2) yielded t h e following e q n s . f r o m w h i c h t h e p u r i n e a n d p y r i m i d i n e c o n c n s , in m o l e s p e r liter c a n be determined. C(cytoslne) = [1.38 A s (280 m t ~ ) - 1.O9 A s (250 mr*)] " 10-4 C(thymlne) ~
[2.59 A s (250m/*) - - 0.804 A s (280 m/~)] " 10-4
(4)
C(guanlne) =
[2.45 As (280m/~)-- 0-875 A s (262mp)] " 10-4
(5)
C(adenine) = [ I . 3 0 A s (262m/,) - - 1.48
A s (280 m/*)] " 10-4
IV. PROCEDURE FOR ANALYSIS OF
DNA
A flow sheet outlining the analytical operations is given in Fig. I. I0 m g D N A , h y d r o l y z e a t i 0 0 ° for I h wifll 2oo ~ I0 N HC104
f A d d 5.o m l H 2 0 , m i x , c e n t r i f u g e
I
I I.O m l s u p e r n a t a n t d i l u t e d to 5 ° m l . Determine absorbancy a t z62 a n d 280 nap. (Total a b s o r b a n c y )
Supernatant
--1 i . o m l s u p e r n a t a n t (in duplicate) is t r e a t e d w i t h i.o m l IO % AgC104, m i x e d , o.75 N N a O H a d d e d ( ~ 0.55 ml) to b r i n g p H to 2.0 to 2. 7. C e n t r i f u g e
Silver-purine precipitate
2.o m l a l i q u o t s (in duplicate), acidified a n d d i l u t e d t o 25 ml. Determine absorbancy a t 250, 262 a n d 280 m/*. (Pyrimidine absorbancy) Subtract pyrimidine absorbancy from total a b s o r b a n c y a t 262 a n d 28o rap. (Purine absorbancy) Fig. I. F l o w s h e e t of a n a l y t i c a l o p e r a t i o n s . Re]erences. p. 5 6 L
(3)
(6)
554
c.F.
EMANUEL, I. L. CHAIKOFF
VOL. 2 8
(1958)
Nucleic acid hydrolysis About IO mg of D N A were weighed in a I5-ml conical centrifuge tube, and 2oo 2 of IO N perchloric acid* were added. After a few minutes, the charred mass was well mixed by careful rotation, and kept at room temperature for 45 rain until the nucleic acid was completely dissolved. The tube was stoppered, and heated for one hour at IOO°. (The charred mass should not be disturbed during heating.) A blank containing only perchloric acid was similarly heated. After the tubes were cooled in a water bath, exactly 5.0 ml of water were added to each tube, and the charred mass was triturated with a stout glass rod.
Nucleic acid analysis The stoppered tubes were centrifuged. I.O ml of the hydrolysate supernatant was removed with a syringe** and transferred to a 5o-ml volumetric flask. After dilution to volume, the total absorbancy was determined, at 262 m/z and 280 m/z, against a properly prepared blank. Two other I.O ml aliquots of the hydrolysate were transferred to 5-ml beakers. Another beaker was prepared with I.O ml of the heated blank solution. To each beaker was then added i.o ml (dispensed with another syringe) of a freshly prepared, IO % solution of silver perchlorate. Mixing was done with a small glass rod. A sufficient a m o u n t of 0.75 N N a O H ( ~ 0.55 ml) was then added (with stirring) to each beaker to bring its contents to p H 2.0 to 2. 7 (checked on a p H meter with the outer surfaces of the electrodes carefully dried to avoid introducing volume errors). (Since the total volume of the m i x t u r e - - i n this case 2.55 m l - - m u s t be known, all volumes added must be measured with care.) The bulk (not quantitatively) of each solution was poured into a dry, conical centrifuge tube (15 ml). The tubes were stoppered and immediately centrifuged. E x a c t l y 2.0 ml of the supernatants, containing the pyrimidines alone, were transferred (by a 2 ml transfer pipette) to 25-ml flasks. After addition of one drop of concentrated perchloric acid, the flask contents were diluted to volume. The absorbancies of these solutions were determined against an appropriate blank at 250, 262, and 280 m/z. These data, representing the pyrimidines alone, were used in eqns. (3) and (4) to determine the pyrimidine content after appropriate dilution corrections. Subtraction of the pyrimidine values (at 262 and 280 m/z) from the total absorption values (at 262 and 28o m/z) gave the absorption derived from purines which was used in eqns. (5) and (6). Although light caused the silver purine precipitate to become mauve-colored, this was not deleterious, and was disregarded.
V. TEST OF METHOD WITH A PURINE AND PYRIMIDINE STANDARD
I. Effect o / p H on the quantitative separation o~ a known base mixture by means o~ silver perchlorate It was necessary to determine the o p t i m u m p H at which a silver perchlorate * The exact strength of this acid was 9.737 N. ** i c c B-D Tuberculin syringe, smallest division, o.oi cc. We have disregarded the difference between cubic centimeters and milliliters.
Re/erences p. 56J:.
VOL. 28 (z958)
SPECTROPHOTOMETRYOF PURINES AND PYRIMIDINES
555
solution precipitates purines quantitatively from solution without at the same time precipitating the pyrimidines. Equimolar mixtures of adenine and guanine were precipitated with silver perchlorate reagent at various p H values. The absorbancy of the supernatants obtained by this treatment was measured against appropriate blank solutions at 262 m/~. Similarly, standard equimolar solutions of pyrimidines (thymine and cytosine) were subjected to the same treatment, and their optical absorbancies were determined at 280 mt~. Fig. 2 shows the result of such experiments in terms of the percentages of heterocyclic absorption still remaining in the supernatants after centrifugation of precipitates that formed. The data obtained in this manner are not precise since most of the absorbancy values fall at the extremes of the absorbancy range. However, it is clear from Fig. 2 that the optimum pH range for purine precipitation lies approximately between p H 2.0 and 2. 7. Optimum pH
,oo
o
[
c
~
r
~
Pyrlmldmes •
1"
'tI g~, [P.rines \~< ~40k
I
2oF
[
•
1/
~~
o
•
1 I
2
,
3
4
,/
- ; 1 ;
pH
Fig. e. Optimum pH for precipitation of purines with silver perchlorate.
2. 'Quantitative separation o[ a standard purine-pyrimidine mixture In Table IV, typical data are given showing the quantitative separation of a known mixture of purines and pyrimidines at p H 2.1 to 2.2. Part A shows the total absorption of the four-component mixture at two wavelengths. These data were obtained in triplicate and are presented with their respective absorbancy ranges. Part B shows the total pyrimidine absorption of a similar standard mixture of pyrimidines (thymine and cytosine), at three wavelengths. Part C gives the absorbancy values found for the supernatant obtained after the same standard mixture of four bases that was used in Part A was treated with silver perchlorate at p H 2.1 to 2.2. A quantitative separation is obtained if the absorbancy values of Part C (supernatant) are identical with those values shown in Part B. Similarly, the absorbancy calculated for the purines (Part D) should equal the absorbancy found (Part E). The data in Table IV show that a quantitative separation of purines from pyrimidines is achieved under the conditions outlined in section IV within the limits of manipulative and spectrophotometric error demonstrated below.
Re/erences p. 56z.
556
C . F . EMANUEL, I. L. CHAIKOFF
VOL. 2 8 ( 1 9 5 8 )
TABLE IV EXPERIMENTAL AND STANDARD ABSORBANCYVALUES OBTAINED FROM THE QUANTITATIVE SEPARATION OF A STANDARD BASE MIXTURE A bsorbancy values 250 ml*
262 mlz
280 ml*
Part A T o t a l a b s o r b a n c y of a g u a n i n e - a d e n i n e thymine-cytosine standard
0.943 ~ o . o o 3 "
0.690 ~ 0.007
Part B A b s o r b a n c y of a s t a n d a r d s o l u t i o n of p y r i m i d i n e s ( t h y m i n e + c y t o s i n e ) of s a m e c o n c e n t r a t i o n s as in P a r t A o.241 4 - 0 . 0 0 3
o.451 ~ o . o o 3
o.419 ± o.oo2
Part C (pyrimidines) A b s o r b a n c y of s u p e r n a t a n t a f t e r a d d i t i o n of AgCIO 4 t o t h e s o l u t i o n u s e d in P a r t A
0.456 ± 0.003
o.421 ± 0.005
Part D S t a n d a r d v a l u e s for p u r i n e s (i.e., d a t a of P a r t A m i n u s d a t a of P a r t B)
0.492
0.272
P a r t E (purines) Total absorption minus supernatant ( p y r i m i d i n e ) a b s o r p t i o n (i.e., d a t a of P a r t A m i n u s d a t a of P a r t C)
0.487
0.269
0.248 ± 0.004
* M a x i m u m r a n g e of a b s o r b a n c y v a l u e s .
3. Precision and accuracy o/recovery o/bases in a standard purine-pyrimidine mixture An approximately equimolar mixture of adenine, guanine, cytosine, and thymine (2.4" IO-s moles of each base per ml) was used to carry out a complete analysis as described in section IV. Particular care was taken to minimize random volumetric errors. The p H of purine precipitation was 2.0. Six separate determinations gave the absorbancy results shown in Table V. I t is evident from these data that the m a x i m u m deviation in accuracy and precision is ± 0.8%. TABLE V PRECISION
AND
ACCURACY
OF THE
PURINE AND PYRIMIDI~/E SEPARATION PROCEDURE Pyrimidine ahsotbancy
250 ml2
Expected* F o u n d (av erag e)** Error
-
-
0.245 0.244 zt- 0.002 0. 4 %
262 ral*
280 mlz
0.452 0.455 4- o.ooi + 0. 7 %
o.419 0.420 :~ 0.002 + 0.2 %
Purine absorbancy
Expected* F o u n d (average)** Error
250 ml*
262 ml*
280 ml~
----
o.497 o.495 ± o.oo2 - - 0. 4 %
o.275 o.274 ~ o.ooi - - 0. 4 %
* E x p e c t e d on t h e b a s i s of t h e m o l a r a b s o r b a n c y coefficients (Table I I I ) a n d k n o w n concentrations. ** Six s e p a r a t e d e t e r m i n a t i o n s . R e [ e r e n c e s p . 562-.
(1958)
VOL. 2 8
SPECTROPHO'IOMETRY OF PURINES AND PYRIMIDINES
557
VI. TEST OF METHOD WITH NUCLEOTIDES AND NUCLEIC ACID 1.
Hydrolysis o/'nucleotides and nucleic acids
To determine whether perchloric acid degrades purines and pyrimidines of nucleic acids, we exposed the crystalline deoxyribonucleotides to varying concentrations of perchloric acid for ~ h, at IOO°, in stoppered tubes. Approximately 1.2. lO -5 moles of each nucleotide were hydrolyzed with i o o ~ of perchloric acid of varying strengths (i to 12 N). The absorbancies of the hydrolysates were then determined at two wavelengths. The results of these experiments are given in Fig. 3(a). The theoretical (calculated) absorbancies are indicated as dashed horizontal lines. It would seem, from these experiments, that i o o 2 of 5 or 6 N perchloric acid are optimum for the recovery of the expected absorbancy. When these hydrolysates were chromatographed on paper, it became evident that acid strengths of 3 N or stronger
0.9 0.8
--
Q2~'~
DEOXYCYTIDYLICACID
_7
0.4 ~,,,~_ __ 2aom~ 02 o
.------
D~OXYTHYMIOYLIC ACID
0.7
O.E
I
>, Q=
DEOXYGUANYLIC ACID
~
2
6
2
o
g
I.
O~
~
•
.
.
.
.
.
.
DEOXYADENYLIC ACID
4 6 8 fO HGI04 Normality
12
I00 200 300 400 500 Micr0~iters 12N HCIO4 per 12xlO "5 f~iole of Nucleofide
Fig. 3 (a) and Fig. 3 (b). The effect of perchloric acid on the absorbancy
of deoxyribonucleotides.
02
I
I
~
I
I
2
3
4
5
6 7 8 9 HC[O4 N0rmol;ty
f
#
I
I
I
I
I0
II
12
m
Fig. 4- T h e effect of v a r i o u s s t r e n g t h s of h o t perchloric acid on t h e optical a b s o r b a n c y of D N A .
caused extensive hydrolysis of the glycosidic bonds. I and 2 N perchloric acid solutions were not very effective in breaking the pyrimidine glycosidic bonds, and considerable amounts of thymidine and cytosine deoxyriboside were left in the hydrolysates. Fig. 3(b) shows the results of experiments in which the strength of the perchloric acid was kept constant, but the volume of acid for each 1.2. IO-s mole of nucleotide was increased from 30 2 to 500 2. In these experiments it was found that excessive perchloric acid caused extensive destruction of both purines and pyrimidines, but particularly the latter. These results indicate that ioo k of perchloric acid per lO -5 mole of nucleotide is about the m a x i m u m amount of 12 N HC104 which will give acceptable recoveries. It is also evident that deoxyribose degradation products contribute no extraneous absorption to the hydrolysate in the region of higher acid strength. In Fig. 4 the results of nucleic acid hydrolysis by different perchloric acid strengths are presented. In these experiments, 4.15 mg of DNA were hydrolyzed with IOO 2 of Re/erences p. 56z.
558
C.F.
E M A N U E L , I. L. C H A I K O F F
VOL. 2 B
(1958)
perchloric acid solution, at IOO°, for 30 rain. In the complete hydrolysis of nucleic acids, three bonds, at least, must be hydrolyzed (for each nucleotide) instead of two bonds as in a free nucleotide. It seems likely that a larger amount of acid per nucleotide will be necessary for nucleic acid hydrolysis than for the free nucleotide. Since the theoretical absorption of a nucleic acid hydrolysate cannot be accurately calculated, it is difficult to determine from Fig. 4 the optimum acid concentration for DNA hydrolysis. However, an examination of these hydrolysates b y paper chromatography revealed that, besides the expected purines and pyrimidines, there were three new components, one with a bright blue fluorescence at RF 0.48 (isopropyl alcohol-water-HC1) ; a second at RF 0.34; and a third at Rv 0.69. The first, which is identical with the LEVY-SNELLBAKER14 substance, gave an orange-tan color with diazotized sulfanilic acid; the second gave a purple-pink color; and the third, an orange color, with this reagent. These substances were especially evident in the hydrolysates produced by intermediate acid strengths (3 N to 8 N), and were absent from the very weak and strongly acid hydrolysates. Cytosine deoxyriboside and thymidine were also evident in the I N and 2 N perchloric acid hydrolysates. I t is not known whether these unknown components are artifacts arising from an intermediate stage of hydrolysis of DNA or are actually integral components of nucleic acids destroyed at the higher acid concentrations. They were present in considerable quantities in nucleic acids obtained from salmon sperm, from mouse liver, and from mouse hepatocarcinoma. Attempts are now being made to isolate them. I t seems probable that the significantly higher absorption observed in the 4 N to 8 N acid hydrolysates is due to the absorption and stray light (fluorescence) contributed b y the unknown materials. Unfortunately, the above experiments do not clearly define the optimum conditions for hydrolysis of nucleic acids. The absorption curves indicate, however, that at least IO N acid is necessary. An incomplete survey of the perchloric acid hydrolysis conditions recommended by other workers is given in Table VI. TABLE VI MICROLITERS OF PERCHLORIC ACID RECOMMENDED FOR EACH IO m g D N A Mieroliters o] acid
143 52
200 8o-15o
Normality
12 12
7.5 12
Literature
15 16
17 I8
2. D N A nitrogen-phosphorus recovery and base ratios obtained under varying conditions o] hydrolysis The criterion for optimum hydrolysis can be either nitrogen recovery, as calculated from the determined purine-pyrimidine content, or phosphorus recovery if one assumes that a strict one-to-one ratio exists between moles of base and moles of phosphorus. DNA samples of io.o mg each (isolated from hepatocareinoma tissue) were hydrolyzed for 1/2 h, at IOO°, with several different strengths of perchloric acid (20o ;~). Purine precipitation with silver perchlorate was carried out at p H 2.o. The results Relerences p. 56L
VOL. 2 8
(1958)
SPECTROPHOTOMETRY
OF PURINES
AND PYRIMIDINES
559
of these experiments are given in Table VII. These data show that hydrolysates prepared with dilute acid contain, in addition to the four desired purines and pyrimidines, other products, presumably of an intermediate stage of hydrolysis. Thus, the calculated nitrogen and phosphorus values (upper parts of columns 9 and io, Table vii) based on eqns. (3), (4), (5), and (6) deviate very widely from the direct nitrogen and phosphorus determinations. It is evident that the use of either IO or 12 N perchloric acid yields a quantitative base recovery and nearly theoretical base ratios (lower parts of columns 6 to IO, Table VII). However, further investigation showed that 12 N perchloric acid sometimes resulted in aberrant base ratios, and could not always be relied upon to yield complete recoveries. The reasons for this are not well defined, but it is certain that base destruction can occur with some nucleic acid samples under what appears to be inadequately controlled hydrolysis conditions. Because of this difficulty, an attempt was made to define the optimum conditions for hydrolysis with IO N perchloric acid. From the data shown in Table VIII, it was TABLE
VII
E F F E C T OF H C I O 4 STRENGTH ON HETEROCYCLIC EASE RECOVERY AND RATIOS Perehloric acid normality
Cytosine
Thymine
4 5 6.5 7 8 IO 12
I3.O 12. 9 14.4 15-3 16.8 19.6 21.2
29.2 32.1 35.4 37.6 36. 5 29.6 29.6
Adenine
Guanine
21. 3 19. 3 2o.6 22.4 25. 3 30.3 28.8
Cytosine
Adenine
Purines
Guanine
thymine
Pyrimidines
Moles o/ phosphorus acc'd /or*
Nitrogen acc'd /or (rag) ~
0.356 0.362 0.489 0.622 o.791 0.96 I.O 4
1.37 1.66 1.72 1.68 1.44 o.98 1.o2
1.38 1.22 I.OI o.89 0.87 1.o3 1.o 3
2 . 9 0 " IO - s 2 . 8 2 . lO -6 2 . 8 3 • IO - s 2 . 6 7 • IO -8 2 . 4 6 . lO 4 2 . 4 6 " IO s 2 . 4 6 . IO s
o.152 o.197 o.145 o.134 o.122 o.128 o.127
28.6 35.8 29.4 24-2 21. 3 2o.5 20. 4
D i r e c t d e t e r m i n a t i o n of p h o s p h o r u s D i r e c t d e t e r m i n a t i o n of n i t r o g e n
2.47" io
s o.i 264
* P h o s p h o r u s and n i t r o g e n " a c c o u n t e d for" were calculated on t h e basis of the mole c o n t e n t s g i v e n in c o l u m n s 2, 3, 4, a n d 5** M o l e s of base × i o ~ [ m g N a D N A . TABLE
VIII
EFFECT OF DIGESTION TIME ON HETEROCYCLIC BASE RECOVERY (IN MOLES OF HETEROCYCLIC BASE PER m g N a D N A ) Digestion limes at too ° !~ h
Cytosine Thymine Adenine Guanine
lO -8 lO -8 IO - 8 IO-S
5.81" 7.o8. 7.26" 5.85 .
lO -8 lO -8 IO -8 IO-S
2h 5.73" 6.8o. 7.38. 5.31"
lO-8 io s IO -8 IO-8
,? h 5 .8o" 6.5o" 7.22" 5.76.
IO-S lO -8 lO -8 lO-8
Total
26.6
• io -s
26.0
• lO -8
25.2
• I n -8
25. 3
• lO -8
Calculated
25. 9
• IO 8
25.9
. lO-8
25.9
. lO-8
25.9
, lO-8
Adenine Thymine Guanine Cytosine Purines l'yrimidines Relerences
5.85" 7.23. 7.76" 5.73"
I h
p. 5 6 I .
t .07
0.99
i.o 7
I.I i
o.98
i .oo
0.93
i.oo
t'°3
I.O2
I.O[
I.O6
560
C. F. E M A N U E L , I. L. C H A I K O F F TABLE MOLE-0/o C O M P O S I T I O N
(1958)
NUCLEIC
ACIDS
IX
OF SALMON
Adenine
Guanine
Purines
Cytosine
Thymine
Adenine
Guanine
Thymine
Cytosine
Pyrimidines
Silver salmon
21.6
27.6
28.8
21.8
1.o 4
I.Oi
1.o 3
2 . 4 8 . lO -5
Chinook salmon
22. 4
27.2
27. 9
22. 5
0.99
i.oo
1.o2
2 . 5 9 . i o -~
Chum salmon
21.9
27.6
28.7
21.9
i,oi
I.()o
1.o2
2 . 3 7 . i o --~
Sockeye salmon
21.8
27.8
28.2
22.0
i .oi
i.ot
i.oi
2 . 5 9 - IO-"
Humpback
22.6
27.5
27.7
22.5
i,o'i
i.oi
i.oi
2.52" IO-"
Sperm sample
salmon
Mote-per cent o] reccmeredbase
VOL. 2 8
Motes phosphor per sro mg DN
decided that optimum conditions obtained when IO mg of DNA were hydrolyzcd with 2o0 ~t of IO N perchloric acid, at IOO°, for I h. Longer periods of digestion caused noticeable destruction of thymine. V I I . A P P L I C A T I O N OF P R O C E D U R E TO A N A L Y S I S OF SALMON S P E R M N U C L E I C ACID
The conditions given in section IV were applied to the analysis of sperm nucleic acids obtained from five species of Pacific Ocean salmon !]'able IX). These data show that unitary base ratios and nearly complete base recovery (98-1oo%) are achieved. VIII. DISCUSSION
The reliability of a procedure for the analysis of purines and pyrimidines of nucleic acids is generally based on nitrogen or phosphorus recoveries. Unfortunately, a check based on these constituents is not completely satisfactory. An accounting of all the nitrogen in terms of the four constituent bases is not possible, usually because almost all nucleic acid samples contain small amounts of foreign nitrogen, mainly protein,_ which is very difficult to remove. For this reason we have not, as a rule, relied upon total nitrogen analyses. Recoveries based on total phosphorus are likewise unsuitable because two assumptions must be made: (I) that no foreign phosphorus-containing substances (e.g., RNA or phospholipids) are present; and (2) that nucleic acids contain, according to theory, a strict one-to-one ratio between moles of heterocyclic base and phosphorus. Few, if any, nucleic acid preparations of mammalian origin have been entirely free of ribonucleic acid. In the second assumption, it is implied that no triply esterified or secondary phosphoryl groups are present. It is well established that fish sperm nuclei contain very small amounts, if any TM, of ribonucleic acid, and the protamine associated with the nucleic acid is readily eliminated by standard procedures. Hence, fish sperm nucleic acids are well suited for testing the quantitativeness of base recoveries. Table IX gives a complete purine and pyrimidine recovery, based on phosphorus analyses, for sperm nucleic acids of five species of salmon. The recoveries shown in the last columnranged from 98 to lOO%. The strongest argument in favor of the WATSON-CRICK model for DNA structure depends upon the adenine-thymine and guanine-cytosine ratios. According to this model, the integrity of the double-stranded DNA molecule depends upon hydrogen bonding between the base pairs that occupy adjacent positions in opposing nncleotide chains. Although such pairing requires that the ratios be unitary, reports of large Re]erences p . 5 6 z .
VOL. 2 8
(1958)
SPECTROPHOTOMETRY OF PURINES AND PYRIMIDINES
561
deviations are still appearing. Because of our initial failure to obtain values of unity for the ratios, we were led to reinvestigate the hydrolysis procedure. It now seems certain Total moles Per cent recovered (basedon phosphorus) that non-unitary ratios obtained in our earliest experiments base/ound resulted from base destruction (particularly of thymine) 2.47" IO -5 IOO during hydrolysis. In this connection it is important to note °-.60" IO -~ IOO that the analyses which gave unitary ratios also gave the most quantitative recovery in terms of total nitrogen or 2-34" IO-5 99 phosphorus. We therefore believe that ratios deviating more 2.54" lO -5 98 than a few % from unity are the result of selective destruc2.51. IO -5 IOO tion of bases during hydrolysis. On the whole, our analyses on sperm nucleic acids do not conflict with the WATSONCRICK theory. The quantitative recovery obtained by our procedure suggests that the three unknown substances that we found in hydrolysates prepared with perchloric acid of intermediate strengths are actually artifacts arising during nucleic acid degradation. If these substances are real D N A components, they must be either triply esterified to phosphorus or united to the nucleic acid in some other way which does not involve phosphorus at all. Although the latter alternatives seems unlikely, we are now attempting to find these unidentified substances in enzymic hydrolysates of nucleic acids. SUMMARY I. A s p e c t r o p h o t o m e t r i c m e t h o d for the analysis of n u c l e i c a c i d purines and p y r i m i d i n e s is d e s c r i b e d . D a t a w h i c h illustrate the o p t i m u m c o n d i t i o n s for p e r c h l o r i c a c i d h y d r o l y s i s of D N A are presented. Heterocyclic base recovery in terms of phosphorus or nitrogen is 9 8 - 1 o o % c o m p l e t e . 2. P u r i n e a n d p y r i m i d i n e c o n t e n t s of five species of s a l m o n s p e r m D N A w e r e s t u d i e d . H y d r o l y s i s c o n d i t i o n s which gave complete recovery in terms of phosphorus also y i e l d e d unitary values for the following base ratios: adenine/thymine, guanine/cytosine, and p u r i n e / p y r i m i d i n e .
REFERENCES 1 E. CHARGAFF AND J. N. DAVlDSON, The Nucleic Acids, Vol. I, A c a d e m i c Press, Inc., N e w Y o r k , 1955. 2 M. DALY, J. Gen. Physiol., 33 (195 o) 497. 3 \ ¥ . E. COHN, Science, lO9 (1949) 377. 4 R. O. HURST, A. ]~. MARKO AND G. C. BUTLER, J. Biol. Chem., 204 (1953) 847. Ii R. L. SINSHEIMER AND J. F. I~OERNER, Science, 114 0 9 5 1 ) 42. 6 R. L. SINSHEIMER AND J. F. KOERNER, J. Biol. Chem., 198 (1952) 293. 7 S. E. KERR, K. SERAIDARIAN AND M. \VARGON, J. Biol. Chem., 181 (1949) 761. 8 H. S. LORING, J. L. FAIRLEY, H. \V. 13ORTNER AND H. L. SEAGRAM, J. Biol. Chem., 197 (1952) 809. 9 C. F. EMANUEL AND I. L. CHAIKOFF, J . Biol. Chem., 203 (1953) 167. 10 C. F. EMANUEL AND 1. L. CHAIKOFF, Biochim. Biophys. Acla, 24 (1957) 25411 G. HUNTER AND I. HLYNKA, Biochem. J., 31 (1937) 486. 12 G. \V. HAUPT, J. Research Natl. Bur. Standards, 48 (1952) 414 . 13 K. S. GIBSON AND M. M. 13ALCOM, J. Research Natl. Bur. Standards, 38 (i947) 6Ol. 14 H. 13. I,Evv A~'D L. F. SI'~ELLBAKER, Arch. Biochem. Biophys., 54 (1949) 549. 15 O. R. \VYATT, Biochem. J., 48 (1951) 584 . 10 A. MARSHAK, J. Biol. Chem., 189 (1951) 607. 17 A. MARSHAK AND H. J. VOGEL, J. Biol. Chem., 189 (1951) 597. IS G. 1C \VYATT, .]. Gelz. Physiol., 36 (1952) 2Ol. I~ t(. I:ELIN, H. FISCHI:.R AN]) .\. KREKELS, Progr. it* Biophys. a~zd t~iophys. Chem., 6 (1956) 2.
Received October 28th, I957
BIOCttIMICA ET BIOPHYSIC& ACTA
A SPECTROPHOTOMETRIC
PROCEDURE
OF NUCLEIC ACID PURINES
VOL. 2 8
(I958)
FOR THE ANALYSIS
AND PYRIMIDINES,
AND ITS APPLICATION TO FISH SPERM NUCLEIC ACIDS* C. F. EMANUEL AND I. L. CHAIKOFF Department o] Physiology o/ the University o! Cali[ornia School o[ Medicine, Berkeley, Cali/. (U,S.A.)
I. INTRODUCTION The quantitative analysis of nucleic acid purines and pyrimidines has been accomplished in several ways. In the most widely used procedure, the purine and pyrimidine components of the hydrolysed nucleic acid are first separated b y paper chromatography, then eluted and measured quantitatively in a spectrophotometer 1. In our hands, this method was found to be of low precision, necessitating m a n y replicate analyses in order to obtain acceptable values. The poor precision resulted from impurities in the filter paper and from extensive manipulations. Furthermore, the recoveries of the heterocyclic bases rarely exceeded 80%, with losses the result of inadequately controlled hydrolysis of the nucleic acid. A few reports have appeared on the use of starch-column chromatography 2 and ion-exchange resins for separating purines and pyrimidines 3 as well as nucleotides 4, ~.6. Although the recoveries are excellent, the methods are tedious and time-consuming, and require at least 5o h for the analysis of a single sample. KERR et al. 7 and, later, LORING et al. s devised a procedure for analysis of 5-1o mg of ribonucleic acid (RNA) based upon differential spectrophotometry. In this method, the RNA was digested for I h, at Ioo °, with N H2SO 4. The p H of the mixture was then adjusted to ~ and the purines were precipitated with silver nitrate solution. The silver-purine precipitate was filtered, washed, and decomposed to the free purines with hot hydrochloric acid. The purine-containing filtrate was then subjected to a twocomponent, differential spectrophotometric analysis by which the exact content of guanine and adenine was obtained. The pyrimidine nucleotide fraction of the hydrolysate (not precipitable with acidic silver solutions) was dephosphorylated with acid phosphatase, purified on an ion-exchange column, and also subjected to a twocomponent spectrophotometric analysis for cytidine and uridine content. Since 99% of the heteroeyclic content of RNA was accounted for in terms of total nitrogen, this procedure has a decided advantage over paper-chromatographic methods in accuracy and probably in time. In its present form, the LORIN6 procedure for RNA is not applicable to deoxyribonucleic acid (DNA) analyses. In order to apply the principles of LORING'S * This work was supported by a contract from the United States Atomic Energy Commission. Re/erences p. 56x.
VOL.
28 (i958)
SPECTROPHOTOMETRY OF PURINES AND PYRIMIDINES
55I
procedure to DNA, it was necessary to determine the optimum conditions for the perchloric acid hydrolysis of DNA. In addition, we determined the molar absorbancy coefficients of the pure purines and pyrimidines under the same experimental conditions as were used in our analyses. A method is described here for the hydrolysis and quantitative analysis of DNA which is precise and accurate. All quantitative manipulations, cooling, etc. of the silver-purine precipitates, and the enzymic hydrolysis and ion-exchange treatment used in the LORING procedure are eliminated. A complete duplicate analysis of at least a dozen samples can be carried out in one working day. II. MATERIALS AND EQUIPMENT The nucleic acid s a m p l e s were isolated f r o m s a l m o n s p e r m * or h e p a t o c a r c i n o m a tissue** b y the a u t h o r s ' procedures 9,1°. I n a f u r t h e r effort to eliminate final traces of protein, we dissolved the highly purified nucleic acid s a m p l e s in physiological saline (in which nucleoprotein is insoluble), and centrifuged t h e solutions at IOO,OOO × g for 6o rain at 7 °. The nucleic acid in the clear s u p e r n a t a n t s w a s precipitated w i t h e t h a n o l b y s t a n d a r d procedure. T h e p u r i n e s and p y r i m i d i n e s were isolated f r o m s a l m o n s p e r m D N A b y the m e t h o d devised for R N A b y HUNTER AND HLYNKA11. Deoxyribonucleotides were purchased from the Biochemical R e s e a r c h F o u n d a t i o n , a n d recrystallized f r o m w a r m w a t e r until suitable p u r i t y was obtained. P h o s p h o r u s and n i t r o g e n analyses were done as previously described 1°. The analytical properties of t h e crystalline s u b s t a n c e s are given in Table I. The nitrogen and p h o s p h o r u s c o n t e n t s of the nucleic acids (dried, i n vacuo, at r o o m t e m p e r a t u r e over Mg(C104) 2) are given in Tables I I and V I I . TABLE I ELEMENI'ARY COMPOSITION OF NUCLEIC ACID DERIVATIVES Compound
Adenine sulfate § (C~H~Ns)2" H~SO4 G u a n i n e hydrochloride CsHaNsO. HC1 T h y m i n e CsH6NzO 2 Cytosine C4HsNsO D e o x y r i b o c y t i d y l i c acid C9H14NzO7P. H 2 0 Calcium t h y m i d y l a t e CIoH14N2OsP- Ca. 2 H 2 0 Diammonium deoxyriboguanylate CIoH1,N~OvP- 2 N H 4. 2H~O Diammonium deoxyriboadenylate C10H14NsO6P. 2 N H 4
Calculated
C
H
29.7 °
3.99
31.99 47.61 43.27
3.20 4.79 4.54
N
Found
P
22.21 37 .82 12.91
9.52
C
H
29.98
3.79
31.89 47.63 43.28
3.34 4.93 4.72
N
P
21.82 37.74 13.37
7 .80
9.22, 9.3 ° 7.72
23.39
7.38
22.95
7.65
26.82
8.44
27.21
8.45
§ P u r i n e s a n d p y r i m i d i n e s were dried over Mg(CIO4) 2 and N a O H at i io ° for 18 11, in vacuo. Nucleotides were dried at r o o m t e m p e r a t u r e , i n vacuo, over Mg(C104) 2 and N a O H . Silver p e r c h l o r a t e w a s p r e p a r e d as follows: silver n i t r a t e was converted to silver hydroxide with a stoichiometric a m o u n t of s o d i u m hydroxide. After the silver hydroxide was t h o r o u g h l y w a s h e d w i t h water, it was mixed with a slight excess of 60 % perchloric acid. The colorless solution w a s filtered t h r o u g h a sintered disk and t h e n slowly c o n c e n t r a t e d over sulfuric acid, in the dark, until c r y s t a l s formed. The silver perchlorate was recrystallized t w o or three times from a small * O b t a i n e d from the I s s a q u a h Fish H a t c h e r y t h r o u g h the c o u r t e s y of the \ V a s h i n g t o n State I ) e p a r t m e n t of Fish and Game. ** C9.~a h e p a t o c a r c i n o m a (obtained f r o m Roscoe B. J a c k s o n Memorial L a b o r a t o r y , Bar H a r b o r , Maine) w a s carried b y serial t r a n s p l a n t a t i o n in C57 leaden mice. Re/erences p. 5 6 L
552
C . F . EMANUEL, I. L. CHAIKOEE
VOL. 2 5
(1958)
TABLE II ANALYTICAL
PROPERTIES
NaDNA source Pink ( H u m p b a c k ) s a l m o n s p e r m (Oncorhynchus gorbuscha) King (Chinook) s a l m o n s p e r m (0. lshawytscha) ( ' h u m (Dog) s a l m o n s p e r m (0. keta) Sockeye (Red) s a l m o n s p e r m (0. nerka) Silver (Coho) s a l m o n s p e r m (0. kisutch)
OF
DNA
SAMPLES
N (%)
P (%)
NIP
15.61
8.55
1.83
i4.78
8.15
1.81
15.19
8.25
1.84
15.1o
8.25
1.83
15.42
--
--
a m o u n t of hot w a t e r until a 20 % solution of it h a d an a b s o r b a n c y at 250 millimicrons of 0.25 or less. The B e c k m a n D U s p e c t r o p h o t o m e t e r was tested b y s t a n d a r d procedures 1~ for w a v e l e n g t h position and a b s o r b a n c y intensity. Deviations in a b s o r b a n c y (.4 s) f r o m National B u r e a u of Standards valnes were 1.2 % (low) at 250 m p , 0.6 % (low) at 260 m#, a n d 1.8 % (low) at 280 m/*. The a b s o r b a n c y values (Table I I I ) used in this w o r k were not corrected for these errors. Reproducibility of a b s o r b a n c y readings a n d w a v e l e n g t h settings required the elimination of p a r a l l a x errors in the m a n n e r described b y GIBSON AND BALCOM13. This is p a r t i c u l a r l y i m p o r t a n t for t h e procedure described here since the w a v e l e n g t h settings often fall on very steep p o r t i o n s of the a b s o r p t i o n curves where a small w a v e l e n g t h error will result in a large a b s o r b a n c y error. The w a v e l e n g t h settings finally selected here are n o t the theoretically o p t i m u m ones. A c o m p r o m i s e was necessary to avoid the s h o r t e r w a v e l e n g t h where interference from u n k n o w n foreign materials in nucleic acid h y d r o l y s a t e s was more likely to occur. The molar a b s o r b a n c y indexes (aM) o b t a i n e d from the analytically p u r e p u r i n e s and pyrimidines are given in Table I I I . The p u r i n e values deviate s o m e w h a t f r o m those r e p o r t e d by LORING et al. The r e a s o n s for this are n o t known. However, e q u a t i o n s derived from our a b s o r b a n c y values gave calculated p u r i n e concentrations, m u c h closer to k n o w n values for a s t a n d a r d m i x t u r e of p u r i n e s t h a n did the e q u a t i o n s given by LORING et al. TABLE III MOLAR ABSORBANCY
Compound
Thymine Cytosine Guanine Adenine
INDEXES
(aM)
FOR
DNA
PURINES
AND PYRIMIDINES
Wavelength 250 ml~
262 ml~
280 ml~
51 oo 2976 ---
~ -7842 1294 °
4030 9600 6866 4608
I I I . GENERAL EQUATIONS FOR TWO-COMPONENT DIFFERENTIAL SPECTROPHOTOMETRY I t was our original i n t e n t i o n to c a r r y out a f o u r - c o m p o n e n t differential analysis of D N A h y d r o lysates, and e q u a t i o n s for such an analysis were developed. H o w e v e r , because of the close overlapping of the a b s o r p t i o n curves for adenine, guanine, cytosine, and t h y m i n e , the precision obtained with such e q u a t i o n s w a s p o o r for several selected w a v e l e n g t h sets tested. F o r this reason we resorted to a t w o - c o m p o n e n t analysis of the t y p e described b y LORING et al. The success of such an analysis dcpeuds upon a q u a n t i t a t i v e separation of the m i x t u r e into t w o pairs of substances, tile purines aml tile pyrinlidines. Solutions to the required s i m u l t a n e o u s e q u a t i o n s yielded the fl)llowing general e q u a t i o n s for a n y t w o - c o m p o n e n t m i x t u r e . Re/erences p. 561.
VOL. 2 8
(I958)
SPECTROPHOTOMETRY OF PURINES AND PYRIMIDINES
553
General e q u a t i o n s / o r determining the concentration o / a n y p a i r o / c o m p o u n d s , x and y
1)., 2
(euw2)(Dwl)
C~ ~
euwl
(i)
(e.wl) (e~2)
exzt,2
eyw 1
(ea:w2) (-DwI)
D w2
ex.wl
Cv =
(2)
(ezw2) (eyw1) exwl
eyw2
D is a b s o r b a n c y (As) w I is w a v e l e n g t h one w 2 is w a v e l e n g t h two x is c o m p o u n d one y is c o m p o u n d two ex is m o l a r a b s o r b a n c y (aM) of c o m p o u n d x eu is m o l a r a b s o r b a n e y (aM) of c o m p o u n d y C~ conc. of c o m p o u n d x in m o l e s p e r liter C v conc. of c o m p o u n d y in m o l e s p e r liter ' S u b s t i t u t i o n of t h e m o l a r a b s o r b a n c y v a l u e s g i v e n in T a b l e I I I into e q n s . (I) a n d (2) yielded t h e following e q n s . f r o m w h i c h t h e p u r i n e a n d p y r i m i d i n e c o n c n s , in m o l e s p e r liter c a n be determined. C(cytoslne) = [1.38 A s (280 m t ~ ) - 1.O9 A s (250 mr*)] " 10-4 C(thymlne) ~
[2.59 A s (250m/*) - - 0.804 A s (280 m/~)] " 10-4
(4)
C(guanlne) =
[2.45 As (280m/~)-- 0-875 A s (262mp)] " 10-4
(5)
C(adenine) = [ I . 3 0 A s (262m/,) - - 1.48
A s (280 m/*)] " 10-4
IV. PROCEDURE FOR ANALYSIS OF
DNA
A flow sheet outlining the analytical operations is given in Fig. I. I0 m g D N A , h y d r o l y z e a t i 0 0 ° for I h wifll 2oo ~ I0 N HC104
f A d d 5.o m l H 2 0 , m i x , c e n t r i f u g e
I
I I.O m l s u p e r n a t a n t d i l u t e d to 5 ° m l . Determine absorbancy a t z62 a n d 280 nap. (Total a b s o r b a n c y )
Supernatant
--1 i . o m l s u p e r n a t a n t (in duplicate) is t r e a t e d w i t h i.o m l IO % AgC104, m i x e d , o.75 N N a O H a d d e d ( ~ 0.55 ml) to b r i n g p H to 2.0 to 2. 7. C e n t r i f u g e
Silver-purine precipitate
2.o m l a l i q u o t s (in duplicate), acidified a n d d i l u t e d t o 25 ml. Determine absorbancy a t 250, 262 a n d 280 m/*. (Pyrimidine absorbancy) Subtract pyrimidine absorbancy from total a b s o r b a n c y a t 262 a n d 28o rap. (Purine absorbancy) Fig. I. F l o w s h e e t of a n a l y t i c a l o p e r a t i o n s . Re]erences. p. 5 6 L
(3)
(6)
554
c.F.
EMANUEL, I. L. CHAIKOFF
VOL. 2 8
(1958)
Nucleic acid hydrolysis About IO mg of D N A were weighed in a I5-ml conical centrifuge tube, and 2oo 2 of IO N perchloric acid* were added. After a few minutes, the charred mass was well mixed by careful rotation, and kept at room temperature for 45 rain until the nucleic acid was completely dissolved. The tube was stoppered, and heated for one hour at IOO°. (The charred mass should not be disturbed during heating.) A blank containing only perchloric acid was similarly heated. After the tubes were cooled in a water bath, exactly 5.0 ml of water were added to each tube, and the charred mass was triturated with a stout glass rod.
Nucleic acid analysis The stoppered tubes were centrifuged. I.O ml of the hydrolysate supernatant was removed with a syringe** and transferred to a 5o-ml volumetric flask. After dilution to volume, the total absorbancy was determined, at 262 m/z and 280 m/z, against a properly prepared blank. Two other I.O ml aliquots of the hydrolysate were transferred to 5-ml beakers. Another beaker was prepared with I.O ml of the heated blank solution. To each beaker was then added i.o ml (dispensed with another syringe) of a freshly prepared, IO % solution of silver perchlorate. Mixing was done with a small glass rod. A sufficient a m o u n t of 0.75 N N a O H ( ~ 0.55 ml) was then added (with stirring) to each beaker to bring its contents to p H 2.0 to 2. 7 (checked on a p H meter with the outer surfaces of the electrodes carefully dried to avoid introducing volume errors). (Since the total volume of the m i x t u r e - - i n this case 2.55 m l - - m u s t be known, all volumes added must be measured with care.) The bulk (not quantitatively) of each solution was poured into a dry, conical centrifuge tube (15 ml). The tubes were stoppered and immediately centrifuged. E x a c t l y 2.0 ml of the supernatants, containing the pyrimidines alone, were transferred (by a 2 ml transfer pipette) to 25-ml flasks. After addition of one drop of concentrated perchloric acid, the flask contents were diluted to volume. The absorbancies of these solutions were determined against an appropriate blank at 250, 262, and 280 m/z. These data, representing the pyrimidines alone, were used in eqns. (3) and (4) to determine the pyrimidine content after appropriate dilution corrections. Subtraction of the pyrimidine values (at 262 and 280 m/z) from the total absorption values (at 262 and 28o m/z) gave the absorption derived from purines which was used in eqns. (5) and (6). Although light caused the silver purine precipitate to become mauve-colored, this was not deleterious, and was disregarded.
V. TEST OF METHOD WITH A PURINE AND PYRIMIDINE STANDARD
I. Effect o / p H on the quantitative separation o~ a known base mixture by means o~ silver perchlorate It was necessary to determine the o p t i m u m p H at which a silver perchlorate * The exact strength of this acid was 9.737 N. ** i c c B-D Tuberculin syringe, smallest division, o.oi cc. We have disregarded the difference between cubic centimeters and milliliters.
Re/erences p. 56J:.
VOL. 28 (z958)
SPECTROPHOTOMETRYOF PURINES AND PYRIMIDINES
555
solution precipitates purines quantitatively from solution without at the same time precipitating the pyrimidines. Equimolar mixtures of adenine and guanine were precipitated with silver perchlorate reagent at various p H values. The absorbancy of the supernatants obtained by this treatment was measured against appropriate blank solutions at 262 m/~. Similarly, standard equimolar solutions of pyrimidines (thymine and cytosine) were subjected to the same treatment, and their optical absorbancies were determined at 280 mt~. Fig. 2 shows the result of such experiments in terms of the percentages of heterocyclic absorption still remaining in the supernatants after centrifugation of precipitates that formed. The data obtained in this manner are not precise since most of the absorbancy values fall at the extremes of the absorbancy range. However, it is clear from Fig. 2 that the optimum pH range for purine precipitation lies approximately between p H 2.0 and 2. 7. Optimum pH
,oo
o
[
c
~
r
~
Pyrlmldmes •
1"
'tI g~, [P.rines \~< ~40k
I
2oF
[
•
1/
~~
o
•
1 I
2
,
3
4
,/
- ; 1 ;
pH
Fig. e. Optimum pH for precipitation of purines with silver perchlorate.
2. 'Quantitative separation o[ a standard purine-pyrimidine mixture In Table IV, typical data are given showing the quantitative separation of a known mixture of purines and pyrimidines at p H 2.1 to 2.2. Part A shows the total absorption of the four-component mixture at two wavelengths. These data were obtained in triplicate and are presented with their respective absorbancy ranges. Part B shows the total pyrimidine absorption of a similar standard mixture of pyrimidines (thymine and cytosine), at three wavelengths. Part C gives the absorbancy values found for the supernatant obtained after the same standard mixture of four bases that was used in Part A was treated with silver perchlorate at p H 2.1 to 2.2. A quantitative separation is obtained if the absorbancy values of Part C (supernatant) are identical with those values shown in Part B. Similarly, the absorbancy calculated for the purines (Part D) should equal the absorbancy found (Part E). The data in Table IV show that a quantitative separation of purines from pyrimidines is achieved under the conditions outlined in section IV within the limits of manipulative and spectrophotometric error demonstrated below.
Re/erences p. 56z.
556
C . F . EMANUEL, I. L. CHAIKOFF
VOL. 2 8 ( 1 9 5 8 )
TABLE IV EXPERIMENTAL AND STANDARD ABSORBANCYVALUES OBTAINED FROM THE QUANTITATIVE SEPARATION OF A STANDARD BASE MIXTURE A bsorbancy values 250 ml*
262 mlz
280 ml*
Part A T o t a l a b s o r b a n c y of a g u a n i n e - a d e n i n e thymine-cytosine standard
0.943 ~ o . o o 3 "
0.690 ~ 0.007
Part B A b s o r b a n c y of a s t a n d a r d s o l u t i o n of p y r i m i d i n e s ( t h y m i n e + c y t o s i n e ) of s a m e c o n c e n t r a t i o n s as in P a r t A o.241 4 - 0 . 0 0 3
o.451 ~ o . o o 3
o.419 ± o.oo2
Part C (pyrimidines) A b s o r b a n c y of s u p e r n a t a n t a f t e r a d d i t i o n of AgCIO 4 t o t h e s o l u t i o n u s e d in P a r t A
0.456 ± 0.003
o.421 ± 0.005
Part D S t a n d a r d v a l u e s for p u r i n e s (i.e., d a t a of P a r t A m i n u s d a t a of P a r t B)
0.492
0.272
P a r t E (purines) Total absorption minus supernatant ( p y r i m i d i n e ) a b s o r p t i o n (i.e., d a t a of P a r t A m i n u s d a t a of P a r t C)
0.487
0.269
0.248 ± 0.004
* M a x i m u m r a n g e of a b s o r b a n c y v a l u e s .
3. Precision and accuracy o/recovery o/bases in a standard purine-pyrimidine mixture An approximately equimolar mixture of adenine, guanine, cytosine, and thymine (2.4" IO-s moles of each base per ml) was used to carry out a complete analysis as described in section IV. Particular care was taken to minimize random volumetric errors. The p H of purine precipitation was 2.0. Six separate determinations gave the absorbancy results shown in Table V. I t is evident from these data that the m a x i m u m deviation in accuracy and precision is ± 0.8%. TABLE V PRECISION
AND
ACCURACY
OF THE
PURINE AND PYRIMIDI~/E SEPARATION PROCEDURE Pyrimidine ahsotbancy
250 ml2
Expected* F o u n d (av erag e)** Error
-
-
0.245 0.244 zt- 0.002 0. 4 %
262 ral*
280 mlz
0.452 0.455 4- o.ooi + 0. 7 %
o.419 0.420 :~ 0.002 + 0.2 %
Purine absorbancy
Expected* F o u n d (average)** Error
250 ml*
262 ml*
280 ml~
----
o.497 o.495 ± o.oo2 - - 0. 4 %
o.275 o.274 ~ o.ooi - - 0. 4 %
* E x p e c t e d on t h e b a s i s of t h e m o l a r a b s o r b a n c y coefficients (Table I I I ) a n d k n o w n concentrations. ** Six s e p a r a t e d e t e r m i n a t i o n s . R e [ e r e n c e s p . 562-.
(1958)
VOL. 2 8
SPECTROPHO'IOMETRY OF PURINES AND PYRIMIDINES
557
VI. TEST OF METHOD WITH NUCLEOTIDES AND NUCLEIC ACID 1.
Hydrolysis o/'nucleotides and nucleic acids
To determine whether perchloric acid degrades purines and pyrimidines of nucleic acids, we exposed the crystalline deoxyribonucleotides to varying concentrations of perchloric acid for ~ h, at IOO°, in stoppered tubes. Approximately 1.2. lO -5 moles of each nucleotide were hydrolyzed with i o o ~ of perchloric acid of varying strengths (i to 12 N). The absorbancies of the hydrolysates were then determined at two wavelengths. The results of these experiments are given in Fig. 3(a). The theoretical (calculated) absorbancies are indicated as dashed horizontal lines. It would seem, from these experiments, that i o o 2 of 5 or 6 N perchloric acid are optimum for the recovery of the expected absorbancy. When these hydrolysates were chromatographed on paper, it became evident that acid strengths of 3 N or stronger
0.9 0.8
--
Q2~'~
DEOXYCYTIDYLICACID
_7
0.4 ~,,,~_ __ 2aom~ 02 o
.------
D~OXYTHYMIOYLIC ACID
0.7
O.E
I
>, Q=
DEOXYGUANYLIC ACID
~
2
6
2
o
g
I.
O~
~
•
.
.
.
.
.
.
DEOXYADENYLIC ACID
4 6 8 fO HGI04 Normality
12
I00 200 300 400 500 Micr0~iters 12N HCIO4 per 12xlO "5 f~iole of Nucleofide
Fig. 3 (a) and Fig. 3 (b). The effect of perchloric acid on the absorbancy
of deoxyribonucleotides.
02
I
I
~
I
I
2
3
4
5
6 7 8 9 HC[O4 N0rmol;ty
f
#
I
I
I
I
I0
II
12
m
Fig. 4- T h e effect of v a r i o u s s t r e n g t h s of h o t perchloric acid on t h e optical a b s o r b a n c y of D N A .
caused extensive hydrolysis of the glycosidic bonds. I and 2 N perchloric acid solutions were not very effective in breaking the pyrimidine glycosidic bonds, and considerable amounts of thymidine and cytosine deoxyriboside were left in the hydrolysates. Fig. 3(b) shows the results of experiments in which the strength of the perchloric acid was kept constant, but the volume of acid for each 1.2. IO-s mole of nucleotide was increased from 30 2 to 500 2. In these experiments it was found that excessive perchloric acid caused extensive destruction of both purines and pyrimidines, but particularly the latter. These results indicate that ioo k of perchloric acid per lO -5 mole of nucleotide is about the m a x i m u m amount of 12 N HC104 which will give acceptable recoveries. It is also evident that deoxyribose degradation products contribute no extraneous absorption to the hydrolysate in the region of higher acid strength. In Fig. 4 the results of nucleic acid hydrolysis by different perchloric acid strengths are presented. In these experiments, 4.15 mg of DNA were hydrolyzed with IOO 2 of Re/erences p. 56z.
558
C.F.
E M A N U E L , I. L. C H A I K O F F
VOL. 2 B
(1958)
perchloric acid solution, at IOO°, for 30 rain. In the complete hydrolysis of nucleic acids, three bonds, at least, must be hydrolyzed (for each nucleotide) instead of two bonds as in a free nucleotide. It seems likely that a larger amount of acid per nucleotide will be necessary for nucleic acid hydrolysis than for the free nucleotide. Since the theoretical absorption of a nucleic acid hydrolysate cannot be accurately calculated, it is difficult to determine from Fig. 4 the optimum acid concentration for DNA hydrolysis. However, an examination of these hydrolysates b y paper chromatography revealed that, besides the expected purines and pyrimidines, there were three new components, one with a bright blue fluorescence at RF 0.48 (isopropyl alcohol-water-HC1) ; a second at RF 0.34; and a third at Rv 0.69. The first, which is identical with the LEVY-SNELLBAKER14 substance, gave an orange-tan color with diazotized sulfanilic acid; the second gave a purple-pink color; and the third, an orange color, with this reagent. These substances were especially evident in the hydrolysates produced by intermediate acid strengths (3 N to 8 N), and were absent from the very weak and strongly acid hydrolysates. Cytosine deoxyriboside and thymidine were also evident in the I N and 2 N perchloric acid hydrolysates. I t is not known whether these unknown components are artifacts arising from an intermediate stage of hydrolysis of DNA or are actually integral components of nucleic acids destroyed at the higher acid concentrations. They were present in considerable quantities in nucleic acids obtained from salmon sperm, from mouse liver, and from mouse hepatocarcinoma. Attempts are now being made to isolate them. I t seems probable that the significantly higher absorption observed in the 4 N to 8 N acid hydrolysates is due to the absorption and stray light (fluorescence) contributed b y the unknown materials. Unfortunately, the above experiments do not clearly define the optimum conditions for hydrolysis of nucleic acids. The absorption curves indicate, however, that at least IO N acid is necessary. An incomplete survey of the perchloric acid hydrolysis conditions recommended by other workers is given in Table VI. TABLE VI MICROLITERS OF PERCHLORIC ACID RECOMMENDED FOR EACH IO m g D N A Mieroliters o] acid
143 52
200 8o-15o
Normality
12 12
7.5 12
Literature
15 16
17 I8
2. D N A nitrogen-phosphorus recovery and base ratios obtained under varying conditions o] hydrolysis The criterion for optimum hydrolysis can be either nitrogen recovery, as calculated from the determined purine-pyrimidine content, or phosphorus recovery if one assumes that a strict one-to-one ratio exists between moles of base and moles of phosphorus. DNA samples of io.o mg each (isolated from hepatocareinoma tissue) were hydrolyzed for 1/2 h, at IOO°, with several different strengths of perchloric acid (20o ;~). Purine precipitation with silver perchlorate was carried out at p H 2.o. The results Relerences p. 56L
VOL. 2 8
(1958)
SPECTROPHOTOMETRY
OF PURINES
AND PYRIMIDINES
559
of these experiments are given in Table VII. These data show that hydrolysates prepared with dilute acid contain, in addition to the four desired purines and pyrimidines, other products, presumably of an intermediate stage of hydrolysis. Thus, the calculated nitrogen and phosphorus values (upper parts of columns 9 and io, Table vii) based on eqns. (3), (4), (5), and (6) deviate very widely from the direct nitrogen and phosphorus determinations. It is evident that the use of either IO or 12 N perchloric acid yields a quantitative base recovery and nearly theoretical base ratios (lower parts of columns 6 to IO, Table VII). However, further investigation showed that 12 N perchloric acid sometimes resulted in aberrant base ratios, and could not always be relied upon to yield complete recoveries. The reasons for this are not well defined, but it is certain that base destruction can occur with some nucleic acid samples under what appears to be inadequately controlled hydrolysis conditions. Because of this difficulty, an attempt was made to define the optimum conditions for hydrolysis with IO N perchloric acid. From the data shown in Table VIII, it was TABLE
VII
E F F E C T OF H C I O 4 STRENGTH ON HETEROCYCLIC EASE RECOVERY AND RATIOS Perehloric acid normality
Cytosine
Thymine
4 5 6.5 7 8 IO 12
I3.O 12. 9 14.4 15-3 16.8 19.6 21.2
29.2 32.1 35.4 37.6 36. 5 29.6 29.6
Adenine
Guanine
21. 3 19. 3 2o.6 22.4 25. 3 30.3 28.8
Cytosine
Adenine
Purines
Guanine
thymine
Pyrimidines
Moles o/ phosphorus acc'd /or*
Nitrogen acc'd /or (rag) ~
0.356 0.362 0.489 0.622 o.791 0.96 I.O 4
1.37 1.66 1.72 1.68 1.44 o.98 1.o2
1.38 1.22 I.OI o.89 0.87 1.o3 1.o 3
2 . 9 0 " IO - s 2 . 8 2 . lO -6 2 . 8 3 • IO - s 2 . 6 7 • IO -8 2 . 4 6 . lO 4 2 . 4 6 " IO s 2 . 4 6 . IO s
o.152 o.197 o.145 o.134 o.122 o.128 o.127
28.6 35.8 29.4 24-2 21. 3 2o.5 20. 4
D i r e c t d e t e r m i n a t i o n of p h o s p h o r u s D i r e c t d e t e r m i n a t i o n of n i t r o g e n
2.47" io
s o.i 264
* P h o s p h o r u s and n i t r o g e n " a c c o u n t e d for" were calculated on t h e basis of the mole c o n t e n t s g i v e n in c o l u m n s 2, 3, 4, a n d 5** M o l e s of base × i o ~ [ m g N a D N A . TABLE
VIII
EFFECT OF DIGESTION TIME ON HETEROCYCLIC BASE RECOVERY (IN MOLES OF HETEROCYCLIC BASE PER m g N a D N A ) Digestion limes at too ° !~ h
Cytosine Thymine Adenine Guanine
lO -8 lO -8 IO - 8 IO-S
5.81" 7.o8. 7.26" 5.85 .
lO -8 lO -8 IO -8 IO-S
2h 5.73" 6.8o. 7.38. 5.31"
lO-8 io s IO -8 IO-8
,? h 5 .8o" 6.5o" 7.22" 5.76.
IO-S lO -8 lO -8 lO-8
Total
26.6
• io -s
26.0
• lO -8
25.2
• I n -8
25. 3
• lO -8
Calculated
25. 9
• IO 8
25.9
. lO-8
25.9
. lO-8
25.9
, lO-8
Adenine Thymine Guanine Cytosine Purines l'yrimidines Relerences
5.85" 7.23. 7.76" 5.73"
I h
p. 5 6 I .
t .07
0.99
i.o 7
I.I i
o.98
i .oo
0.93
i.oo
t'°3
I.O2
I.O[
I.O6
560
C. F. E M A N U E L , I. L. C H A I K O F F TABLE MOLE-0/o C O M P O S I T I O N
(1958)
NUCLEIC
ACIDS
IX
OF SALMON
Adenine
Guanine
Purines
Cytosine
Thymine
Adenine
Guanine
Thymine
Cytosine
Pyrimidines
Silver salmon
21.6
27.6
28.8
21.8
1.o 4
I.Oi
1.o 3
2 . 4 8 . lO -5
Chinook salmon
22. 4
27.2
27. 9
22. 5
0.99
i.oo
1.o2
2 . 5 9 . i o -~
Chum salmon
21.9
27.6
28.7
21.9
i,oi
I.()o
1.o2
2 . 3 7 . i o --~
Sockeye salmon
21.8
27.8
28.2
22.0
i .oi
i.ot
i.oi
2 . 5 9 - IO-"
Humpback
22.6
27.5
27.7
22.5
i,o'i
i.oi
i.oi
2.52" IO-"
Sperm sample
salmon
Mote-per cent o] reccmeredbase
VOL. 2 8
Motes phosphor per sro mg DN
decided that optimum conditions obtained when IO mg of DNA were hydrolyzcd with 2o0 ~t of IO N perchloric acid, at IOO°, for I h. Longer periods of digestion caused noticeable destruction of thymine. V I I . A P P L I C A T I O N OF P R O C E D U R E TO A N A L Y S I S OF SALMON S P E R M N U C L E I C ACID
The conditions given in section IV were applied to the analysis of sperm nucleic acids obtained from five species of Pacific Ocean salmon !]'able IX). These data show that unitary base ratios and nearly complete base recovery (98-1oo%) are achieved. VIII. DISCUSSION
The reliability of a procedure for the analysis of purines and pyrimidines of nucleic acids is generally based on nitrogen or phosphorus recoveries. Unfortunately, a check based on these constituents is not completely satisfactory. An accounting of all the nitrogen in terms of the four constituent bases is not possible, usually because almost all nucleic acid samples contain small amounts of foreign nitrogen, mainly protein,_ which is very difficult to remove. For this reason we have not, as a rule, relied upon total nitrogen analyses. Recoveries based on total phosphorus are likewise unsuitable because two assumptions must be made: (I) that no foreign phosphorus-containing substances (e.g., RNA or phospholipids) are present; and (2) that nucleic acids contain, according to theory, a strict one-to-one ratio between moles of heterocyclic base and phosphorus. Few, if any, nucleic acid preparations of mammalian origin have been entirely free of ribonucleic acid. In the second assumption, it is implied that no triply esterified or secondary phosphoryl groups are present. It is well established that fish sperm nuclei contain very small amounts, if any TM, of ribonucleic acid, and the protamine associated with the nucleic acid is readily eliminated by standard procedures. Hence, fish sperm nucleic acids are well suited for testing the quantitativeness of base recoveries. Table IX gives a complete purine and pyrimidine recovery, based on phosphorus analyses, for sperm nucleic acids of five species of salmon. The recoveries shown in the last columnranged from 98 to lOO%. The strongest argument in favor of the WATSON-CRICK model for DNA structure depends upon the adenine-thymine and guanine-cytosine ratios. According to this model, the integrity of the double-stranded DNA molecule depends upon hydrogen bonding between the base pairs that occupy adjacent positions in opposing nncleotide chains. Although such pairing requires that the ratios be unitary, reports of large Re]erences p . 5 6 z .
VOL. 2 8
(1958)
SPECTROPHOTOMETRY OF PURINES AND PYRIMIDINES
561
deviations are still appearing. Because of our initial failure to obtain values of unity for the ratios, we were led to reinvestigate the hydrolysis procedure. It now seems certain Total moles Per cent recovered (basedon phosphorus) that non-unitary ratios obtained in our earliest experiments base/ound resulted from base destruction (particularly of thymine) 2.47" IO -5 IOO during hydrolysis. In this connection it is important to note °-.60" IO -~ IOO that the analyses which gave unitary ratios also gave the most quantitative recovery in terms of total nitrogen or 2-34" IO-5 99 phosphorus. We therefore believe that ratios deviating more 2.54" lO -5 98 than a few % from unity are the result of selective destruc2.51. IO -5 IOO tion of bases during hydrolysis. On the whole, our analyses on sperm nucleic acids do not conflict with the WATSONCRICK theory. The quantitative recovery obtained by our procedure suggests that the three unknown substances that we found in hydrolysates prepared with perchloric acid of intermediate strengths are actually artifacts arising during nucleic acid degradation. If these substances are real D N A components, they must be either triply esterified to phosphorus or united to the nucleic acid in some other way which does not involve phosphorus at all. Although the latter alternatives seems unlikely, we are now attempting to find these unidentified substances in enzymic hydrolysates of nucleic acids. SUMMARY I. A s p e c t r o p h o t o m e t r i c m e t h o d for the analysis of n u c l e i c a c i d purines and p y r i m i d i n e s is d e s c r i b e d . D a t a w h i c h illustrate the o p t i m u m c o n d i t i o n s for p e r c h l o r i c a c i d h y d r o l y s i s of D N A are presented. Heterocyclic base recovery in terms of phosphorus or nitrogen is 9 8 - 1 o o % c o m p l e t e . 2. P u r i n e a n d p y r i m i d i n e c o n t e n t s of five species of s a l m o n s p e r m D N A w e r e s t u d i e d . H y d r o l y s i s c o n d i t i o n s which gave complete recovery in terms of phosphorus also y i e l d e d unitary values for the following base ratios: adenine/thymine, guanine/cytosine, and p u r i n e / p y r i m i d i n e .
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Received October 28th, I957