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Reactions of glyoxal with nucleic acids, nucleotides and their component bases
23
BIOCHIMICA ET BIOPHYSICA ACTA
g g A 95907
REACTIONS OF GLYOXAL W I T H NUCLEIC ACIDS, NUCLEOTIDES AND T H E I R COMPONENT BASES K A Z U Y A S U N A K A Y A , OSAMU T A K E N A K A , H I R O O H O R I N I S H I AND K A Z U O S H I B A T A
The Tokugawa Institute [or Biological Research, Me]iromachi, Tokyo", and Tokyo Institute o[ Technology, Meguroku, Tokyo (Japan) (Received J a n u a r y 3oth, 1968)
SUMMARY
The reactions of glyoxal with nucleic acids, nucleotides and their bases were examined by measuring spectral changes. The spectra of all of the bases and nucleotides in RNA and DNA were changed appreciably by treatment with a high concentration of glyoxal. When treated with a low concentration of glyoxal, however, only guanine or guanylic acid underwent a specific spectral change. The reaction of glyoxal with this base or its nucleotide proceeded to completion within 30 rain between pH 8. 3 and 9.8 as estimated from the spectral change at room temperature. This phenomenon was applied for determination of the guanosine content of nucleic acids and the results agreed well with previous data obtained by chromatography. As judged from the spectral change, glyoxal did not react with native calf thymus DNA, while it reacted considerably with heat-denatured DNA. The spectral change obtained with denatured DNA was a composite of two qualitatively different changes, the change being due solely to the reaction between glyoxal and guanosine residues and the hyperchromic shift of the 26o-m# band, which may be a secondary effect of the reaction. From the analysis of these two effects, the degree of reaction of the guanosine residues in denatured DNA was estimated to be 72 %.
INTRODUCTION
It was demonstrated in a previous study I that glyoxal specifically modifies the guanidyl group of arginine residues in proteins in aqueous solution. For example, the II arginine residues in the lysozyme molecule reacted to the extent of approx. 80 % with this reagent, while other amino acids including amino groups of lysine were not appreciably modified as determined by amino acid analysis of the hydrolyzate of the modified protein. This is in contrast with high reactivity found with amino groups and much lower reactivity with arginine residues of formaldehyde z. Such a specificity of glyoxal stimulated the authors to use glyoxal in the present study with nucleic acids, nucleotides and their component bases or nucleosides. A highly specific reaction of glyoxal with guanine, guanosine or guanylic acid in a weakly alkaline solution at room temperature was thus found spectrophotometrically, and the spectral change due to this reaction was applied to determination of the guanine contents * P o s t a l address.
Biochim. Biophys. Acta, 161 (1968) 23-31
24
K. NAKAYAet al.
of several kinds of RNA and DNA and to examination of the difference in reactivity between guanine groups in native DNA and those after denaturation b y heat treatment. Described herein are the results obtained in these experiments.
EXPERIMENTAL
Materials An aqueous solution of 4o % glyoxal was obtained from Tokyo Kasei Kogyo Co. Herring sperm DNA was purchased from Sigma Chemical Co., and calf thymus DNA was prepared b y the method of KAY,SIMMONSAND DOUNCE3 and recrystallized three times. Sodium ribonucleate of yeast as well as purines, pyrimidines and ribonucleotides were purchased from Tokyo Kasei Kogyo Co. The concentrations of bases, nucleosides and nucleotides were determined spectrophotometrically a. The molar extinction coefficients at 260 m# for native and denatured DNA were assumed to be 6000 and 8500 cm -1 • M -1, respectively.
Procedure An aqueous solution (i ml) of glyoxal was added to the mixture of a sample solution (i ml) of nucleotide, nucleoside or base and 0.5 M bicarbonate-NaOH buffer (3 ml) at p H 8.6. The p H value of the reaction mixture increased slightly to about 8. 9 on the addition of glyoxal and therefore was adjusted to exactly 8. 9. In an experiment designed to show the effect of p H on the reactivity of glyoxal, the above buffer was replaced either by the same buffer of different p H values, to obtain mixtures above p H 8.6, or b y 0.2 M phosphate buffer, to obtain mixtures below p H 8.6. The reaction mixture was left standing at room temperature ( I 5 ~ 5 °) for 2 h and then the absorption spectrum was observed with a Shimadzu recording spectrophotometer model SV-5o, using I.o-cm cells. The same reagent solution without the sample was used as the reference in this measurement.
RESULTS
Reactions o~ glyoxal with the nucleotides o/ R N A The spectrum of guanylic acid was markedly changed by treatment with glyoxah Curve A in Fig. I is the spectrum of 42.4/,M guanylic acid at pH 8.9 and Curves B, C and D are the spectra of this sample treated at the same p H with 0.08, 0.32 and 1.88 mlV[ glyoxal, respectively. By this treatment, the band of guanylic acid at 250 m/~ was enhanced and shifted to 248 m/~ and, in addition, the shoulder around 270 m/~ was lowered but was more pronounced. Curve D is regarded as the spectrum of the reaction product because no further spectral change occurred above 1.88 mM glyoxal. Clear isosbestic points were observed at 254 and 284 m/~ in this spectral change, which indicates that no side reaction is involved in the reaction. Curve G in Fig. 2 is the difference spectrum for this reaction which was calculated from Curves A and D in Fig. I. The spectrum shows a negative band at 269 mt,. Biochim. Biophys. ~tcta, I 6 I (x968) 23-31
REACTIONS OF GLYOXAL WITH NUCLEIC ACIDS r
,
,
0£
Ouanylic
25
i
acid
'
OI
'C ,'~ "',\
A
[
o~
<3 2~ <~
02.
-01 1
/
0 230
250
270 290 Wavelength in m~
I
2~0
310
250
I
I
270 290 Wavelength in mp
310
Fig. i. T h e s p e c t r a l c h a n g e of 42.4 btM g u a n y l i c acid t r e a t e d w i t h g l y o x a l a t p H 8.9. C u r v e A, t h e s p e c t r u m before t r e a t m e n t ; C u r v e s B, C a n d D, t h e s p e c t r a o b t a i n e d b y t r e a t m e n t with 0.08, 0.32 a n d 1.88 m M g l y o x a l for 2 h, r e s p e c t i v e l y . Fig. 2. Difference s p e c t r a before a n d a f t e r t r e a t m e n t of 42. 4/zM n u c l e o t i d e s w i t h glyoxal. C u r v e G, t h e s p e c t r u m o b t a i n e d o n t h e t r e a t m e n t of g u a n y l i c acid w i t h 1.88 m M glyoxa]. C u r v e s A, C a n d U, t h e s p e c t r a o b t a i n e d w i t h 145 m M g l y o x a l for adenylic, cytidylic a n d uridylic acids, respectively.
The effect of pH was examined with 4.o mM glyoxal for 47.8/zM guanylic acid. The drop (--AA) of absorbance at 269 m # after 2 h of incubation is plotted against pH in Fig. 3, which indicates that the reaction proceeds to completion between pH 8.3 and 9.8. Curve A in Fig. 4 shows the degree of reaction plotted against glyoxal concentration which was observed after 2 h of incubation of 42.4/zM guanylic acid with the reagent at pH 8. 9. The curve rises uniformly and reaches a plateau of - - A A ~ o.134 at 2.2 mM glyoxal. The spectral change at such an excess concentration of glyoxal was completed after 30 rain of incubation and stayed constant at least for 3 days. The reaction time in further experiments was therefore fixed at 2 h.
i
i
r
i
L
0.15 ,
Ol5
i B
~
~ OlO c~
Olyoxal
I
I
7
8
X
f
•
---
0.10 p //
i0.05
I
9
pH
I~3
It~
o
Glyoml Concentralionin mM
Fig. 3. T h e effect of p H on t h e degree of r e a c t i o n of 47.8/zM g u a n y l i c acid b y 4.o m M g l y o x a l a f t e r 2 h of i n c u b a t i o n , as e s t i m a t e d f r o m t h e lowering of a b s o r b a n c e a t 269 m F. Fig. 4. T h e v a l u e s of --z]A o b t a i n e d a t pI-I 8.9 for 42.4 # M g u a n y l i c acid (Curve A), a n equim o l a r m i x t u r e of 42.4/zM n u c l e o t i d e s (Curve B) a n d 6 5 . 1 / , M g u a n i n e (Curve C) p l o t t e d as a f u n c t i o n of g l y o x a l c o n c e n t r a t i o n .
Biochim. Biophys. Mcta, 161 (1968) 23-31
K. NAKAYAet al.
26
From the above value of - - A A , the value of zle (the change of the molar extinction coefficient due to the reaction) was calculated to be 32oo cm -1 • M-1, which was used in fllrther experiments to calculate the moles of guanylic acid reacted with glyoxal. Adenylic and cytidylic acids underwent a spectral change on treatment with glyoxal, but a much higher concentration of glyoxal was required to obtain a measurable spectral change. The difference spectra obtained on the treatment of 42.4 ~M adenylic and cytidylic acids with 145 mM glyoxal are shown by Curves A and C, respectively, in Fig. 2. The spectrum of uridylic acid also changed at this high concentration of reagent but the change took place at shorter wavelengths, below 25o mF, as shown by curve U in Fig. 2. The spectral changes of these 3 mononucleotides were, however, not observable when treated with glyoxal below 4 raM, the concentration being sufficient to cause the change of guanylic acid. This difference in reactivity between guanylic acid and the other 3 mononucleotides in RNA provides the experimental basis for determination of the guanylic acid content of RNA. As a preliminary application, the guanylic acid content in an equimolar mixture of 42.4 ~M guanylic, cytidylic, uridylic and adenylic acids was determined from the spectral change by glyoxal. Curve B in Fig. 4, which is the reaction curve obtained for this mixture, was identical with Curve A in the same figure, the result obtained for guanylic acid alone. TABLE
l
GUANINE
OR G U A N O S I N E
CONTENTS
Gg in mole of g u a n i n e p e r mole of p h o s p h a t e , of R N A or D N A d e t e r m i n e d w i t h g l y o x a l as c o m p a r e d w i t h Ge, t h e c o n t e n t s b e i n g in t h e s a m e u n i t d e t e r m i n e d p r e v i o u s l y b y c h r o m a t o g r a p h y .
Sample
(;~
G¢
Go/G~
0.254
o.246 (ref. 5), o.280 (ref. 6)
lO3.3, 91.9,
o.194
o.193 (ref. 12)
lOO.5
0.203 o o. I59
o.212 (ref. I1)
96.5 o 72.3
(%)
Yeast R N A Hydrolyzate Oligonucleotides
o.226
90.7 80. 7
Herring sperm DNA Hydrolyzate
Call thymus DNA Hydrolyzate Native Denatured
The base composition of yeast RNA has been determined chromatographically by ELSON AND CHARGAFF5 and CRESTFIELD,SMITH AND ALLEN~ as listed in Table I. The above spectral change was applied to determine the guanylic acid content of yeast RNA. The RNA preparation was hydrolyzed with o.3 IV[ NaOH at 37 ° for 18 h according to the method of MOLDANE AND H E I D E L B E R G E R 7, and the hydrolyzate solution was neutralized with HC1 and diluted Io-fold with water. A glyoxal solution was added to this diluted solution, and the absorbance change at 269 my was observed. The phosphate concentration was determined for the same solution by the method of NAKAMURAs after treatment with perchloric acid 9. The change in the molar Biochim. Biophys. Acta, 16i (1968) 23 31
27
REACTIONS OF GLYOXAL WITH NUCLEIC ACIDS
extinction coefficient, AeM, at 269 m/~ per g atom of phosphorous of yeast RNA was calculated from the data to be 8o2 cm -1. M -1. Dividing this value b y 32oo, which is the Ae2n9 value for guanylic acid, we obtained o.254 as the guanylic acid content of yeast RNA, which agrees well with the values shown in Table I.
Reaction o/ glyoxal with the bases o/DNA Guanine also undergoes great spectral changes when treated with glyoxal. Curve A in Fig. 5, which is the spectrum of 65.1/*M guanine, changed to Curves B and C on the treatment with o.45 and 1.8i mM glyoxal, respectively. The difference spect r u m of Curve A minus Curve C in Fig. 5 gave a negative band at 269 m/~ as shown by Curve A in Fig. 6. This difference spectrum is similar to that obtained for guanylic acid (curve A in Fig. 2), especially in the position of the negative band. No spectral change was observed for the other 3 bases of DNA with 1.8I mM glyoxal, although their spectra changed greatly at a higher concentration (145 raM) of glyoxal as seen from Curves A, C and T in Fig. 6. Curve C in Fig. 4 is the reaction curve obtained for 65.1 yM guanine, from which the Ae value at 269 m/, was calculated to be 199o cm -1. M -~. The reaction curve obtained for a mixture of the same concentration of guanine, adenine, thymine and cytosine was identical with that obtained for guanine alone. It is evident from these results that none of the other DNA bases interfere with the determination of guanine in mixtures of these 4 bases.
//
AGuanine
06
Glyoxal
+
/
02 -0.1 270
290
Wavelength .in mp
310
23O
.
//// ~111111~_ ~ /
o
250
.
¢
o~
0 230
/
/
//
OI
<~
/
--~/G [ 250
I 270
Wavelength in mp
I 290
310
Fig. 5. T h e s p e c t r a l c h a n g e of 65.1 # M g u a n i n e t r e a t e d w i t h g l y o x a l a t p H 8. 9. C u r v e A, t h e spect r u m before t r e a t m e n t ; C u r v e s B a n d C, t h e s p e c t r a o b t a i n e d b y t r e a t m e n t w i t h o.45 a n d i .8I m M glyoxal, r e s p e c t i v e l y . Fig. 6. Difference s p e c t r a before a n d a f t e r t r e a t m e n t of 65.1/,1~1 b a s e s w i t h glyoxal. C u r v e G, t h e s p e c t r u m o b t a i n e d w i t h 1.81 m M g l y o x a l for g u a n i n e , a n d C u r v e s A, C a n d T o b t a i n e d w i t h 145 m M g l y o x a l for a d e n i n e , c y t o s i n e a n d t h y m i n e , r e s p e c t i v e l y .
Calf thymus and herring sperm DNA preparations were hydrolyzed with 6 M HC1 at IOO° for 3 h (ref. IO) in order to determine the guanine contents. The solutions were neutralized and diluted lo-fold, and the contents were determined using the Ae269 value obtained for guanine. The results were o.2o3 for calf thymus DNA and o.194 for herring sperm DNA, which agree well with those reported by BURTONn and CHARGAFF AND DAVIDSON TM, respectively, as seen from Table I. It was thus demBiochim. Biophys. -dcta, 161 (r968) 23-31
28
K. NAKAYAel al.
onstrated that the guanine content can be determined easily and precisely by means of the spectral change on treatment with glyoxal. Guanosine, deoxyguanosine and guanosine 5-monophosphate gave spectral changes which were almost identical with that obtained for guanylic acid with respect to the position and the zJe269 value of the negative band as well as the reaction curve, the degree of reaction plotted versus glyoxal concentration. For example, Curve A in Fig. 7 is the reaction curve obtained for 24.0 pM guanosine, which is very much like Curve B for guanylic acid in the same figure. The value of Ae269 for guanosine was 3200 cm -1 • M -1 which agrees with the value obtained for guanylic acid. This agreement between guanosine and guanylic acid and the finding of a considerably lower value described above for guanine indicate that the addition of a phosphate group to the sugar moiety does not affect the Ae2~0 value but the addition of a sugar, ribose or deoxyribose, to the 9th nitrogen atom of purine does affect the Ae269 value.
0.08 i
= o.o6~
i
~ _
A
i
i
~
.
0
0 0
o
2 I
.
~ 2
3
GlyoxalConcenlmfioinmM
Fig. 7. The values of --zlA obtained for 24.0/~M guanosine (Curve A), the s a m e molar c o n c e n tration of guanylic acid (Curve B), oligonucleotides from yeast R N A (Curve C) and h e a t - d e n a t u r e d calf t h y m u s D N A (Curve D).
Glyoxal was next applied to nucleotides with low molecular weights. Such oligonucleotides were obtained b y degradation of yeast RNA with bovine pancreatic ribonuclease, and more than 9 ° ~o of the nucleotides in the digest was obtained as a fraction of oligonucleotides b y chromatography with a Sephadex G-25 column. Curve C in Fig. 7 is the reaction curve obtained for this sample of nucleotides. The guanine content was calculated from the --AA value on the plateau of this reaction curve and the phosphorus content to be 0.226, using the Ae26o value for guanylic acid. This guanine content is 92 °/o of that reported b y ELSON AND CHARGAFF5 and 81 °/o of that reported by CRESTFIELD, SMITH AND ALLEN6, as listed in Table I. The deviation of 8 - I 9 % from the previously reported values is certainly greater than the errors in the measurement by the glyoxal method. The deviation m a y be due to the difference in the samples or due to the errors included in the previous chromatographic assay. The systematically lower value obtained b y the glyoxal method may, however, support the view that the reaction of glyoxal with the guanine group in oligonucleotides proceeds to the extent of 81-92 % but not to completion, or that the Ae269 value for the guanosine residue in oligonucleotides is somewhat lower. Biochim. Biophys. Acta, 161 (1968) 23 31
REACTIONS OF GLYOXALWITH NUCLEICACIDS
29
No spectral change was observed for native calf thymus DNA on the treatment with 5.o mM glyoxal, the concentration being sufficient to modify free guanine groups. This indicates that all of the guanine groups in native DNA are in a nonreactive state, probably due to hydrogen bonding. Native calf thymus DNA was heated at IOO° for 15 rain and then cooled rapidly within 3 rain to obtain heatdenatured DNA. This preparation when treated with glyoxal underwent an intricate spectral change composed of two different phenomena: a spectral change due solely to the reaction with guanosine residues, and an enhancement of the ultraviolet nucleotide band, the hyperchromic shift, which was superimposed on the former spectral change. The hyperchromic shift suggests the presence of some structures causing hypochromicity in the denatured DNA and the reaction of glyoxal with guanosine residues in such a structure releases this hypochromic effect. The overall spectral change was analyzed, assuming that the change results from overlapping of these two effects. The following equation was derived based on this assumption, and was used for determination of glyoxal-reactive guanosine residues in the denatured DNA: --AA269 = A (D+G)269--A (N)269--~(A (D+G)254--A (N)2~a) where ~. -- (A (D)269--A (N)269)/(A (D)254--A (N)254); A (N)254 or A (N)269 = the absorbance value of native DNA at 254 m/,, the wavelength of the isosbestic point, or at 26 9 m/~, the wavelength of the maximal spectral change due to the reaction; A (D)254 or A (D)269 = the absorbance value of denatured DNA at 254 m/~ or 26 9 m/~ before the treatment; A (D+G)254 or A (D+G)260 = the absorbance value of denatured DNA at 254 m/~ or 26 9 m# after the treatment with glyoxal. In the above equation, the hyperchromic effect is estimated from the change of absorbance at 254 m~, the wavelength of the isosbestic point on the spectral change due solely to the reaction, and the hyperchromic effect at a different wavelength of 26 9 m/~ is estimated from the ratio of the hyperchromic effects at these wavelengths determined with native and denatured DNA. The overall change at 26 9 m F is corrected by the hyperchromic effect thus estimated to obtain the change due solely to the reaction. Curve D in Fig. 7 shows the change due solely to the reaction with the denatured calf thymus DNA as a function of glyoxal concentration, from which 72 % of the guanine groups was found to react with glyoxal (Table I). The hyperchromic increment on the reaction was 19 %. It took about 12 h to obtain a constant reading of absorbance on this reaction, the time being longer than that required for guanine or its mononucleotide. Considering this fact, the guanine groups in the denatured DNA are in a less reactive state than free base or the base in mononucleotide. These data are compatible with those obtained by HAYATSU,TAKEISHI AND UKITAla who found that 54 % of cytidine in denatured calf thymus DNA reacted with semicarbazide while only 2.5 % of cytidine in native DNA reacted with this reagent.
DISCUSSION The specific reaction of glyoxal at a low concentration with free guanine or guanylic acid or those in nucleic acids may be related to a similar specificity of glyoxal to arginine 1, considering that both guanine and arginine have two amino groups. The product obtained by the reaction between the two aldehyde groups of glyoxal Biochim. Biophys. Acta, 161 (1968) 23-3I
30
t~. NAKAYAel al.
and the two amino groups of the substrate molecule m a y be more stable than the product derived from a single amino group of a substrate and a single aldehyde group of a reagent such as formaldehyde. Recently, SHAPIROAND HACHMANN14 studied the reaction between guanosine and glyoxal at pH 4, and determined the structure of the product to be a cyclic compound resulting from the reaction of the two amino groups. This may support the above interpretation. STAEHELIN15 reported that only the spectrum of guanylic acid changed when treated overnight with I °/o (36 raM) glyoxal at pH 6.8 whereas the spectra of other mononucleotides were not changed by glyoxal at such a high concentration. This conflicts with our data that the other three bases as well as guanylie acid undergo spectral changes at this high concentration of glyoxal. In STAEHELIN'Sexperiment, however, after incubation the reaction mixture was diluted IOO times before measurement, so that tile products, once formed from other mononucleotides, may have been decomposed into the original nucleotides by the effect of dilution. This interpretation is supported by the above-mentioned instability of the product formed from a base with a single amino group. Several reagents for guanine have been explored by various investigators. Diazotized 2-amino-p-benzenedisulfonic acid was used by MOUDRIANAKISAND BEER1G as a selective marker of guanine for electron microscopic determination of base sequences of nucleic acids. Guanylic acid reacts at pH 9 with this reagent 60 times faster than any of the other common nucleotides. KOCHETKOV, BUDOWSKYAND SHIBAENA17 demonstrated that propiohydroxamic acid reacts at pH 9 both with uridine and with guanine but the rates of reaction with these bases were different. They found that the difference in the rate is greater at pH 8 and this characteristic can be used for selective modification of guanine. The treatment of nucleic acids with N-2fluorenyl hydroxalamine TM and N-acetoxy-2-acetylaminofluorenea9 at neutral pH caused a marked decrease in the guanine content. The guanine content was also decreased by photooxidation in the presence of riboflavin z°, methylene blue 21,22 or lumichrome 23. Glyoxal found in the present study as a reagent for guanine or its nucleotide may be superior to these other reagents in that glyoxal satisfies all of the following conditions. (a) Glyoxal at a low concentration reacts specifically with guanine groups in nucleic acids at room temperature and near neutral pH. (b) The reaction proceeds rather rapidly and yet the reactivity of the reagent is moderate, such that hydrogen-bonded guanine groups in native DNA do not react with glyoxal. (c) The degree of reaction can be determined by the simple means of absorption photometry. The application of glyoxal to microdetermination of guanine in various materials may be useful. The Folin phenol reagent, which was applied for the determination of guanine in tissue extracts or urine by \VILLIAMS 24, reacts both with guanine and with xanthine. The glyoxal method is free from such interference by xanthine.
REFEI{ENCES I 2 3 4
K . NAKAYA, H . HORINISHI AND K . SHIBATA, J. Biochem., Tokyo, 6~ (1967) 3 4 5 . a . FRAENKEL-CONRAT AND H . S. OLCOTT, J. Apt. Chem. Soc., 68 (1946) 34E . R . M. KAY, N . S. SIMMONS AND A . L. DOUNCE, J. A~Iz. Chem. Soc., 74 (1952) 174. tL. VOLKIN AND V~r. ]~. COHN, Methods o/ Biochemical Analysis, Vol. i, [ n t c r s c i e n c e , N e w Y o r k : I 9 5 4 , p. 287.
Biochim. t3iophys. Acla, 1 6 I (1968) 2 3 - 3 I
REACTIONS OF GLYOXAL WITH NUCLEIC ACIDS 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24
31
D. ELSON AND E. CHARGAFF, Biochim. Biophys. Acta, 17 (1955) 367 • A. M. CRESTFIELD, 1~. C. SMITH AND V. W. ALLEN, J. Biol. Chem., 216 (1955) 185. K. MOLDANE AND C. HEIDELBERGER, J. Am. Chem. Soc., 76 (1954) 679. M. NAKAMURA, J. Agr. Chem. Soc. Japan, 24 (195 o) i. R. J. L. ALLEN, Biochem. J., 34 (194 ° ) 858. A. D. HERSHEY, J. DIxoN AND M. CHASE, J. Gen. Physiol., 36 (1953) 777. K. BURTON, Biochem. J., 77 (196o) 547. E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids. Vol. I, Academic Press, New York, 1955, p. 3o7 • H. HAYATSU, K. TAKEISHI AND T. UKITA, Biochim. Biophys. Acta, 123 (1966) 445. R. SHAPIRO AND J. I-[ACHMANN, Biochemistry, 5 (1966) 2799. M. STAEHELIN, Biochim. Biophys. Acla, 31 (1959) 448. E. lET. MOUDRIANAKIS AND M. BEER, Biochim. Biophys. Acta, 95 (1965) 23. N. K. KOCHETKOV, E. I. BUDO~,VSKYAND ]{. P. SHIBAENA, Biochim. Biophys. Acta, 87 (I964) 515 • E. KRIEK, Biochem. Biophys. Res. Commun., 2o (1965) 793. E. C. MILLER, U. JUHL AND J. A. MILLER, Science, 153 (1966) 1125. A. TSUGITA, Y. OKADA AND K. UEHARA, Biochim. Biophys. Acta, lO 3 (1965) 360. J. S. BELLIN AND G. OSTER, Biochim. Biophys. Acta, 42 (196o) 533. M. I. SIMON AND H. VAN VUNAKIS, Arch. Bioehem. Biophys., lO 5 (1964) 197. J. S. SUSSENBACH AND W'. BERENDS, Biochim. Biophys. Aela, 76 (1963) 154. J. N. WILLIAMS. JR., J. Biol., Chem. 184 (195 o) 627.
Biochim. Biophys. Acta, 161 (i968) 23-31
BIOCHIMICA ET BIOPHYSICA ACTA
g g A 95907
REACTIONS OF GLYOXAL W I T H NUCLEIC ACIDS, NUCLEOTIDES AND T H E I R COMPONENT BASES K A Z U Y A S U N A K A Y A , OSAMU T A K E N A K A , H I R O O H O R I N I S H I AND K A Z U O S H I B A T A
The Tokugawa Institute [or Biological Research, Me]iromachi, Tokyo", and Tokyo Institute o[ Technology, Meguroku, Tokyo (Japan) (Received J a n u a r y 3oth, 1968)
SUMMARY
The reactions of glyoxal with nucleic acids, nucleotides and their bases were examined by measuring spectral changes. The spectra of all of the bases and nucleotides in RNA and DNA were changed appreciably by treatment with a high concentration of glyoxal. When treated with a low concentration of glyoxal, however, only guanine or guanylic acid underwent a specific spectral change. The reaction of glyoxal with this base or its nucleotide proceeded to completion within 30 rain between pH 8. 3 and 9.8 as estimated from the spectral change at room temperature. This phenomenon was applied for determination of the guanosine content of nucleic acids and the results agreed well with previous data obtained by chromatography. As judged from the spectral change, glyoxal did not react with native calf thymus DNA, while it reacted considerably with heat-denatured DNA. The spectral change obtained with denatured DNA was a composite of two qualitatively different changes, the change being due solely to the reaction between glyoxal and guanosine residues and the hyperchromic shift of the 26o-m# band, which may be a secondary effect of the reaction. From the analysis of these two effects, the degree of reaction of the guanosine residues in denatured DNA was estimated to be 72 %.
INTRODUCTION
It was demonstrated in a previous study I that glyoxal specifically modifies the guanidyl group of arginine residues in proteins in aqueous solution. For example, the II arginine residues in the lysozyme molecule reacted to the extent of approx. 80 % with this reagent, while other amino acids including amino groups of lysine were not appreciably modified as determined by amino acid analysis of the hydrolyzate of the modified protein. This is in contrast with high reactivity found with amino groups and much lower reactivity with arginine residues of formaldehyde z. Such a specificity of glyoxal stimulated the authors to use glyoxal in the present study with nucleic acids, nucleotides and their component bases or nucleosides. A highly specific reaction of glyoxal with guanine, guanosine or guanylic acid in a weakly alkaline solution at room temperature was thus found spectrophotometrically, and the spectral change due to this reaction was applied to determination of the guanine contents * P o s t a l address.
Biochim. Biophys. Acta, 161 (1968) 23-31
24
K. NAKAYAet al.
of several kinds of RNA and DNA and to examination of the difference in reactivity between guanine groups in native DNA and those after denaturation b y heat treatment. Described herein are the results obtained in these experiments.
EXPERIMENTAL
Materials An aqueous solution of 4o % glyoxal was obtained from Tokyo Kasei Kogyo Co. Herring sperm DNA was purchased from Sigma Chemical Co., and calf thymus DNA was prepared b y the method of KAY,SIMMONSAND DOUNCE3 and recrystallized three times. Sodium ribonucleate of yeast as well as purines, pyrimidines and ribonucleotides were purchased from Tokyo Kasei Kogyo Co. The concentrations of bases, nucleosides and nucleotides were determined spectrophotometrically a. The molar extinction coefficients at 260 m# for native and denatured DNA were assumed to be 6000 and 8500 cm -1 • M -1, respectively.
Procedure An aqueous solution (i ml) of glyoxal was added to the mixture of a sample solution (i ml) of nucleotide, nucleoside or base and 0.5 M bicarbonate-NaOH buffer (3 ml) at p H 8.6. The p H value of the reaction mixture increased slightly to about 8. 9 on the addition of glyoxal and therefore was adjusted to exactly 8. 9. In an experiment designed to show the effect of p H on the reactivity of glyoxal, the above buffer was replaced either by the same buffer of different p H values, to obtain mixtures above p H 8.6, or b y 0.2 M phosphate buffer, to obtain mixtures below p H 8.6. The reaction mixture was left standing at room temperature ( I 5 ~ 5 °) for 2 h and then the absorption spectrum was observed with a Shimadzu recording spectrophotometer model SV-5o, using I.o-cm cells. The same reagent solution without the sample was used as the reference in this measurement.
RESULTS
Reactions o~ glyoxal with the nucleotides o/ R N A The spectrum of guanylic acid was markedly changed by treatment with glyoxah Curve A in Fig. I is the spectrum of 42.4/,M guanylic acid at pH 8.9 and Curves B, C and D are the spectra of this sample treated at the same p H with 0.08, 0.32 and 1.88 mlV[ glyoxal, respectively. By this treatment, the band of guanylic acid at 250 m/~ was enhanced and shifted to 248 m/~ and, in addition, the shoulder around 270 m/~ was lowered but was more pronounced. Curve D is regarded as the spectrum of the reaction product because no further spectral change occurred above 1.88 mM glyoxal. Clear isosbestic points were observed at 254 and 284 m/~ in this spectral change, which indicates that no side reaction is involved in the reaction. Curve G in Fig. 2 is the difference spectrum for this reaction which was calculated from Curves A and D in Fig. I. The spectrum shows a negative band at 269 mt,. Biochim. Biophys. ~tcta, I 6 I (x968) 23-31
REACTIONS OF GLYOXAL WITH NUCLEIC ACIDS r
,
,
0£
Ouanylic
25
i
acid
'
OI
'C ,'~ "',\
A
[
o~
<3 2~ <~
02.
-01 1
/
0 230
250
270 290 Wavelength in m~
I
2~0
310
250
I
I
270 290 Wavelength in mp
310
Fig. i. T h e s p e c t r a l c h a n g e of 42.4 btM g u a n y l i c acid t r e a t e d w i t h g l y o x a l a t p H 8.9. C u r v e A, t h e s p e c t r u m before t r e a t m e n t ; C u r v e s B, C a n d D, t h e s p e c t r a o b t a i n e d b y t r e a t m e n t with 0.08, 0.32 a n d 1.88 m M g l y o x a l for 2 h, r e s p e c t i v e l y . Fig. 2. Difference s p e c t r a before a n d a f t e r t r e a t m e n t of 42. 4/zM n u c l e o t i d e s w i t h glyoxal. C u r v e G, t h e s p e c t r u m o b t a i n e d o n t h e t r e a t m e n t of g u a n y l i c acid w i t h 1.88 m M glyoxa]. C u r v e s A, C a n d U, t h e s p e c t r a o b t a i n e d w i t h 145 m M g l y o x a l for adenylic, cytidylic a n d uridylic acids, respectively.
The effect of pH was examined with 4.o mM glyoxal for 47.8/zM guanylic acid. The drop (--AA) of absorbance at 269 m # after 2 h of incubation is plotted against pH in Fig. 3, which indicates that the reaction proceeds to completion between pH 8.3 and 9.8. Curve A in Fig. 4 shows the degree of reaction plotted against glyoxal concentration which was observed after 2 h of incubation of 42.4/zM guanylic acid with the reagent at pH 8. 9. The curve rises uniformly and reaches a plateau of - - A A ~ o.134 at 2.2 mM glyoxal. The spectral change at such an excess concentration of glyoxal was completed after 30 rain of incubation and stayed constant at least for 3 days. The reaction time in further experiments was therefore fixed at 2 h.
i
i
r
i
L
0.15 ,
Ol5
i B
~
~ OlO c~
Olyoxal
I
I
7
8
X
f
•
---
0.10 p //
i0.05
I
9
pH
I~3
It~
o
Glyoml Concentralionin mM
Fig. 3. T h e effect of p H on t h e degree of r e a c t i o n of 47.8/zM g u a n y l i c acid b y 4.o m M g l y o x a l a f t e r 2 h of i n c u b a t i o n , as e s t i m a t e d f r o m t h e lowering of a b s o r b a n c e a t 269 m F. Fig. 4. T h e v a l u e s of --z]A o b t a i n e d a t pI-I 8.9 for 42.4 # M g u a n y l i c acid (Curve A), a n equim o l a r m i x t u r e of 42.4/zM n u c l e o t i d e s (Curve B) a n d 6 5 . 1 / , M g u a n i n e (Curve C) p l o t t e d as a f u n c t i o n of g l y o x a l c o n c e n t r a t i o n .
Biochim. Biophys. Mcta, 161 (1968) 23-31
K. NAKAYAet al.
26
From the above value of - - A A , the value of zle (the change of the molar extinction coefficient due to the reaction) was calculated to be 32oo cm -1 • M-1, which was used in fllrther experiments to calculate the moles of guanylic acid reacted with glyoxal. Adenylic and cytidylic acids underwent a spectral change on treatment with glyoxal, but a much higher concentration of glyoxal was required to obtain a measurable spectral change. The difference spectra obtained on the treatment of 42.4 ~M adenylic and cytidylic acids with 145 mM glyoxal are shown by Curves A and C, respectively, in Fig. 2. The spectrum of uridylic acid also changed at this high concentration of reagent but the change took place at shorter wavelengths, below 25o mF, as shown by curve U in Fig. 2. The spectral changes of these 3 mononucleotides were, however, not observable when treated with glyoxal below 4 raM, the concentration being sufficient to cause the change of guanylic acid. This difference in reactivity between guanylic acid and the other 3 mononucleotides in RNA provides the experimental basis for determination of the guanylic acid content of RNA. As a preliminary application, the guanylic acid content in an equimolar mixture of 42.4 ~M guanylic, cytidylic, uridylic and adenylic acids was determined from the spectral change by glyoxal. Curve B in Fig. 4, which is the reaction curve obtained for this mixture, was identical with Curve A in the same figure, the result obtained for guanylic acid alone. TABLE
l
GUANINE
OR G U A N O S I N E
CONTENTS
Gg in mole of g u a n i n e p e r mole of p h o s p h a t e , of R N A or D N A d e t e r m i n e d w i t h g l y o x a l as c o m p a r e d w i t h Ge, t h e c o n t e n t s b e i n g in t h e s a m e u n i t d e t e r m i n e d p r e v i o u s l y b y c h r o m a t o g r a p h y .
Sample
(;~
G¢
Go/G~
0.254
o.246 (ref. 5), o.280 (ref. 6)
lO3.3, 91.9,
o.194
o.193 (ref. 12)
lOO.5
0.203 o o. I59
o.212 (ref. I1)
96.5 o 72.3
(%)
Yeast R N A Hydrolyzate Oligonucleotides
o.226
90.7 80. 7
Herring sperm DNA Hydrolyzate
Call thymus DNA Hydrolyzate Native Denatured
The base composition of yeast RNA has been determined chromatographically by ELSON AND CHARGAFF5 and CRESTFIELD,SMITH AND ALLEN~ as listed in Table I. The above spectral change was applied to determine the guanylic acid content of yeast RNA. The RNA preparation was hydrolyzed with o.3 IV[ NaOH at 37 ° for 18 h according to the method of MOLDANE AND H E I D E L B E R G E R 7, and the hydrolyzate solution was neutralized with HC1 and diluted Io-fold with water. A glyoxal solution was added to this diluted solution, and the absorbance change at 269 my was observed. The phosphate concentration was determined for the same solution by the method of NAKAMURAs after treatment with perchloric acid 9. The change in the molar Biochim. Biophys. Acta, 16i (1968) 23 31
27
REACTIONS OF GLYOXAL WITH NUCLEIC ACIDS
extinction coefficient, AeM, at 269 m/~ per g atom of phosphorous of yeast RNA was calculated from the data to be 8o2 cm -1. M -1. Dividing this value b y 32oo, which is the Ae2n9 value for guanylic acid, we obtained o.254 as the guanylic acid content of yeast RNA, which agrees well with the values shown in Table I.
Reaction o/ glyoxal with the bases o/DNA Guanine also undergoes great spectral changes when treated with glyoxal. Curve A in Fig. 5, which is the spectrum of 65.1/*M guanine, changed to Curves B and C on the treatment with o.45 and 1.8i mM glyoxal, respectively. The difference spect r u m of Curve A minus Curve C in Fig. 5 gave a negative band at 269 m/~ as shown by Curve A in Fig. 6. This difference spectrum is similar to that obtained for guanylic acid (curve A in Fig. 2), especially in the position of the negative band. No spectral change was observed for the other 3 bases of DNA with 1.8I mM glyoxal, although their spectra changed greatly at a higher concentration (145 raM) of glyoxal as seen from Curves A, C and T in Fig. 6. Curve C in Fig. 4 is the reaction curve obtained for 65.1 yM guanine, from which the Ae value at 269 m/, was calculated to be 199o cm -1. M -~. The reaction curve obtained for a mixture of the same concentration of guanine, adenine, thymine and cytosine was identical with that obtained for guanine alone. It is evident from these results that none of the other DNA bases interfere with the determination of guanine in mixtures of these 4 bases.
//
AGuanine
06
Glyoxal
+
/
02 -0.1 270
290
Wavelength .in mp
310
23O
.
//// ~111111~_ ~ /
o
250
.
¢
o~
0 230
/
/
//
OI
<~
/
--~/G [ 250
I 270
Wavelength in mp
I 290
310
Fig. 5. T h e s p e c t r a l c h a n g e of 65.1 # M g u a n i n e t r e a t e d w i t h g l y o x a l a t p H 8. 9. C u r v e A, t h e spect r u m before t r e a t m e n t ; C u r v e s B a n d C, t h e s p e c t r a o b t a i n e d b y t r e a t m e n t w i t h o.45 a n d i .8I m M glyoxal, r e s p e c t i v e l y . Fig. 6. Difference s p e c t r a before a n d a f t e r t r e a t m e n t of 65.1/,1~1 b a s e s w i t h glyoxal. C u r v e G, t h e s p e c t r u m o b t a i n e d w i t h 1.81 m M g l y o x a l for g u a n i n e , a n d C u r v e s A, C a n d T o b t a i n e d w i t h 145 m M g l y o x a l for a d e n i n e , c y t o s i n e a n d t h y m i n e , r e s p e c t i v e l y .
Calf thymus and herring sperm DNA preparations were hydrolyzed with 6 M HC1 at IOO° for 3 h (ref. IO) in order to determine the guanine contents. The solutions were neutralized and diluted lo-fold, and the contents were determined using the Ae269 value obtained for guanine. The results were o.2o3 for calf thymus DNA and o.194 for herring sperm DNA, which agree well with those reported by BURTONn and CHARGAFF AND DAVIDSON TM, respectively, as seen from Table I. It was thus demBiochim. Biophys. -dcta, 161 (r968) 23-31
28
K. NAKAYAel al.
onstrated that the guanine content can be determined easily and precisely by means of the spectral change on treatment with glyoxal. Guanosine, deoxyguanosine and guanosine 5-monophosphate gave spectral changes which were almost identical with that obtained for guanylic acid with respect to the position and the zJe269 value of the negative band as well as the reaction curve, the degree of reaction plotted versus glyoxal concentration. For example, Curve A in Fig. 7 is the reaction curve obtained for 24.0 pM guanosine, which is very much like Curve B for guanylic acid in the same figure. The value of Ae269 for guanosine was 3200 cm -1 • M -1 which agrees with the value obtained for guanylic acid. This agreement between guanosine and guanylic acid and the finding of a considerably lower value described above for guanine indicate that the addition of a phosphate group to the sugar moiety does not affect the Ae2~0 value but the addition of a sugar, ribose or deoxyribose, to the 9th nitrogen atom of purine does affect the Ae269 value.
0.08 i
= o.o6~
i
~ _
A
i
i
~
.
0
0 0
o
2 I
.
~ 2
3
GlyoxalConcenlmfioinmM
Fig. 7. The values of --zlA obtained for 24.0/~M guanosine (Curve A), the s a m e molar c o n c e n tration of guanylic acid (Curve B), oligonucleotides from yeast R N A (Curve C) and h e a t - d e n a t u r e d calf t h y m u s D N A (Curve D).
Glyoxal was next applied to nucleotides with low molecular weights. Such oligonucleotides were obtained b y degradation of yeast RNA with bovine pancreatic ribonuclease, and more than 9 ° ~o of the nucleotides in the digest was obtained as a fraction of oligonucleotides b y chromatography with a Sephadex G-25 column. Curve C in Fig. 7 is the reaction curve obtained for this sample of nucleotides. The guanine content was calculated from the --AA value on the plateau of this reaction curve and the phosphorus content to be 0.226, using the Ae26o value for guanylic acid. This guanine content is 92 °/o of that reported b y ELSON AND CHARGAFF5 and 81 °/o of that reported by CRESTFIELD, SMITH AND ALLEN6, as listed in Table I. The deviation of 8 - I 9 % from the previously reported values is certainly greater than the errors in the measurement by the glyoxal method. The deviation m a y be due to the difference in the samples or due to the errors included in the previous chromatographic assay. The systematically lower value obtained b y the glyoxal method may, however, support the view that the reaction of glyoxal with the guanine group in oligonucleotides proceeds to the extent of 81-92 % but not to completion, or that the Ae269 value for the guanosine residue in oligonucleotides is somewhat lower. Biochim. Biophys. Acta, 161 (1968) 23 31
REACTIONS OF GLYOXALWITH NUCLEICACIDS
29
No spectral change was observed for native calf thymus DNA on the treatment with 5.o mM glyoxal, the concentration being sufficient to modify free guanine groups. This indicates that all of the guanine groups in native DNA are in a nonreactive state, probably due to hydrogen bonding. Native calf thymus DNA was heated at IOO° for 15 rain and then cooled rapidly within 3 rain to obtain heatdenatured DNA. This preparation when treated with glyoxal underwent an intricate spectral change composed of two different phenomena: a spectral change due solely to the reaction with guanosine residues, and an enhancement of the ultraviolet nucleotide band, the hyperchromic shift, which was superimposed on the former spectral change. The hyperchromic shift suggests the presence of some structures causing hypochromicity in the denatured DNA and the reaction of glyoxal with guanosine residues in such a structure releases this hypochromic effect. The overall spectral change was analyzed, assuming that the change results from overlapping of these two effects. The following equation was derived based on this assumption, and was used for determination of glyoxal-reactive guanosine residues in the denatured DNA: --AA269 = A (D+G)269--A (N)269--~(A (D+G)254--A (N)2~a) where ~. -- (A (D)269--A (N)269)/(A (D)254--A (N)254); A (N)254 or A (N)269 = the absorbance value of native DNA at 254 m/,, the wavelength of the isosbestic point, or at 26 9 m/~, the wavelength of the maximal spectral change due to the reaction; A (D)254 or A (D)269 = the absorbance value of denatured DNA at 254 m/~ or 26 9 m/~ before the treatment; A (D+G)254 or A (D+G)260 = the absorbance value of denatured DNA at 254 m/~ or 26 9 m# after the treatment with glyoxal. In the above equation, the hyperchromic effect is estimated from the change of absorbance at 254 m~, the wavelength of the isosbestic point on the spectral change due solely to the reaction, and the hyperchromic effect at a different wavelength of 26 9 m/~ is estimated from the ratio of the hyperchromic effects at these wavelengths determined with native and denatured DNA. The overall change at 26 9 m F is corrected by the hyperchromic effect thus estimated to obtain the change due solely to the reaction. Curve D in Fig. 7 shows the change due solely to the reaction with the denatured calf thymus DNA as a function of glyoxal concentration, from which 72 % of the guanine groups was found to react with glyoxal (Table I). The hyperchromic increment on the reaction was 19 %. It took about 12 h to obtain a constant reading of absorbance on this reaction, the time being longer than that required for guanine or its mononucleotide. Considering this fact, the guanine groups in the denatured DNA are in a less reactive state than free base or the base in mononucleotide. These data are compatible with those obtained by HAYATSU,TAKEISHI AND UKITAla who found that 54 % of cytidine in denatured calf thymus DNA reacted with semicarbazide while only 2.5 % of cytidine in native DNA reacted with this reagent.
DISCUSSION The specific reaction of glyoxal at a low concentration with free guanine or guanylic acid or those in nucleic acids may be related to a similar specificity of glyoxal to arginine 1, considering that both guanine and arginine have two amino groups. The product obtained by the reaction between the two aldehyde groups of glyoxal Biochim. Biophys. Acta, 161 (1968) 23-3I
30
t~. NAKAYAel al.
and the two amino groups of the substrate molecule m a y be more stable than the product derived from a single amino group of a substrate and a single aldehyde group of a reagent such as formaldehyde. Recently, SHAPIROAND HACHMANN14 studied the reaction between guanosine and glyoxal at pH 4, and determined the structure of the product to be a cyclic compound resulting from the reaction of the two amino groups. This may support the above interpretation. STAEHELIN15 reported that only the spectrum of guanylic acid changed when treated overnight with I °/o (36 raM) glyoxal at pH 6.8 whereas the spectra of other mononucleotides were not changed by glyoxal at such a high concentration. This conflicts with our data that the other three bases as well as guanylie acid undergo spectral changes at this high concentration of glyoxal. In STAEHELIN'Sexperiment, however, after incubation the reaction mixture was diluted IOO times before measurement, so that tile products, once formed from other mononucleotides, may have been decomposed into the original nucleotides by the effect of dilution. This interpretation is supported by the above-mentioned instability of the product formed from a base with a single amino group. Several reagents for guanine have been explored by various investigators. Diazotized 2-amino-p-benzenedisulfonic acid was used by MOUDRIANAKISAND BEER1G as a selective marker of guanine for electron microscopic determination of base sequences of nucleic acids. Guanylic acid reacts at pH 9 with this reagent 60 times faster than any of the other common nucleotides. KOCHETKOV, BUDOWSKYAND SHIBAENA17 demonstrated that propiohydroxamic acid reacts at pH 9 both with uridine and with guanine but the rates of reaction with these bases were different. They found that the difference in the rate is greater at pH 8 and this characteristic can be used for selective modification of guanine. The treatment of nucleic acids with N-2fluorenyl hydroxalamine TM and N-acetoxy-2-acetylaminofluorenea9 at neutral pH caused a marked decrease in the guanine content. The guanine content was also decreased by photooxidation in the presence of riboflavin z°, methylene blue 21,22 or lumichrome 23. Glyoxal found in the present study as a reagent for guanine or its nucleotide may be superior to these other reagents in that glyoxal satisfies all of the following conditions. (a) Glyoxal at a low concentration reacts specifically with guanine groups in nucleic acids at room temperature and near neutral pH. (b) The reaction proceeds rather rapidly and yet the reactivity of the reagent is moderate, such that hydrogen-bonded guanine groups in native DNA do not react with glyoxal. (c) The degree of reaction can be determined by the simple means of absorption photometry. The application of glyoxal to microdetermination of guanine in various materials may be useful. The Folin phenol reagent, which was applied for the determination of guanine in tissue extracts or urine by \VILLIAMS 24, reacts both with guanine and with xanthine. The glyoxal method is free from such interference by xanthine.
REFEI{ENCES I 2 3 4
K . NAKAYA, H . HORINISHI AND K . SHIBATA, J. Biochem., Tokyo, 6~ (1967) 3 4 5 . a . FRAENKEL-CONRAT AND H . S. OLCOTT, J. Apt. Chem. Soc., 68 (1946) 34E . R . M. KAY, N . S. SIMMONS AND A . L. DOUNCE, J. A~Iz. Chem. Soc., 74 (1952) 174. tL. VOLKIN AND V~r. ]~. COHN, Methods o/ Biochemical Analysis, Vol. i, [ n t c r s c i e n c e , N e w Y o r k : I 9 5 4 , p. 287.
Biochim. t3iophys. Acla, 1 6 I (1968) 2 3 - 3 I
REACTIONS OF GLYOXAL WITH NUCLEIC ACIDS 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24
31
D. ELSON AND E. CHARGAFF, Biochim. Biophys. Acta, 17 (1955) 367 • A. M. CRESTFIELD, 1~. C. SMITH AND V. W. ALLEN, J. Biol. Chem., 216 (1955) 185. K. MOLDANE AND C. HEIDELBERGER, J. Am. Chem. Soc., 76 (1954) 679. M. NAKAMURA, J. Agr. Chem. Soc. Japan, 24 (195 o) i. R. J. L. ALLEN, Biochem. J., 34 (194 ° ) 858. A. D. HERSHEY, J. DIxoN AND M. CHASE, J. Gen. Physiol., 36 (1953) 777. K. BURTON, Biochem. J., 77 (196o) 547. E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids. Vol. I, Academic Press, New York, 1955, p. 3o7 • H. HAYATSU, K. TAKEISHI AND T. UKITA, Biochim. Biophys. Acta, 123 (1966) 445. R. SHAPIRO AND J. I-[ACHMANN, Biochemistry, 5 (1966) 2799. M. STAEHELIN, Biochim. Biophys. Acla, 31 (1959) 448. E. lET. MOUDRIANAKIS AND M. BEER, Biochim. Biophys. Acta, 95 (1965) 23. N. K. KOCHETKOV, E. I. BUDO~,VSKYAND ]{. P. SHIBAENA, Biochim. Biophys. Acta, 87 (I964) 515 • E. KRIEK, Biochem. Biophys. Res. Commun., 2o (1965) 793. E. C. MILLER, U. JUHL AND J. A. MILLER, Science, 153 (1966) 1125. A. TSUGITA, Y. OKADA AND K. UEHARA, Biochim. Biophys. Acta, lO 3 (1965) 360. J. S. BELLIN AND G. OSTER, Biochim. Biophys. Acta, 42 (196o) 533. M. I. SIMON AND H. VAN VUNAKIS, Arch. Bioehem. Biophys., lO 5 (1964) 197. J. S. SUSSENBACH AND W'. BERENDS, Biochim. Biophys. Aela, 76 (1963) 154. J. N. WILLIAMS. JR., J. Biol., Chem. 184 (195 o) 627.
Biochim. Biophys. Acta, 161 (i968) 23-31