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Studies on the enzymic oxidation of aminopurines
POLYNUCLEOTIDES AND METAL IONS, AMINO ACIDS, POLYAMINES
43~
REFERENCES I R. C. WARNER, J. Biol. Chem., 229 (I957) 711. 2 G. FELSENF~LD AND A. RICH, Biochim. Biophys. Acta, 26 (1957) 457. 8 G. FELSESFELD, D. R. DAVmS AND A. RICH, ]. Am. Chem. Soc., 79 (1957) 2023. 4 G. FELSENFELD AND S. HUANG, Biochim. Biophys. Acta, 34 (1959) 234. 8 j . R. FRESCO AND P. DorY, J. Am. Chem. Soc., 79 (1957) 3928. 6 R. F. BEERS AND R. F. SrEINER, Nature, 179 (1957) lO76. 7 A. M. MICH~LSON, Nature, 182 (1958) 15o2. s j . SHACK, D. J. JENKINS AND J. M. THOMPSETT, ]. Biol. Chem., 203 (I953) 373. 9 R. THOMAS, Biochim. Biophys. Acta, 14 (1954) 231. t0 F. E. HARRIS AND S. A. RICE, J. Phys. Chem., 61 (1957) 136o. 11 C. D. JARDErZKY, J. Am. Chem. Soc., 80 (1958) 1125. 18 j . S. WIBERG AND W. F. NEUMAN, Arch. Biochem. Biophys., 72 (1957) 66. 18 G. ZUBAYAND P. DoTe, Biochim. Biophys. Acta, 29 (1958) 47. It B. N. AMES, D. T. DUBIN AND S. M. ROSENXHAL, Science, 127 (1958) 814.
Biochim. Biophys. Aaa, 37 (196o) 425-433
STUDIES ON T H E ENZYMIC OXIDATION OF AMINOPURINES F E L I X BERGMANN, HANNA KWIETNY*, GERSHON LEVIN* AND HANNA E N G E L B E R G
Department o[ Pharmacology, The Hebrew University, Hadassah Medical School, Jerusalem (Israel) (Received April 24th, I959)
SUMMARY
I. 8-Aminopurine is oxidised by mammalian xanthine oxidase along the following pathway: 8-Aminopurine ---> 6-hydroxy-8-aminopurine ~
2,6-dihydroxy-8-aminopurine
It is thus evident that oxidation of all three aminopurines takes a course different from the corresponding hydroxypurines. 2. Adenine and 6-methylaminopurine are oxidised along the same pathway, but all other methyl derivatives of adenine are refractory. 3-2-Aminopurine is converted by a bacterial enzyme system into 2-amino8-hydroxypurine. These results are discussed in terms of specific enzyme--substrate complexes.
INTRODUCTION WYNGAA~DEN AND DUNN I h a v e r e c e n t l y e s t a b l i s h e d t h a t a d e n i n e is a t t a c k e d b y m a m m a l i a n x a n t h i n e o x i d a s e (XO) first a t p o s i t i o n 8 a n d t h e r e a f t e r a t c a r b o n a t o m ~. T h e o r d e r of s t e p s is t h u s a t v a r i a n c e w i t h t h e s e q u e n c e , a p p l y i n g t o t h e e n z y m i c o x i d a t i o n of h y p o x a n t l f i n e : Hypoxanthine --~ xanthine ~ uric acid Adenine ~ 8-hydroxyadenine ~ 2,8-dihydroxyadenine. * The results of this investigation are parts of Ph.D. theses, submitted to the Faculty of Science, The Hebrew University, Jerusalem.
Biochim. Biophys. Aaa, 37 (196o) 433-441
434
F. BERGMANN, H. KWIETNY, G. LEVIN, H. ENGELBERG
Analogously, 2-aminopurine is converted into guanine, as shown b y LORZ AND I-IITCHINGS2 and the present authors a, in contrast to the behavior of 2-hydroxypurine: 2-Hydroxypurine --+ 2,8-dihydroxypurine --+ uric acid 2-Aminopurine ~ guanine (= 2-amino-6-hydroxypurine) In the present study, we find that 8-aminopurine (I) too differs characteristically from 8-hydroxypurine in its behavior towards XO: 8-Hydroxypurine ~ 2,8-dihydroxypurine ~ uric acid4 8-Aminopurine ~ 6-hydroxy-8-aminopurine --+ 2,6-dihydroxy-8-aminopurine Thus, a systematic difference exists between these two series of purines, which m a y be due either to a common structural difference, responsible for the specific electron distribution and the polarity in each group, or to a different mode of attachment of hydroxy and amino derivatives to the active center of the enzyme. An answer to this problem was sought b y testing the oxidation of aminopurines by a bacterial enzyme system, described previously ~. In the formation of the enzyme-substrate (ES) complex, hydrogen bonding plays a decisive role. The groups participating in the binding of adenine to XO have been determined b y systematically substituting methyl for hydrogen at all available nitrogen atoms. The results obtained give some information on the "active form" of this substrate, actually involved in the enzymic process. MATERIALS AND METHODS
Purine derivatives 6-Hydroxy-8-aminopurine e was a gift of Dr. R. K. ROBINS, Arizona State College, Tempe, Arizona. 2,6-Dihydroxy-8-aminopurine 7 was obtained through the courtesy of Dr. A. BENDICH, the Sloan-Kettering Institute for Cancer Research, New York. 6-Methylamino- and 6-dimethylaminopurine were synthetic products, prepared b y Dr. G. B. ELION of the Wellcome Research Laboratories, Tuckahoe, New York s. Likewise, 9-methyladenine 9 was supplied by Dr. ELION. Finally, 7-methyladenine 1° was a gift from Dr. E. C. TAYLOR, Princeton University.
Enzymes Highly purified milk xanthine oxidase was supplied by Prof. F. BERGEL and Dr. R. C. BRAY of the Chester Beatty Institute of Cancer Research, London, England n. This preparation, when diluted I:8OO, produced I/zg/ml of uric acid/rain, when the reaction was carried out at 28 ° and p H 8.0 with 6.5" IO-s M xanthine as substrate. As source of the bacterial enzyme system, whole cells of Pseudomonas aeruginosa BDH, an air-borne strain described previously s, were used. Identical results were obtained with resting cells or with a culture growing in a hqnid medium lz. However, growing cells exhibited a faster turnover and thus facihtated analysis of the oxidation products. They were therefore used preferentially.
Methods o/analysis The progress of the reactions with mammalian XO was followed spectrophotometrically, using a Beckman thermospacer for temperature control. With the bacterial Biochim. Biophys. Acta, 37 (196o) 433-441
435
ENZYMIC OXIDATION OF AMINOPURINES
system, this method is applicable only for fast reactions since the cells slowly secrete a greenish pigment, which exhibits unspecific absorption. At suitable intervals, samples of the bacterial reaction mixtures, kept at 3°0 , were centrifuged at 3000 rev./min and the supernatant used for spectral measurements. All runs were submitted to paperchromatographic analysis. After development, the individual spots were located by means of their fluorescence under a Mineralight u.v. lamp, emitting light of about 255 m/~ wave length. They were then extracted with water and their spectrum measured. When necessary, the shift of Slmaxwith pH was also determined (see under RESULTS). RESULTS
Reaction o/8-aminopurine with mammalian xanthine oxidase Preliminary experiments indicated that oxidation of (I) passes through the 6-hydroxy derivative (II). The formation of the alternative intermediate, viz. 2-hydroxy-8-arninopurine--which is unknown--, would have been recognised easily by a temporary shift of the absorption maximum to higher wave lengths, because all 2,8-disubstituted purines exhibit maxima above 3oo mr*. Fig. I shows the absorption
4.1
3.9
t! 240
260
280
A (rap)
300
Fig. I. U.v. absorption spectra'of 8-aminopunne and its oxidation products at pH 8.o. A 8-Aminopurine (I). 0
0 6 - H y d r o x y - 8 - a m i n o p u r i n c (II). • purine (III).
A • 2,6-Dihydroxy-8 -amino-
spectra of (I), the intermediate (II) and the end-product (III). At 276 m/~, the isosbestic point of (II) and (III), further oxidation of the intermediate produces no change. Therefore, the initial decrease of optical density (O.D.) at this wave length O
H /N
H
(i)
O +
H
N~_
H
(ii)
all) Biochim. Biopkys. Aaa, 37 (196o) 433-441
436
F. B E R G M A N N , H. K W l E T N Y , G. L E V I N , H. E N G E L B E R G
measures the first oxidation step (see Table I). The experimental data also demonstrate that at 273 m/~, the isosbestic point of (I) and (II), the 0.D. remains constant for the first half hour and decreases only later, when sufficient intermediate has accumulated to give measurable rates of formation of (III). At 260 m/~, tile O.D. increases for the first one and a half hour, as required by the curves in Fig. I, and then again decreases. The reverse behavior is found at 290 mix. All data taken together establish beyond doubt that (II) is the main, if not the only intermediate in the enzymic oxidation of (I). The rate of the conversion (I) --~ (II) is compared in Table II with that of the second step, (II) --~ (III), which was determined at 268 and 290 mtz. The latter conversion proceeds at a rate more than IO times higher than that of the first step, when equimolar substrate concentrations are used. TABLE I SPRCTI~kL CHANGES DURING THE OXIDATION OF 8-AMINOPURINE
S u b s t r a t e : 7 . 4 . i o -~ 21//; XO, i : i o o ; i o -3 M p h o s p h a t e buffer, p H 8.o. T e m p e r a t u r e 28 °. All readings represent t h e difference b e t w e e n this e x p e r i m e n t and a second vessel, containing o n l y s u b s t r a t e and buffer. Catalase, i :50o, was added to b o t h vessels. Time
Change o/ O.D. at
rain
#60 rot*
~73 ra,u
276 mtJ
z9o ra.u
1 3 5 7 9 II I3 25 17 19 22 25 3° 40 55 7° 95 14o 195
0.400
o.915 0.920 0.920 o.915 0.920 o.9I 5 o.915 0.920 0.920 o.9I 5 0.920 o.91o o.9I 5 0.900 0.890 0.900
i.o9o I.O85 1.o8o i.o75 1.065 I.O65 I.O65 I.o68 I.O62 1.o6o 1.o35 I.o25 I.OI8 I.o25 1.O2O I.OI 5
I.O95 I.O9O I.O8 5 1.o75 1.o8o I.O75 I.O65 I.O6Z 1.060 I.O5O 1.o4o I.OI8 I.O2O I.O25 I.O32 1.o4o 1.o6o 1.o85 1.2oo
0.405 o.4Io o.415
0.420 0.430 0.440 0.453 0.465 0.480 o.465 0.44o
TABLE II CHARACTERISATION OF 8-AMINOPURINE AND ITS OXIDATION PRODUCTS Substance
8-Aminopurine 6 - H y d r o x y - 8 - a m i n o p u r i n e (II) 2,6-Dihydroxy-8-aminopurine ( I I I ) 6,8-Dihydroxypurine Uric acid
2raax a~ p H 8.0 (rat*)
243 283 268 289 26i 292
R F value in solvent A*
Fluorescence
0.71
Blue-violet
0.43 0.25 0.45 0.26
Black-violet Black-violet Black-violet Black-violet
Rdative ra~e** o/oxidation ~xanthine = zoo)
i .3 I8 ioo
* Solvent A: 95 ~o alcohol-acetic a c i d - w a t e r (85 : 5 : io) v/v. ** All s u b s t r a t e s were used at 6.5-7.5" i o -5 M ; XO, x :800; p H 8.0. Biochim.
Biophys.
Aaa,
37 (196o) 433-441
437
ENZYMIC OXIDATION OF AMINOPURINES
Chromatographic analysis revealed the presence of 3 spots after a reaction period of I h ('Fig. 2). All 3 components were identified by absorption measurements. 6-Hydroxy-8-aminopurine (II) is clearly distinguished from 6,8-dihydroxypurine by its ~max, although the Re values in the solvents used are practically identical (see Table II). On the other hand, the absorption maximum of (III) is close to that of uric acid and the same is true for the RF values of this pair. Since purified XO may include occasionally small amounts of desaminaseszs, it was deemed necessary to prove that during the oxidation of (I) the amino group had not been lost. Unequivocal evidence for the structure of (III) could be obtained by following the shift of ~max with pH changes. As shown in Fig. 3, the most characteristic difference between (III) and uric acid appears in the acid range, because only the former can give a cation in acid solution, whereas the absorption maximum of uric acid does not change below pH 4.
0 ~uz
c--)
8NHz*XO"
(---)
8NI"~4'XO "°
0
8NI'~
0
end-.6
0
00--
SNl..~..2,6 6,8
:L6,B
Fig. 2. Paper chromatogram of the reaction mixture of 8aminopurine with mammalian xanthine oxidase. Compari- .~ 295 Ison with synthetic products. Substrate: 6. 5. io -s M ; XO, ~ 1 z:IOO; catalase (Worthington), i :500. Temperature 28 °, ~ 1 p H 8.0. Solvent used for development: 95% ethanol| water-glacial acetic acid (85 : IO: 5) V/V. 290 ~* Reaction stopped after i h, by immersing into boiling water for io min. ** Reaction stopped after 24 h. (Numbers indicate position of hydroxyl groups, e.g. 6,8 = 6,8-dihydroxypurine)
285
280
Fig. 3. The long-wave absorption maxima of 2,6-dihydroxy8-aminopurine (III) ( O - - O ) and of uric acid ( 0 - - 0 ) as a function of pH. Between p H o and 4, note the large shift of )[max for (III), in contrast to the unchanged position of the absorption maximum of uric acid.
2
4
6
8
10
12
pH
14
Biochim. Biophys. ,4aa, 37 (196o) 433-441
438
F. BERGMANN, H. KWIETNY, G. LEVIN, H. ENGELBERG
Oxidation o/aminopurines by a bacterial enzyme system 8-Aminopurine was not attacked during 72 h of incubation at 3 o°. Adenine, on the other hand, underwent a slow change. When the supernatant of the bacterial reaction mixture was analysed chromatographically after a 24-h incubation, hypoxanthine was found in amounts roughly equivalent to the decrease in adenine concentration. The desamination product was separated from the starting material by the method of WEISSMANN, BROMBERG AND GUTMAN14. After extraction from the paper, hypoxanthine was identified by measuring its 2max at 3 different pH values (see Table III). TABLE III CONVERSION OF ADENINE INTO HYPOXANTHINE BY P s e u d o m o n a s a e r u g i n o s a BDH A bacterial suspension of 5" lO9 cells was incubated with ioo/zg/ml adenine. Temperature 3 o°, pH 7.5. After 24 h, the mixture was centrifuged and the supernatant subjected to chromatography, using the solvent: n-butanol-i % ammonia (6:1) v/v li. The spots were then extracted and their spectrum measured at 3 different p H values. Absorption maximum (m#) at pH Substa~we
Adenine Hypoxanthine Product, obtained from chromatogram
RF
o.55 o.47 0.46
5.5
xo.5
22.7
259.5 25I 251
256 26o 26o
256 263 262
TABLE IV OXIDATION OF 2-AMINOPLTRINE TO 2-AMINO-8-HYDROXYPURINEBY P s e u d o m o n a s a e r u g i n o s a BDH Experimental conditions as in Table III. Solvent used for chromatography : 95 ~o ethanol-waterglacial acetic acid (85:i o:5) v/v (solvent A); 95 % ethanol-i 2.5 ~o ammonia (8o:2o) v/v (solvent B). RF in solvent
Absorption maximum (mt~) at pH
S~sta~e
2-Aminopurine 2-Amino-8-hydroxypurine Product, obtained from chromatogram Guanine
A
B
5.5
8.0
0.63 o.51
0.62 o.39
304 3o5
304 3o5
o.5o o.46
0.36 o. 37
3o5 246 275
305 246 275
2-Aminopurine was slowly oxidised to its 8-hydroxy derivative. The latter accumulated in the medium, as it is resistant to attack by the bacterial enzyme, in sharp contrast to its behavior towards mammalian XO 3. The oxidation product was identified by its RF value and by the fact that its absorption spectrum is close to that of the starting material 3 (see Table IV). No trace of the alternative oxidation product guanine was found. Methylated adenines as substrates o/ mammalian XO 6-Methylaminopurine (IV), like adenine and kinetin xS, is attacked first at position 8. This is demonstrated in Fig. 4, where a peak at 273 m/~ appears in the B i o c h i m . B i o p h y s . A c t a , 37 (196o) 433-441
439
ENZYMIC OXIDATION. OF AMINOPURINES
intermediary stages of the enzymic reaction and vanishes again towards the end of the oxidation. If the corresponding isoguanine derivative would have been formed, its presence would have been noted by a temporary peak at about 285 m/z1. The results of Fig. 4 are therefore completely analogous to those with kinetin and establish the main course of oxidation, although they do not exclude the possibility that the alternative pathway may be used to a very small extent. When the reaction of (IV) with XO was stopped at an intermediary stage, chromatography indicated the presence of 3 components, corresponding to (IV), a single intermediate (V) and the end-product, 2,8-dihydroxy-6-methylaminopurine (VI) (see Table V). The change of O.D. at 284 m/z, the isosbestic point of (IV) and (VI), is plotted in Fig. 5 as function 1.101~--
OOi( 0.550
>0.90C
. ~ ' O.SO0
f¢ "00.70 C
0.450 0.40
"~0.50C
0/0 2
0.30C
s
o.2ooY I
°
20
~ I
60
I
I
140
I00 Time
I
180
I
220
(rnin)
O. IOC
I 240
280
320
I 360
A (m~) _ Fig. 4. Spectral changes during the enzymic oxidation of 6-methylaminopurine (IV). Substrafe: 6.7- i o -~ M ; other conditions as in Fig. 2. A - - A Zero time: Spectrum of (IV). O - - 0 After 35 rain: Two new peaks have appeared, at 273 m F (V) and 3o2 m p (VI), respectively. O - - O After 9o min: Reaction completed; spectrum of VI.
Fig. 5. Spectroscopic evidence for the formation of an intermediate, bearing an 8-hydroxyl group, during the enzymic oxidation of 6methylaminopurine ( 0 - - 0 ) and adenine ( O - - O ). The changes in O.D. at the isosbestic points of 6-methylaminopurine (IV) and its 2,8-dihydroxy derivative (VI) (284 mp) and of adenine and its final oxidation product (277 m#) are plotted as function of time. XO, i : 2oo; catalase, i : see. Temperature 28 ° ; p H 8.o. Substrates: 6-Methylaminopurine, 6. 7. Io 4 M ; adenine, 4.7" IO-* M.
TABLE V PATHWAY
OF OXIDATION
OF 6-METHYI*AMINOPURINE
BY
MAMMALIAN
XANTHINE
OXIDASE
Substrate, 1.3.io -4 M ; XO, i :2o0; catalase, 1:5oo; IO-8 M phosphate buffer, p H 8.o. Temperature 28 °. The reaction was stopped after 55 and 2oo min respectively by immersion into boiling water for 5 rain. The paper chromatograms were developed with solvent A (see Table IV). Since the reaction products did not fluoresce on paper, each strip was cut into IO sections, parallel to the starting line; each section was extracted with water and the u.v. spectrum of all extracts measured. Therefore the RF values are only approximate. Substance
Starting material (IV) Intermediate (V) End-product (VI)
RF
o.8 o. 7 o.3
Absor~ion maximum (m/~) at pH 5.0
8.0
xo.o
267 272 3o4
267 273 3o2
269 281 3o0
B i o c h i m . B i o p h y s . A c t a , 37 (i96o) 433-441
44 °
F. BERGMANN, H. KWlETNY, G. LEVIN, H. ENGELBERG
of time, for comparison with the analogous curve for adenine. The latter is shown here again, in order to correct a technical error in Fig. 2 of our previous paper is. 6-Dimethylaminopurine, like 7- or 9-methyladenine, was found refractory to XO. Similar observations have been made previously by Dr. G. B. ELIONis. CHaNH ÷xo
H
N
I
I
H
(IV)
H
(v)
CH3NH
H
N\_ °
+xo
I
H
I
H
(vI)
DISCUSSION
All three aminopurines are attacked by mammalian XO in a manner, different from the corresponding hydroxy derivatives. This fact could be explained as follows: I. Due to a systematical difference in structure, the polarity of the aminopurines is reversed as compared to the oxy derivatives. Indeed it has been shown b y BROWN AND MASON17 that the latter exist in the lactam form, whilst the aminopurines all bear free amino groups and thus possess a completely aromatic ring system is. Therefore, if we assume hydration of a CH = N bond to represent the first reaction step, it is conceivable that a carbonyl could direct the attachment of the hydroxyl ion to a point, different from that to which it would cling under the polar influence of an amino group. 2. This interpretation of the experimental data is, however, contradicted by the observation that the pathway of 2-aminopurine in the bacterie 1 oxidation system differs from that with mammalian X0, but is identical with the course, taken by the oxidation of 2-hydroxypurine with the latter enzyme. Clearly, it is not so much the intrinsic polarity of the substrate as the polarity induced by complex formation with the enzyme, that determines the point of attack. Aminopurines, e.g., could combine with X 0 by attaching their amino groups to a negatively charged phosphate group, present in the flavin-adenine-dinucleotiden, or by forming a hydrogen bridge, e.g. with a carbonyl group in the active center. The assumption that the specific mode of attachment of a substrate to the active surface determines the pathway of oxidation is also supported by earlier observations revealing characteristic differences between mammalian and bacterial enzyme. Thus, 7-methylhypoxanthine is refractory towards the former, but is oxidised by the latter to 7-methylxanthine s. Similarly, 3-methylxanthine is converted only by the bacterial enzyme to 3-methyluric acid. Adenine is oxidised by XO only if specific structural requirements are fulfilled: (a) At least one hydrogen atom must be present in the 6-amino group. (b) The imidazole ring must contain a free N H group. The resistance of 6-dimethylaminopurine to enzymic attack may be ascribed to steric hindrance, but such an explanation appears unsatisfactory in view of the fact that the large furfuryl group of kinetin does not prevent reaction at C-8. More probably, requirement (a) indicates hydrogen bonding either to an acceptor group in the enzyme surface or to N-7 in the purine system
(see MASON18): Biochim. Biophys. Aata, 37 (x90o) 433-441
ENZYMIC OXIDATION OF AMINOPURINES
441
N
~N~,,N/ + I
H •
8
7
In this representation of the adenine structure, polarisation of the C = N double bond would make C-8 susceptible to attack by an hydroxyl ion. A choice between the two modes of hydrogen bonding--whether inter- or intramolecularly--could be made by introducing mono- and dimethylamino substituents into positions 2 or 8, where an intramolecular bridge is no longer possible. Experiments in this direction will be described at a later occasion. ACKNOWLEDGEMENTS
The authors wish to express their sincere thanks to Prof. F. BERGEL and Dr. R. C. BRAY for the supply of purified milk xanthine oxidase and to Drs. R. K. ROBINS, A. BENDICH, G. B. ELION and E. C. TAYLORfor the generous gift of purine derivatives. They are also obliged to Mr. M. CHAIMOVlTZand R. KNAFO for their expert technical help. REFERENCES 1 j . B. WYNGAARDEN AND J. T. DUNN, Arch. Biochem. Biophys., 7° (1957) 15o. z D. C. LORZ AND G. H. HITCHINGS, Abstr. z29th Meeting Am. Chem. Soc., (1956) 3o0. 3 F. BERGMANN, G. LEVlN AND H. KWlETNY, Biochim. Biophys. Acta, 3 ° (1958) 509. 4 F. BERGMANN AND S. DIXSTEIN, J. Biol. Chem., 223 (i956) 765 . 5 S. DIKSTEIN, F. BERGMANN AND Y. HENIS, J. Biol. Chem., 224 (1957) 67. 6 R. K. ROBINS, J. Am. Chem. Soc., 8o (1958) 6671. 7 E. FISCHER, Bet., 3 ° (1897) 222o. 8 G. B. ELIgN, E. BLrRGI AND G. H. HITCHINGS, J. Am. Chem. SON., 74 (1952) 411. 9 j . W. DALY AND B. E. CHRISTENSEN, J. Org. Chem., 21 (1956) 177. 1o j . M. GULLAND AND E. R. HOLIDAY, J. Chem. Sot., (1936) 765 • 11 p. G. Avis, F. BERGEL AND R. C. BRAY, J. Chem. Sot., (1956) 1219. 18 B. D. DAVlS AND E. S. MINGIOLI, J. Bacteriol., 6o (195 o) i2. 18 J. B. WYNGAARDRN, J. Biol. Chem., 224 (1957) 453. 14 B. WEISSMANN, P. A. BROMBERG AND A. B. GUTMAN, Proc. Soc. Exptl. Biol. Med., 87 (1954) 257. 1~ F. BERGMANN AND H. KWlETNY, Biochim. Biophys. Acta, 28 (1958) IOO. xe G-. B. ELION, p r i v a t e c o m m u n i c a t i o n . 17 D. J. BROWN AND S. F. MASON, J. Chem. Sot., (1957) 682. 18 S. F. MASON, J. Chem. Soc., (1954) 2o71.
Biochim. Biophys. Aaa, 37 (196o) 433-441
43~
REFERENCES I R. C. WARNER, J. Biol. Chem., 229 (I957) 711. 2 G. FELSENF~LD AND A. RICH, Biochim. Biophys. Acta, 26 (1957) 457. 8 G. FELSESFELD, D. R. DAVmS AND A. RICH, ]. Am. Chem. Soc., 79 (1957) 2023. 4 G. FELSENFELD AND S. HUANG, Biochim. Biophys. Acta, 34 (1959) 234. 8 j . R. FRESCO AND P. DorY, J. Am. Chem. Soc., 79 (1957) 3928. 6 R. F. BEERS AND R. F. SrEINER, Nature, 179 (1957) lO76. 7 A. M. MICH~LSON, Nature, 182 (1958) 15o2. s j . SHACK, D. J. JENKINS AND J. M. THOMPSETT, ]. Biol. Chem., 203 (I953) 373. 9 R. THOMAS, Biochim. Biophys. Acta, 14 (1954) 231. t0 F. E. HARRIS AND S. A. RICE, J. Phys. Chem., 61 (1957) 136o. 11 C. D. JARDErZKY, J. Am. Chem. Soc., 80 (1958) 1125. 18 j . S. WIBERG AND W. F. NEUMAN, Arch. Biochem. Biophys., 72 (1957) 66. 18 G. ZUBAYAND P. DoTe, Biochim. Biophys. Acta, 29 (1958) 47. It B. N. AMES, D. T. DUBIN AND S. M. ROSENXHAL, Science, 127 (1958) 814.
Biochim. Biophys. Aaa, 37 (196o) 425-433
STUDIES ON T H E ENZYMIC OXIDATION OF AMINOPURINES F E L I X BERGMANN, HANNA KWIETNY*, GERSHON LEVIN* AND HANNA E N G E L B E R G
Department o[ Pharmacology, The Hebrew University, Hadassah Medical School, Jerusalem (Israel) (Received April 24th, I959)
SUMMARY
I. 8-Aminopurine is oxidised by mammalian xanthine oxidase along the following pathway: 8-Aminopurine ---> 6-hydroxy-8-aminopurine ~
2,6-dihydroxy-8-aminopurine
It is thus evident that oxidation of all three aminopurines takes a course different from the corresponding hydroxypurines. 2. Adenine and 6-methylaminopurine are oxidised along the same pathway, but all other methyl derivatives of adenine are refractory. 3-2-Aminopurine is converted by a bacterial enzyme system into 2-amino8-hydroxypurine. These results are discussed in terms of specific enzyme--substrate complexes.
INTRODUCTION WYNGAA~DEN AND DUNN I h a v e r e c e n t l y e s t a b l i s h e d t h a t a d e n i n e is a t t a c k e d b y m a m m a l i a n x a n t h i n e o x i d a s e (XO) first a t p o s i t i o n 8 a n d t h e r e a f t e r a t c a r b o n a t o m ~. T h e o r d e r of s t e p s is t h u s a t v a r i a n c e w i t h t h e s e q u e n c e , a p p l y i n g t o t h e e n z y m i c o x i d a t i o n of h y p o x a n t l f i n e : Hypoxanthine --~ xanthine ~ uric acid Adenine ~ 8-hydroxyadenine ~ 2,8-dihydroxyadenine. * The results of this investigation are parts of Ph.D. theses, submitted to the Faculty of Science, The Hebrew University, Jerusalem.
Biochim. Biophys. Aaa, 37 (196o) 433-441
434
F. BERGMANN, H. KWIETNY, G. LEVIN, H. ENGELBERG
Analogously, 2-aminopurine is converted into guanine, as shown b y LORZ AND I-IITCHINGS2 and the present authors a, in contrast to the behavior of 2-hydroxypurine: 2-Hydroxypurine --+ 2,8-dihydroxypurine --+ uric acid 2-Aminopurine ~ guanine (= 2-amino-6-hydroxypurine) In the present study, we find that 8-aminopurine (I) too differs characteristically from 8-hydroxypurine in its behavior towards XO: 8-Hydroxypurine ~ 2,8-dihydroxypurine ~ uric acid4 8-Aminopurine ~ 6-hydroxy-8-aminopurine --+ 2,6-dihydroxy-8-aminopurine Thus, a systematic difference exists between these two series of purines, which m a y be due either to a common structural difference, responsible for the specific electron distribution and the polarity in each group, or to a different mode of attachment of hydroxy and amino derivatives to the active center of the enzyme. An answer to this problem was sought b y testing the oxidation of aminopurines by a bacterial enzyme system, described previously ~. In the formation of the enzyme-substrate (ES) complex, hydrogen bonding plays a decisive role. The groups participating in the binding of adenine to XO have been determined b y systematically substituting methyl for hydrogen at all available nitrogen atoms. The results obtained give some information on the "active form" of this substrate, actually involved in the enzymic process. MATERIALS AND METHODS
Purine derivatives 6-Hydroxy-8-aminopurine e was a gift of Dr. R. K. ROBINS, Arizona State College, Tempe, Arizona. 2,6-Dihydroxy-8-aminopurine 7 was obtained through the courtesy of Dr. A. BENDICH, the Sloan-Kettering Institute for Cancer Research, New York. 6-Methylamino- and 6-dimethylaminopurine were synthetic products, prepared b y Dr. G. B. ELION of the Wellcome Research Laboratories, Tuckahoe, New York s. Likewise, 9-methyladenine 9 was supplied by Dr. ELION. Finally, 7-methyladenine 1° was a gift from Dr. E. C. TAYLOR, Princeton University.
Enzymes Highly purified milk xanthine oxidase was supplied by Prof. F. BERGEL and Dr. R. C. BRAY of the Chester Beatty Institute of Cancer Research, London, England n. This preparation, when diluted I:8OO, produced I/zg/ml of uric acid/rain, when the reaction was carried out at 28 ° and p H 8.0 with 6.5" IO-s M xanthine as substrate. As source of the bacterial enzyme system, whole cells of Pseudomonas aeruginosa BDH, an air-borne strain described previously s, were used. Identical results were obtained with resting cells or with a culture growing in a hqnid medium lz. However, growing cells exhibited a faster turnover and thus facihtated analysis of the oxidation products. They were therefore used preferentially.
Methods o/analysis The progress of the reactions with mammalian XO was followed spectrophotometrically, using a Beckman thermospacer for temperature control. With the bacterial Biochim. Biophys. Acta, 37 (196o) 433-441
435
ENZYMIC OXIDATION OF AMINOPURINES
system, this method is applicable only for fast reactions since the cells slowly secrete a greenish pigment, which exhibits unspecific absorption. At suitable intervals, samples of the bacterial reaction mixtures, kept at 3°0 , were centrifuged at 3000 rev./min and the supernatant used for spectral measurements. All runs were submitted to paperchromatographic analysis. After development, the individual spots were located by means of their fluorescence under a Mineralight u.v. lamp, emitting light of about 255 m/~ wave length. They were then extracted with water and their spectrum measured. When necessary, the shift of Slmaxwith pH was also determined (see under RESULTS). RESULTS
Reaction o/8-aminopurine with mammalian xanthine oxidase Preliminary experiments indicated that oxidation of (I) passes through the 6-hydroxy derivative (II). The formation of the alternative intermediate, viz. 2-hydroxy-8-arninopurine--which is unknown--, would have been recognised easily by a temporary shift of the absorption maximum to higher wave lengths, because all 2,8-disubstituted purines exhibit maxima above 3oo mr*. Fig. I shows the absorption
4.1
3.9
t! 240
260
280
A (rap)
300
Fig. I. U.v. absorption spectra'of 8-aminopunne and its oxidation products at pH 8.o. A 8-Aminopurine (I). 0
0 6 - H y d r o x y - 8 - a m i n o p u r i n c (II). • purine (III).
A • 2,6-Dihydroxy-8 -amino-
spectra of (I), the intermediate (II) and the end-product (III). At 276 m/~, the isosbestic point of (II) and (III), further oxidation of the intermediate produces no change. Therefore, the initial decrease of optical density (O.D.) at this wave length O
H /N
H
(i)
O +
H
N~_
H
(ii)
all) Biochim. Biopkys. Aaa, 37 (196o) 433-441
436
F. B E R G M A N N , H. K W l E T N Y , G. L E V I N , H. E N G E L B E R G
measures the first oxidation step (see Table I). The experimental data also demonstrate that at 273 m/~, the isosbestic point of (I) and (II), the 0.D. remains constant for the first half hour and decreases only later, when sufficient intermediate has accumulated to give measurable rates of formation of (III). At 260 m/~, tile O.D. increases for the first one and a half hour, as required by the curves in Fig. I, and then again decreases. The reverse behavior is found at 290 mix. All data taken together establish beyond doubt that (II) is the main, if not the only intermediate in the enzymic oxidation of (I). The rate of the conversion (I) --~ (II) is compared in Table II with that of the second step, (II) --~ (III), which was determined at 268 and 290 mtz. The latter conversion proceeds at a rate more than IO times higher than that of the first step, when equimolar substrate concentrations are used. TABLE I SPRCTI~kL CHANGES DURING THE OXIDATION OF 8-AMINOPURINE
S u b s t r a t e : 7 . 4 . i o -~ 21//; XO, i : i o o ; i o -3 M p h o s p h a t e buffer, p H 8.o. T e m p e r a t u r e 28 °. All readings represent t h e difference b e t w e e n this e x p e r i m e n t and a second vessel, containing o n l y s u b s t r a t e and buffer. Catalase, i :50o, was added to b o t h vessels. Time
Change o/ O.D. at
rain
#60 rot*
~73 ra,u
276 mtJ
z9o ra.u
1 3 5 7 9 II I3 25 17 19 22 25 3° 40 55 7° 95 14o 195
0.400
o.915 0.920 0.920 o.915 0.920 o.9I 5 o.915 0.920 0.920 o.9I 5 0.920 o.91o o.9I 5 0.900 0.890 0.900
i.o9o I.O85 1.o8o i.o75 1.065 I.O65 I.O65 I.o68 I.O62 1.o6o 1.o35 I.o25 I.OI8 I.o25 1.O2O I.OI 5
I.O95 I.O9O I.O8 5 1.o75 1.o8o I.O75 I.O65 I.O6Z 1.060 I.O5O 1.o4o I.OI8 I.O2O I.O25 I.O32 1.o4o 1.o6o 1.o85 1.2oo
0.405 o.4Io o.415
0.420 0.430 0.440 0.453 0.465 0.480 o.465 0.44o
TABLE II CHARACTERISATION OF 8-AMINOPURINE AND ITS OXIDATION PRODUCTS Substance
8-Aminopurine 6 - H y d r o x y - 8 - a m i n o p u r i n e (II) 2,6-Dihydroxy-8-aminopurine ( I I I ) 6,8-Dihydroxypurine Uric acid
2raax a~ p H 8.0 (rat*)
243 283 268 289 26i 292
R F value in solvent A*
Fluorescence
0.71
Blue-violet
0.43 0.25 0.45 0.26
Black-violet Black-violet Black-violet Black-violet
Rdative ra~e** o/oxidation ~xanthine = zoo)
i .3 I8 ioo
* Solvent A: 95 ~o alcohol-acetic a c i d - w a t e r (85 : 5 : io) v/v. ** All s u b s t r a t e s were used at 6.5-7.5" i o -5 M ; XO, x :800; p H 8.0. Biochim.
Biophys.
Aaa,
37 (196o) 433-441
437
ENZYMIC OXIDATION OF AMINOPURINES
Chromatographic analysis revealed the presence of 3 spots after a reaction period of I h ('Fig. 2). All 3 components were identified by absorption measurements. 6-Hydroxy-8-aminopurine (II) is clearly distinguished from 6,8-dihydroxypurine by its ~max, although the Re values in the solvents used are practically identical (see Table II). On the other hand, the absorption maximum of (III) is close to that of uric acid and the same is true for the RF values of this pair. Since purified XO may include occasionally small amounts of desaminaseszs, it was deemed necessary to prove that during the oxidation of (I) the amino group had not been lost. Unequivocal evidence for the structure of (III) could be obtained by following the shift of ~max with pH changes. As shown in Fig. 3, the most characteristic difference between (III) and uric acid appears in the acid range, because only the former can give a cation in acid solution, whereas the absorption maximum of uric acid does not change below pH 4.
0 ~uz
c--)
8NHz*XO"
(---)
8NI"~4'XO "°
0
8NI'~
0
end-.6
0
00--
SNl..~..2,6 6,8
:L6,B
Fig. 2. Paper chromatogram of the reaction mixture of 8aminopurine with mammalian xanthine oxidase. Compari- .~ 295 Ison with synthetic products. Substrate: 6. 5. io -s M ; XO, ~ 1 z:IOO; catalase (Worthington), i :500. Temperature 28 °, ~ 1 p H 8.0. Solvent used for development: 95% ethanol| water-glacial acetic acid (85 : IO: 5) V/V. 290 ~* Reaction stopped after i h, by immersing into boiling water for io min. ** Reaction stopped after 24 h. (Numbers indicate position of hydroxyl groups, e.g. 6,8 = 6,8-dihydroxypurine)
285
280
Fig. 3. The long-wave absorption maxima of 2,6-dihydroxy8-aminopurine (III) ( O - - O ) and of uric acid ( 0 - - 0 ) as a function of pH. Between p H o and 4, note the large shift of )[max for (III), in contrast to the unchanged position of the absorption maximum of uric acid.
2
4
6
8
10
12
pH
14
Biochim. Biophys. ,4aa, 37 (196o) 433-441
438
F. BERGMANN, H. KWIETNY, G. LEVIN, H. ENGELBERG
Oxidation o/aminopurines by a bacterial enzyme system 8-Aminopurine was not attacked during 72 h of incubation at 3 o°. Adenine, on the other hand, underwent a slow change. When the supernatant of the bacterial reaction mixture was analysed chromatographically after a 24-h incubation, hypoxanthine was found in amounts roughly equivalent to the decrease in adenine concentration. The desamination product was separated from the starting material by the method of WEISSMANN, BROMBERG AND GUTMAN14. After extraction from the paper, hypoxanthine was identified by measuring its 2max at 3 different pH values (see Table III). TABLE III CONVERSION OF ADENINE INTO HYPOXANTHINE BY P s e u d o m o n a s a e r u g i n o s a BDH A bacterial suspension of 5" lO9 cells was incubated with ioo/zg/ml adenine. Temperature 3 o°, pH 7.5. After 24 h, the mixture was centrifuged and the supernatant subjected to chromatography, using the solvent: n-butanol-i % ammonia (6:1) v/v li. The spots were then extracted and their spectrum measured at 3 different p H values. Absorption maximum (m#) at pH Substa~we
Adenine Hypoxanthine Product, obtained from chromatogram
RF
o.55 o.47 0.46
5.5
xo.5
22.7
259.5 25I 251
256 26o 26o
256 263 262
TABLE IV OXIDATION OF 2-AMINOPLTRINE TO 2-AMINO-8-HYDROXYPURINEBY P s e u d o m o n a s a e r u g i n o s a BDH Experimental conditions as in Table III. Solvent used for chromatography : 95 ~o ethanol-waterglacial acetic acid (85:i o:5) v/v (solvent A); 95 % ethanol-i 2.5 ~o ammonia (8o:2o) v/v (solvent B). RF in solvent
Absorption maximum (mt~) at pH
S~sta~e
2-Aminopurine 2-Amino-8-hydroxypurine Product, obtained from chromatogram Guanine
A
B
5.5
8.0
0.63 o.51
0.62 o.39
304 3o5
304 3o5
o.5o o.46
0.36 o. 37
3o5 246 275
305 246 275
2-Aminopurine was slowly oxidised to its 8-hydroxy derivative. The latter accumulated in the medium, as it is resistant to attack by the bacterial enzyme, in sharp contrast to its behavior towards mammalian XO 3. The oxidation product was identified by its RF value and by the fact that its absorption spectrum is close to that of the starting material 3 (see Table IV). No trace of the alternative oxidation product guanine was found. Methylated adenines as substrates o/ mammalian XO 6-Methylaminopurine (IV), like adenine and kinetin xS, is attacked first at position 8. This is demonstrated in Fig. 4, where a peak at 273 m/~ appears in the B i o c h i m . B i o p h y s . A c t a , 37 (196o) 433-441
439
ENZYMIC OXIDATION. OF AMINOPURINES
intermediary stages of the enzymic reaction and vanishes again towards the end of the oxidation. If the corresponding isoguanine derivative would have been formed, its presence would have been noted by a temporary peak at about 285 m/z1. The results of Fig. 4 are therefore completely analogous to those with kinetin and establish the main course of oxidation, although they do not exclude the possibility that the alternative pathway may be used to a very small extent. When the reaction of (IV) with XO was stopped at an intermediary stage, chromatography indicated the presence of 3 components, corresponding to (IV), a single intermediate (V) and the end-product, 2,8-dihydroxy-6-methylaminopurine (VI) (see Table V). The change of O.D. at 284 m/z, the isosbestic point of (IV) and (VI), is plotted in Fig. 5 as function 1.101~--
OOi( 0.550
>0.90C
. ~ ' O.SO0
f¢ "00.70 C
0.450 0.40
"~0.50C
0/0 2
0.30C
s
o.2ooY I
°
20
~ I
60
I
I
140
I00 Time
I
180
I
220
(rnin)
O. IOC
I 240
280
320
I 360
A (m~) _ Fig. 4. Spectral changes during the enzymic oxidation of 6-methylaminopurine (IV). Substrafe: 6.7- i o -~ M ; other conditions as in Fig. 2. A - - A Zero time: Spectrum of (IV). O - - 0 After 35 rain: Two new peaks have appeared, at 273 m F (V) and 3o2 m p (VI), respectively. O - - O After 9o min: Reaction completed; spectrum of VI.
Fig. 5. Spectroscopic evidence for the formation of an intermediate, bearing an 8-hydroxyl group, during the enzymic oxidation of 6methylaminopurine ( 0 - - 0 ) and adenine ( O - - O ). The changes in O.D. at the isosbestic points of 6-methylaminopurine (IV) and its 2,8-dihydroxy derivative (VI) (284 mp) and of adenine and its final oxidation product (277 m#) are plotted as function of time. XO, i : 2oo; catalase, i : see. Temperature 28 ° ; p H 8.o. Substrates: 6-Methylaminopurine, 6. 7. Io 4 M ; adenine, 4.7" IO-* M.
TABLE V PATHWAY
OF OXIDATION
OF 6-METHYI*AMINOPURINE
BY
MAMMALIAN
XANTHINE
OXIDASE
Substrate, 1.3.io -4 M ; XO, i :2o0; catalase, 1:5oo; IO-8 M phosphate buffer, p H 8.o. Temperature 28 °. The reaction was stopped after 55 and 2oo min respectively by immersion into boiling water for 5 rain. The paper chromatograms were developed with solvent A (see Table IV). Since the reaction products did not fluoresce on paper, each strip was cut into IO sections, parallel to the starting line; each section was extracted with water and the u.v. spectrum of all extracts measured. Therefore the RF values are only approximate. Substance
Starting material (IV) Intermediate (V) End-product (VI)
RF
o.8 o. 7 o.3
Absor~ion maximum (m/~) at pH 5.0
8.0
xo.o
267 272 3o4
267 273 3o2
269 281 3o0
B i o c h i m . B i o p h y s . A c t a , 37 (i96o) 433-441
44 °
F. BERGMANN, H. KWlETNY, G. LEVIN, H. ENGELBERG
of time, for comparison with the analogous curve for adenine. The latter is shown here again, in order to correct a technical error in Fig. 2 of our previous paper is. 6-Dimethylaminopurine, like 7- or 9-methyladenine, was found refractory to XO. Similar observations have been made previously by Dr. G. B. ELIONis. CHaNH ÷xo
H
N
I
I
H
(IV)
H
(v)
CH3NH
H
N\_ °
+xo
I
H
I
H
(vI)
DISCUSSION
All three aminopurines are attacked by mammalian XO in a manner, different from the corresponding hydroxy derivatives. This fact could be explained as follows: I. Due to a systematical difference in structure, the polarity of the aminopurines is reversed as compared to the oxy derivatives. Indeed it has been shown b y BROWN AND MASON17 that the latter exist in the lactam form, whilst the aminopurines all bear free amino groups and thus possess a completely aromatic ring system is. Therefore, if we assume hydration of a CH = N bond to represent the first reaction step, it is conceivable that a carbonyl could direct the attachment of the hydroxyl ion to a point, different from that to which it would cling under the polar influence of an amino group. 2. This interpretation of the experimental data is, however, contradicted by the observation that the pathway of 2-aminopurine in the bacterie 1 oxidation system differs from that with mammalian X0, but is identical with the course, taken by the oxidation of 2-hydroxypurine with the latter enzyme. Clearly, it is not so much the intrinsic polarity of the substrate as the polarity induced by complex formation with the enzyme, that determines the point of attack. Aminopurines, e.g., could combine with X 0 by attaching their amino groups to a negatively charged phosphate group, present in the flavin-adenine-dinucleotiden, or by forming a hydrogen bridge, e.g. with a carbonyl group in the active center. The assumption that the specific mode of attachment of a substrate to the active surface determines the pathway of oxidation is also supported by earlier observations revealing characteristic differences between mammalian and bacterial enzyme. Thus, 7-methylhypoxanthine is refractory towards the former, but is oxidised by the latter to 7-methylxanthine s. Similarly, 3-methylxanthine is converted only by the bacterial enzyme to 3-methyluric acid. Adenine is oxidised by XO only if specific structural requirements are fulfilled: (a) At least one hydrogen atom must be present in the 6-amino group. (b) The imidazole ring must contain a free N H group. The resistance of 6-dimethylaminopurine to enzymic attack may be ascribed to steric hindrance, but such an explanation appears unsatisfactory in view of the fact that the large furfuryl group of kinetin does not prevent reaction at C-8. More probably, requirement (a) indicates hydrogen bonding either to an acceptor group in the enzyme surface or to N-7 in the purine system
(see MASON18): Biochim. Biophys. Aata, 37 (x90o) 433-441
ENZYMIC OXIDATION OF AMINOPURINES
441
N
~N~,,N/ + I
H •
8
7
In this representation of the adenine structure, polarisation of the C = N double bond would make C-8 susceptible to attack by an hydroxyl ion. A choice between the two modes of hydrogen bonding--whether inter- or intramolecularly--could be made by introducing mono- and dimethylamino substituents into positions 2 or 8, where an intramolecular bridge is no longer possible. Experiments in this direction will be described at a later occasion. ACKNOWLEDGEMENTS
The authors wish to express their sincere thanks to Prof. F. BERGEL and Dr. R. C. BRAY for the supply of purified milk xanthine oxidase and to Drs. R. K. ROBINS, A. BENDICH, G. B. ELION and E. C. TAYLORfor the generous gift of purine derivatives. They are also obliged to Mr. M. CHAIMOVlTZand R. KNAFO for their expert technical help. REFERENCES 1 j . B. WYNGAARDEN AND J. T. DUNN, Arch. Biochem. Biophys., 7° (1957) 15o. z D. C. LORZ AND G. H. HITCHINGS, Abstr. z29th Meeting Am. Chem. Soc., (1956) 3o0. 3 F. BERGMANN, G. LEVlN AND H. KWlETNY, Biochim. Biophys. Acta, 3 ° (1958) 509. 4 F. BERGMANN AND S. DIXSTEIN, J. Biol. Chem., 223 (i956) 765 . 5 S. DIKSTEIN, F. BERGMANN AND Y. HENIS, J. Biol. Chem., 224 (1957) 67. 6 R. K. ROBINS, J. Am. Chem. Soc., 8o (1958) 6671. 7 E. FISCHER, Bet., 3 ° (1897) 222o. 8 G. B. ELIgN, E. BLrRGI AND G. H. HITCHINGS, J. Am. Chem. SON., 74 (1952) 411. 9 j . W. DALY AND B. E. CHRISTENSEN, J. Org. Chem., 21 (1956) 177. 1o j . M. GULLAND AND E. R. HOLIDAY, J. Chem. Sot., (1936) 765 • 11 p. G. Avis, F. BERGEL AND R. C. BRAY, J. Chem. Sot., (1956) 1219. 18 B. D. DAVlS AND E. S. MINGIOLI, J. Bacteriol., 6o (195 o) i2. 18 J. B. WYNGAARDRN, J. Biol. Chem., 224 (1957) 453. 14 B. WEISSMANN, P. A. BROMBERG AND A. B. GUTMAN, Proc. Soc. Exptl. Biol. Med., 87 (1954) 257. 1~ F. BERGMANN AND H. KWlETNY, Biochim. Biophys. Acta, 28 (1958) IOO. xe G-. B. ELION, p r i v a t e c o m m u n i c a t i o n . 17 D. J. BROWN AND S. F. MASON, J. Chem. Sot., (1957) 682. 18 S. F. MASON, J. Chem. Soc., (1954) 2o71.
Biochim. Biophys. Aaa, 37 (196o) 433-441