- Email: [email protected]
Estimation of trace amounts of inosinic acid formed by the deamination of adenylic acid with alkali
BIOCHIMICAET BIOPHYSICAACTA
167
BBA 97062
E S T I M A T I O N OF T R A C E AMOUNTS OF INOSINIC ACID FORMED BY T H E D E A M I N A T I O N OF A D E N Y L I C ACID W I T H ALKALI* P. I{. GANGULI, A. REINER AND L. GYENES Department o/ Microbiology and Immunology, Faculty o/ Medicine, University o/ Montreal, Montreal, Quebec (Canada)
(Received August 9th, I97 I)
SUMMARY The deamination of 2'(3')-AMP with alkali under the conditions used for the hydrolysis of RNA (o.I M KOH, IOO°, 20 min or 0.3 M KOH, 37 °, 18 h) was estimated after separating IMP from AMP by cation-exchange chromatography on Dowex 5o(H+). Before treatment with alkali, the commercial prepalation of AMP was purified b y this chromatographic method to remove impurities. Within the concentration range of o.13-14.o IV[,the extent of deamination of AMP was o.I4~-o.o3 %, as determined spectrophotometrically from the recovered nucleotides.
INTRODUCTION Many of the minor ribonucleotides which were first reported to b~ present in the t R N A ' s 1,~ have been subsequently found in the higher molecular weight cellular RNA fractions 3. Of these, however, the presence of IMP has not yet been reported in any RNA other than some of the tRNA's. As has been proposed recently4, the presence of IMP in mRNA could explain, through a variable translational mechanism, the synthesis of polymorphic proteins, such as the immunoglobulins. Since IMP, if present, could be expected only in trace amounts, its separation, identification and quantitation would require large scale hydrolysis of RNA. For this purpose alkaline hydrolysis into monoribonucleotides 5 is a convenient method. However, KAMMEN AND SPENGLER6 have recently reported that under the conditions of alkaline hydrolysis 0.3-0.35 % deamination of the adenosine moiety occurred, irrespective of whether it was present as a nucleoside, nucleotide or polynucleotide. In their study the deamination of AMP was estimated by an indirect method, using isotopically labelled compounds which were fractionated by anion-exchange chromatography after the alkali treatment. In view of the importance of the accurate estimation of the deamination of AMP for the detection and/or quantitation of IMP in the hydrolysate of high molecular weight mammalian RNA, the present work was carried out to verify the extent of deamination of AMP. The direct spectrophotometric determination of IMP, separated by cation-exchange chromatography, showed a lesser extent of deamination of 2'(3')-AMP with alkali than reported by KAMMEN AND SPENGLER6. * Preliminary results of this study were presented at the i4th Annual Meeting of the Canadian Federation of Biological Societies, Toronto, 1971. Biochim. Biophys. Acta, 254 (1971) 167-171
168
P.K.
GANGULI
Cl al.
EXPERIMENTAL
As described earlier v, I M P can be recovered in a small volume, clearly separated from the four m a j o r ribonucleotides, b y c h r o m a t o g r a p h y on the strong-acid cation-exchanger Dowex 5o(H+). In this procedure the sample is applied in o.o5 M HC1 (or HC104) , UlV[P is eluted with o.o 5 M acid, I M P with o.oo5 M acid and GMP, followed b y the mixture of CMP and AMP, with water. In the present s t u d y highly reproducible separation between I M P and A M P was obtained with this technique under the conditions described in Table I and the separation was similar on the three different columns. TABLE I CONDITIONS OF CATION EXCHANG~ CHROMATOGRAPHY
From the Dowex 5o(FI+) (2OO-4OOmesh, 4 % cross-linked) columns of increasing size IMP was recovered within 4, IO and 25 ml and AMP within 4o, 200 and 7o0 ml of effluent, respectively. The volume of 0.05 M HC104 includes the sample volume. Column size l(cm) × d(cm)
Amount o] sample
(A260nm)
Sample volume in 0.05 M HCIO 4 (ml)
i-2
Volume o[ eluant (ml) 0.05 M HCIO 4
0.005 M HCIO,
Water
Flow rate by gravity (ml/min)
io × 0.9
3o-60
7
io
60
24 )< 0 . 9
500-800
Up to 4
15
20
250
o.2-o. 3 o . I o.15
15 × 2
98o-13oo
Up to 20
55
7°
9°0
0.5-0.7
RESULTS AND DISCUSSION
The commercial preparation (Calbiochem) of 2'(3')-AMP used in this s t u d y contained about i % of impurities, represented b y the two minor components in the c h r o m a t o g r a m (Fig. i, top). The first component, corresponding to 0.32 o~, of the total, was eluted in the UMP region and showed spectral characteristics of UMP (ref. 8). The second component, corresponding to 0.66 % of the total, emerged in the I ~ P region, i.e. near the Io-ml effluent volume, a n d showed spectral characteristics of A M P (ref. 8). This component was further resolved into two constituents b y descending paper c h r o m a t o g r a p h y in the (NH4)2SO4-ethanol systemg: the m a j o r constituent (about 9 ° %) had AMP-like spectra at p H 3, 7, and 13 b u t m o v e d much slower t h a n 2' (3')-AMP, i.e. in the position of 2' (3')-CMP, and the minor constituent (about IO %) h a d the properties characteristic for 2'(3')-IMP. The AMP-like material was precipitable with ZnSO4-Ba(OH)z (ref. IO) and was stable to t r e a t m e n t with I M K O H (23 °, 24 h), o.I IV[HC1 (23 °, 4 h) and snake v e n o m phosphodiestelase, as d e m o n s t r a t e d b y its u n c h a n g e d chromatographic behaviour following these treatments. F r o m these results is can be concluded t h a t this c o m p o u n d is not cyclic AMP (refs. IO, I I ) . The orcinol test 12 gave 1.12 times higher colour intensity at 660 nm v ith this c o m p o u n d t h a n with A M P used at the same concentration (same A,59 nm units). Therefore, it would appear t h a t this c o m p o u n d might have an e at 259 nm about IO % lower t h a n AMP and is not identifiable as 9-E2'(3')-0-fibosyl-fl-D-ribofuranosyl]adenine which also has the spectral characteristics of AMP (ref. 13). Biochim. Biophys. Acta, 254 (i97 I) i67-17i
ESTIMATION OF IMP
0.2 0.05M.O.O05M
o tll o/
0.5 0.4 0.3 E
0.2
, /1
0
10
169
FROM DEAMINATION OF AMP
'--'/2 20
30
H20
).05MO.O05M
40
50
~
'
~
1,30
40
50
60
1
,~ 0.1 "
20
60
70
0.5 0.4 0.3 0.2
).05M
O.OC6M
2O
1 0
10 2'0 " 30 Volume of effluent (ml)
40
50
F i g . I. C h r o m a t o g r a p h y of A M P on D o w e x 5 o ( H +) c o l u m n ( i o c m X o . 9 cm) before and after t r e a t m e n t with alkali. The chromatograms were recorded with an L K B U v i c o r d at 253. 7 n m using a 2 m m flow cell. Top: Commercial A M P showing impurities in the U M P and I M P regions; the large double p e a k eluted with water represents 2'(3')-AMP. Middle: Purified A M P showing the absence of c o n t a m i n a n t s in the I M P region. B o t t o m : D e t e c t i o n of I M P formed b y the alkali t r e a t m e n t of the purified A M P .
Moreover, preliminary results obtained with chromatography on Sephadex G-Io and G-25 indicated that the elution volume of this compound was identical with that of AMP. The identification of this compound is under further investigation. The commercial preparation of 2'(3')-AMP was purified on a large scale using the 15 cm × 2 cm column and the purified preparation was concentrated by freezedrying and clarified. This preparation was devoid of the contaminant in the IMP region, as observed by chromatography on a IO cm × 0. 9 cm column (Fig. I, middle); the small peak eluted with 0.05 M acid represented non-nucleotide material, presumably soluble particles of resin, carried over from the previous chromatographic step. This material showed a high A320 nm and a continuous linear increase at lower wavelengths which is characteristic of light scattering. When the purified AMP preparation was treated with alkali under the conditions indicated in Table n and analyzed by chromatography on a IO cm x 0. 9 cm column, the presence of a trace amount of IMP, formed by the deamination of AMP, was visible in the chromatogram (Fig. I, bottom). To obtain sufficient amount of IMP for quantitative estimation, chromatography of the alkali-treated AMP was carried out on a larger column. The eluate containing IMP was neutralized with KOH, concentrated by freeze-drying and clarified by centrifugation to remove KCI04. Biochim. Biophys. Acta, 2 5 4 ( 1 9 7 1 ) 1 6 7 - 1 7 1
17o TABLE
P.K. GANGULIel al. ii
E S T I M A T I O N O F T t I E D E A M I N A T I O N OF 2 ' ( 3 ' ) - A M P
Chromatographically pure AMP, after treatment with KOH, was brought to a n d a f t e r c l a r i f i c a t i o n i t w a s c h r o m a t o g r a p h e d o n e i t h e r (a) 24 c m × 0.9 cnl c o l u m n . A M P a n d I3,{P w e r e e s t i m a t e d u s i n g ~(259 n m ) = i5.4/mmole at pH i 2 . 2 / m m o l e a t p H 3, r e s p e c t i v e l y . T h e p e r c e n t d e a m i n a t i o n w a s c a l c u l a t e d n u c l e o t i d e s . F o r e x p e r i m e n t a l d e t a i l s see t h e t e x t .
Conditions o / a l k a l i treatment A M P ~oncn. K O H conch. (M ) (M ) 14.o 4 4.88 4.29 3.87 o.I 3
o.i 0. 3 o.I 0. 3 0. 3
( i o o °, ( 37 °, (IOO °, ( 37 '~, ( 37 ° ,
2o I8 20 18 18
Recovery after chromatography
rain) h) nlin) h) h)
.4MP (mmoles )
IMP (mmole )
29.oo 85.11 42.36 46.76 63.16
o.o4o O.lO 3 o.o66 0.088 0.063
(a) (b) (a) (a) (b)
p H 1. 7 w i t h H C 1 0 a o r (b) 15 c m x 2 c m 7 a n d e (248 n m ) = from the recovered
Mole 0,6 deamination o[ d M P
o.i37 o.125 o.156 o.188 o.ioo Yfean ± S . E . o . i 4 i i o . o 3
This concentrated material showed an ultraviolet spectrum characteristic of I1VfP (2max = 248 nm) which, however, was modified by the light-scattering contribution from the resin particles. The latter were removed b y rechromatography on a io cm × 0. 9 cm column and IMP was estimated spcctrophotometrically, taking into account the 20 % loss due to manipulation (see below). On the other hand, AMP was estimated directly from the large columns; recovery of AMP was of the order of 87-95 %. The results of five experiments indicated that the two conditions of alkaline hydrolysis of RNA did not produce a significantly different degree of deamination of AMP (Table II). Moreover, the concentration of AMP during the treatment with alkali did not significantly influence the extent of deamination. Since the average value of o.14 % deamination of AMP was less than half of that reported by KAMMEN AND SPENGLER 6, the possibility of underestimating IMP in the present study was carefully considered. 2' (3')-IMP was prepared from 2' (3')-A~P b y deamination with HNO., according to the method of KAPLAN14. This preparation was then subjected to all the handling and experimental conditions used in this study, such as storage at 4 ° in o.oo 5 IV[ HC1Oa, neutralization with KOH, freeze-drying, clarification and rechromatography in the presence or absence of AMP. Except for about 2o % loss during the steps of freeze-drying and clarification, there was no significant loss of IMP. Moreover, a n y partial conversion of 2'(3')-IMP to inosine or hypoxanthine would have been detected in the present study either as a lower recovery of IMP in the cation-exchange chromatography or b y the appearance of additional spots in paper chromatography. As no breakdown of IMP was detectable b y these two methods, the possibility of any significant degradation of IMP leading to an underestimation of the deamination of AMP can be ruled out. Due to its high sensitivity, the present method of cation exchange chromatography is capable of detecting trace amounts of impurities. The component having AMP-like spectra but chromatographic propeIties different from those of AMP is of particular interest. The results of preliminary experiments performed with the alkali hydrolysate of m a m m a l i a n r R N A also indicated the presence of a similar component in the IMP region. B i o c h i m . B i o p h y s . ,4cta, 254 (1971) i 6 7 - i 7 1
ESTIMATION OF IMP FROM DEAMINATION OF A M P
171
A l t h o u g h hydrolysis of R N A w i t h T~-ribonuclease was r e p o r t e d to cause a lower e x t e n t of d e a m i n a t i o n of A M P t h a n t h a t w i t h alkali e, large scale hydrolysis w i t h this e n z y m e has c e l t a i n drawbacks. Thus, t h e hydrolysis of R N A is often inc om p l et e, it is costly and t h e e n z y m e m u s t be a b s o l u t e l y free of deaminases or of o t h e r e n z y m e s wh i ch affect t h e nucleotides. Therefore, in spite of t h e low degree of d e a m i n a t i o n b r o u g h t a b o u t d u r i n g alkaline hydrolysis, one could still o b t a i n m e a n i n g f u l i n f o r m a t i o n concerning t h e presence of I M P in R N A . Since m a m m a l i a n R N A gene~ally c o n t a i n s a b o u t 20 °/o AMP, th e e s t i m a t e d d e a m i n a t i o n of A M P d u r i n g alkaline hydrolysis w o u l d p r o d u c e less t h a n 0.03 % IMP. Therefore, w i t h t h e sensitive c h r o m a t o g r a p h i c t e c h n i q u e used in this s t u d y one could easily d e t e c t o.I °/o IMP, i.e. one I M P per IOOO nucleotides.
ACKNOWLEDGEMENTS This work was s u p p o r t e d b y a g r a n t from t h e Medical Research Council of Ca n ad a. One of us (L.G.) was holder of a Scholarship from the Medical R e s e a r c h Council of Canada.
REFERENCES i R. H. HALL, in L. GROSSMANAND K. MOLDAVE,Methods in Enzymology, Vol. XII, Part A, Academic Press, New York, 1967, p. 3o5. 2 J. T. MADISON,Annu. Rev. Biochem., 37 (1968) 131. 3 G. ATTARDIAND F. AMALDI,Annu. Rev. Biochem., 39 (197o) 183. 4 L. GYENES, Rev. Can. Biol., 28 (1969) 179. 5 R. M. BOCK, in L. GROSSMANANn K. MOLDAVE,Methods in Enzymology, Vol. XlI, Part A, Academic Press, New York, 1967, p. 224. 6 H. O. KAMMENAND S. J. SPENGLER, Biochim. Biophys. dcta, 213 (197o) 352. 7 P. K. G-ANGULI,A. REINER AND L. GYENES, Anal. Biochem., 42 (1971) 91. 8 H. A. SOBER, Handbook o/ Biochemistry, The Chemical Rubber Co., Cleveland, Ohio, 197o. 9 ]3. G. LANE, Biochim. Biophys. Acta, 72 (1963) IiO. IO G. KRISHNA, ]3. WEISS AND ]3. B. ]3RODIE, J. Pharmacol. Exp. Therap., 163 (1968) 379. i i R. MARKHAM,in S. P. COLOWICKANn N. O. KAPLAN, Methods in Enzymology, Vol. III, Academic Press, New York, 1957, p. 8o5. 12 E. VOLKIN AND W. E. COHN, in D. GLICK, Methods o[ Biochemical Analysis, Vol. I, Interscience, New York, 1954, P. 287. 13 R. H. HALL, Biochemistry, 4 (1965) 661. 14 N. O. KAPLAN, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. Ili, Academic Press, New York, 1957, p. 873. Biochim. Biophys. Acta, 254 (1971) 167-171
167
BBA 97062
E S T I M A T I O N OF T R A C E AMOUNTS OF INOSINIC ACID FORMED BY T H E D E A M I N A T I O N OF A D E N Y L I C ACID W I T H ALKALI* P. I{. GANGULI, A. REINER AND L. GYENES Department o/ Microbiology and Immunology, Faculty o/ Medicine, University o/ Montreal, Montreal, Quebec (Canada)
(Received August 9th, I97 I)
SUMMARY The deamination of 2'(3')-AMP with alkali under the conditions used for the hydrolysis of RNA (o.I M KOH, IOO°, 20 min or 0.3 M KOH, 37 °, 18 h) was estimated after separating IMP from AMP by cation-exchange chromatography on Dowex 5o(H+). Before treatment with alkali, the commercial prepalation of AMP was purified b y this chromatographic method to remove impurities. Within the concentration range of o.13-14.o IV[,the extent of deamination of AMP was o.I4~-o.o3 %, as determined spectrophotometrically from the recovered nucleotides.
INTRODUCTION Many of the minor ribonucleotides which were first reported to b~ present in the t R N A ' s 1,~ have been subsequently found in the higher molecular weight cellular RNA fractions 3. Of these, however, the presence of IMP has not yet been reported in any RNA other than some of the tRNA's. As has been proposed recently4, the presence of IMP in mRNA could explain, through a variable translational mechanism, the synthesis of polymorphic proteins, such as the immunoglobulins. Since IMP, if present, could be expected only in trace amounts, its separation, identification and quantitation would require large scale hydrolysis of RNA. For this purpose alkaline hydrolysis into monoribonucleotides 5 is a convenient method. However, KAMMEN AND SPENGLER6 have recently reported that under the conditions of alkaline hydrolysis 0.3-0.35 % deamination of the adenosine moiety occurred, irrespective of whether it was present as a nucleoside, nucleotide or polynucleotide. In their study the deamination of AMP was estimated by an indirect method, using isotopically labelled compounds which were fractionated by anion-exchange chromatography after the alkali treatment. In view of the importance of the accurate estimation of the deamination of AMP for the detection and/or quantitation of IMP in the hydrolysate of high molecular weight mammalian RNA, the present work was carried out to verify the extent of deamination of AMP. The direct spectrophotometric determination of IMP, separated by cation-exchange chromatography, showed a lesser extent of deamination of 2'(3')-AMP with alkali than reported by KAMMEN AND SPENGLER6. * Preliminary results of this study were presented at the i4th Annual Meeting of the Canadian Federation of Biological Societies, Toronto, 1971. Biochim. Biophys. Acta, 254 (1971) 167-171
168
P.K.
GANGULI
Cl al.
EXPERIMENTAL
As described earlier v, I M P can be recovered in a small volume, clearly separated from the four m a j o r ribonucleotides, b y c h r o m a t o g r a p h y on the strong-acid cation-exchanger Dowex 5o(H+). In this procedure the sample is applied in o.o5 M HC1 (or HC104) , UlV[P is eluted with o.o 5 M acid, I M P with o.oo5 M acid and GMP, followed b y the mixture of CMP and AMP, with water. In the present s t u d y highly reproducible separation between I M P and A M P was obtained with this technique under the conditions described in Table I and the separation was similar on the three different columns. TABLE I CONDITIONS OF CATION EXCHANG~ CHROMATOGRAPHY
From the Dowex 5o(FI+) (2OO-4OOmesh, 4 % cross-linked) columns of increasing size IMP was recovered within 4, IO and 25 ml and AMP within 4o, 200 and 7o0 ml of effluent, respectively. The volume of 0.05 M HC104 includes the sample volume. Column size l(cm) × d(cm)
Amount o] sample
(A260nm)
Sample volume in 0.05 M HCIO 4 (ml)
i-2
Volume o[ eluant (ml) 0.05 M HCIO 4
0.005 M HCIO,
Water
Flow rate by gravity (ml/min)
io × 0.9
3o-60
7
io
60
24 )< 0 . 9
500-800
Up to 4
15
20
250
o.2-o. 3 o . I o.15
15 × 2
98o-13oo
Up to 20
55
7°
9°0
0.5-0.7
RESULTS AND DISCUSSION
The commercial preparation (Calbiochem) of 2'(3')-AMP used in this s t u d y contained about i % of impurities, represented b y the two minor components in the c h r o m a t o g r a m (Fig. i, top). The first component, corresponding to 0.32 o~, of the total, was eluted in the UMP region and showed spectral characteristics of UMP (ref. 8). The second component, corresponding to 0.66 % of the total, emerged in the I ~ P region, i.e. near the Io-ml effluent volume, a n d showed spectral characteristics of A M P (ref. 8). This component was further resolved into two constituents b y descending paper c h r o m a t o g r a p h y in the (NH4)2SO4-ethanol systemg: the m a j o r constituent (about 9 ° %) had AMP-like spectra at p H 3, 7, and 13 b u t m o v e d much slower t h a n 2' (3')-AMP, i.e. in the position of 2' (3')-CMP, and the minor constituent (about IO %) h a d the properties characteristic for 2'(3')-IMP. The AMP-like material was precipitable with ZnSO4-Ba(OH)z (ref. IO) and was stable to t r e a t m e n t with I M K O H (23 °, 24 h), o.I IV[HC1 (23 °, 4 h) and snake v e n o m phosphodiestelase, as d e m o n s t r a t e d b y its u n c h a n g e d chromatographic behaviour following these treatments. F r o m these results is can be concluded t h a t this c o m p o u n d is not cyclic AMP (refs. IO, I I ) . The orcinol test 12 gave 1.12 times higher colour intensity at 660 nm v ith this c o m p o u n d t h a n with A M P used at the same concentration (same A,59 nm units). Therefore, it would appear t h a t this c o m p o u n d might have an e at 259 nm about IO % lower t h a n AMP and is not identifiable as 9-E2'(3')-0-fibosyl-fl-D-ribofuranosyl]adenine which also has the spectral characteristics of AMP (ref. 13). Biochim. Biophys. Acta, 254 (i97 I) i67-17i
ESTIMATION OF IMP
0.2 0.05M.O.O05M
o tll o/
0.5 0.4 0.3 E
0.2
, /1
0
10
169
FROM DEAMINATION OF AMP
'--'/2 20
30
H20
).05MO.O05M
40
50
~
'
~
1,30
40
50
60
1
,~ 0.1 "
20
60
70
0.5 0.4 0.3 0.2
).05M
O.OC6M
2O
1 0
10 2'0 " 30 Volume of effluent (ml)
40
50
F i g . I. C h r o m a t o g r a p h y of A M P on D o w e x 5 o ( H +) c o l u m n ( i o c m X o . 9 cm) before and after t r e a t m e n t with alkali. The chromatograms were recorded with an L K B U v i c o r d at 253. 7 n m using a 2 m m flow cell. Top: Commercial A M P showing impurities in the U M P and I M P regions; the large double p e a k eluted with water represents 2'(3')-AMP. Middle: Purified A M P showing the absence of c o n t a m i n a n t s in the I M P region. B o t t o m : D e t e c t i o n of I M P formed b y the alkali t r e a t m e n t of the purified A M P .
Moreover, preliminary results obtained with chromatography on Sephadex G-Io and G-25 indicated that the elution volume of this compound was identical with that of AMP. The identification of this compound is under further investigation. The commercial preparation of 2'(3')-AMP was purified on a large scale using the 15 cm × 2 cm column and the purified preparation was concentrated by freezedrying and clarified. This preparation was devoid of the contaminant in the IMP region, as observed by chromatography on a IO cm × 0. 9 cm column (Fig. I, middle); the small peak eluted with 0.05 M acid represented non-nucleotide material, presumably soluble particles of resin, carried over from the previous chromatographic step. This material showed a high A320 nm and a continuous linear increase at lower wavelengths which is characteristic of light scattering. When the purified AMP preparation was treated with alkali under the conditions indicated in Table n and analyzed by chromatography on a IO cm x 0. 9 cm column, the presence of a trace amount of IMP, formed by the deamination of AMP, was visible in the chromatogram (Fig. I, bottom). To obtain sufficient amount of IMP for quantitative estimation, chromatography of the alkali-treated AMP was carried out on a larger column. The eluate containing IMP was neutralized with KOH, concentrated by freeze-drying and clarified by centrifugation to remove KCI04. Biochim. Biophys. Acta, 2 5 4 ( 1 9 7 1 ) 1 6 7 - 1 7 1
17o TABLE
P.K. GANGULIel al. ii
E S T I M A T I O N O F T t I E D E A M I N A T I O N OF 2 ' ( 3 ' ) - A M P
Chromatographically pure AMP, after treatment with KOH, was brought to a n d a f t e r c l a r i f i c a t i o n i t w a s c h r o m a t o g r a p h e d o n e i t h e r (a) 24 c m × 0.9 cnl c o l u m n . A M P a n d I3,{P w e r e e s t i m a t e d u s i n g ~(259 n m ) = i5.4/mmole at pH i 2 . 2 / m m o l e a t p H 3, r e s p e c t i v e l y . T h e p e r c e n t d e a m i n a t i o n w a s c a l c u l a t e d n u c l e o t i d e s . F o r e x p e r i m e n t a l d e t a i l s see t h e t e x t .
Conditions o / a l k a l i treatment A M P ~oncn. K O H conch. (M ) (M ) 14.o 4 4.88 4.29 3.87 o.I 3
o.i 0. 3 o.I 0. 3 0. 3
( i o o °, ( 37 °, (IOO °, ( 37 '~, ( 37 ° ,
2o I8 20 18 18
Recovery after chromatography
rain) h) nlin) h) h)
.4MP (mmoles )
IMP (mmole )
29.oo 85.11 42.36 46.76 63.16
o.o4o O.lO 3 o.o66 0.088 0.063
(a) (b) (a) (a) (b)
p H 1. 7 w i t h H C 1 0 a o r (b) 15 c m x 2 c m 7 a n d e (248 n m ) = from the recovered
Mole 0,6 deamination o[ d M P
o.i37 o.125 o.156 o.188 o.ioo Yfean ± S . E . o . i 4 i i o . o 3
This concentrated material showed an ultraviolet spectrum characteristic of I1VfP (2max = 248 nm) which, however, was modified by the light-scattering contribution from the resin particles. The latter were removed b y rechromatography on a io cm × 0. 9 cm column and IMP was estimated spcctrophotometrically, taking into account the 20 % loss due to manipulation (see below). On the other hand, AMP was estimated directly from the large columns; recovery of AMP was of the order of 87-95 %. The results of five experiments indicated that the two conditions of alkaline hydrolysis of RNA did not produce a significantly different degree of deamination of AMP (Table II). Moreover, the concentration of AMP during the treatment with alkali did not significantly influence the extent of deamination. Since the average value of o.14 % deamination of AMP was less than half of that reported by KAMMEN AND SPENGLER 6, the possibility of underestimating IMP in the present study was carefully considered. 2' (3')-IMP was prepared from 2' (3')-A~P b y deamination with HNO., according to the method of KAPLAN14. This preparation was then subjected to all the handling and experimental conditions used in this study, such as storage at 4 ° in o.oo 5 IV[ HC1Oa, neutralization with KOH, freeze-drying, clarification and rechromatography in the presence or absence of AMP. Except for about 2o % loss during the steps of freeze-drying and clarification, there was no significant loss of IMP. Moreover, a n y partial conversion of 2'(3')-IMP to inosine or hypoxanthine would have been detected in the present study either as a lower recovery of IMP in the cation-exchange chromatography or b y the appearance of additional spots in paper chromatography. As no breakdown of IMP was detectable b y these two methods, the possibility of any significant degradation of IMP leading to an underestimation of the deamination of AMP can be ruled out. Due to its high sensitivity, the present method of cation exchange chromatography is capable of detecting trace amounts of impurities. The component having AMP-like spectra but chromatographic propeIties different from those of AMP is of particular interest. The results of preliminary experiments performed with the alkali hydrolysate of m a m m a l i a n r R N A also indicated the presence of a similar component in the IMP region. B i o c h i m . B i o p h y s . ,4cta, 254 (1971) i 6 7 - i 7 1
ESTIMATION OF IMP FROM DEAMINATION OF A M P
171
A l t h o u g h hydrolysis of R N A w i t h T~-ribonuclease was r e p o r t e d to cause a lower e x t e n t of d e a m i n a t i o n of A M P t h a n t h a t w i t h alkali e, large scale hydrolysis w i t h this e n z y m e has c e l t a i n drawbacks. Thus, t h e hydrolysis of R N A is often inc om p l et e, it is costly and t h e e n z y m e m u s t be a b s o l u t e l y free of deaminases or of o t h e r e n z y m e s wh i ch affect t h e nucleotides. Therefore, in spite of t h e low degree of d e a m i n a t i o n b r o u g h t a b o u t d u r i n g alkaline hydrolysis, one could still o b t a i n m e a n i n g f u l i n f o r m a t i o n concerning t h e presence of I M P in R N A . Since m a m m a l i a n R N A gene~ally c o n t a i n s a b o u t 20 °/o AMP, th e e s t i m a t e d d e a m i n a t i o n of A M P d u r i n g alkaline hydrolysis w o u l d p r o d u c e less t h a n 0.03 % IMP. Therefore, w i t h t h e sensitive c h r o m a t o g r a p h i c t e c h n i q u e used in this s t u d y one could easily d e t e c t o.I °/o IMP, i.e. one I M P per IOOO nucleotides.
ACKNOWLEDGEMENTS This work was s u p p o r t e d b y a g r a n t from t h e Medical Research Council of Ca n ad a. One of us (L.G.) was holder of a Scholarship from the Medical R e s e a r c h Council of Canada.
REFERENCES i R. H. HALL, in L. GROSSMANAND K. MOLDAVE,Methods in Enzymology, Vol. XII, Part A, Academic Press, New York, 1967, p. 3o5. 2 J. T. MADISON,Annu. Rev. Biochem., 37 (1968) 131. 3 G. ATTARDIAND F. AMALDI,Annu. Rev. Biochem., 39 (197o) 183. 4 L. GYENES, Rev. Can. Biol., 28 (1969) 179. 5 R. M. BOCK, in L. GROSSMANANn K. MOLDAVE,Methods in Enzymology, Vol. XlI, Part A, Academic Press, New York, 1967, p. 224. 6 H. O. KAMMENAND S. J. SPENGLER, Biochim. Biophys. dcta, 213 (197o) 352. 7 P. K. G-ANGULI,A. REINER AND L. GYENES, Anal. Biochem., 42 (1971) 91. 8 H. A. SOBER, Handbook o/ Biochemistry, The Chemical Rubber Co., Cleveland, Ohio, 197o. 9 ]3. G. LANE, Biochim. Biophys. Acta, 72 (1963) IiO. IO G. KRISHNA, ]3. WEISS AND ]3. B. ]3RODIE, J. Pharmacol. Exp. Therap., 163 (1968) 379. i i R. MARKHAM,in S. P. COLOWICKANn N. O. KAPLAN, Methods in Enzymology, Vol. III, Academic Press, New York, 1957, p. 8o5. 12 E. VOLKIN AND W. E. COHN, in D. GLICK, Methods o[ Biochemical Analysis, Vol. I, Interscience, New York, 1954, P. 287. 13 R. H. HALL, Biochemistry, 4 (1965) 661. 14 N. O. KAPLAN, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. Ili, Academic Press, New York, 1957, p. 873. Biochim. Biophys. Acta, 254 (1971) 167-171