The photosensitized degradation of guanosine by acridine orange

42

BIOCHIMICA ET BIOPHYSICA ACTA

BBB 95483

T H E P H O T O S E N S I T I Z E D DEGRADATION OF GUANOSINE BY ACRIDINE ORANGE

K. S I V A R A M A S A S T R Y * AND M I L T O N P. G O R D O N * *

Department o/ Biochemistry, University of Washington, Seattle, Wash. (U.S.A.) ( R e c e i v e d D e c e m b e r 2oth, 1965)

SUMMARY

A high concentration of acridine orange (IO-4 M) and intense illumination for prolonged periods of time result in photochemical degradation of guanosine. Adenosine, cytidine and uridine were unaffected under similar conditions. This selective disruption of guanosine has been shown to involve both ring systems in guanosine. The chief products of the reaction have been characterized to be ribosylurea, ribose, urea and guanidine. The significance of these findings in the photodynamic inactivation of the ribonucleic acid of tobacco mosaic virus has been discussed.

INTRODUCTION

The photodynamic inactivation of viruses and viral nucleic acids by dyes has been studied in the past few years in order to elucidate the basic mechanisms involved. SIMON AND VAN VUNAKIS1,1z showed recently that one probable modification of polynucleotides was a photosensitized destruction of guanine residues. SUSSENBACH AND BERENDS2 have observed the degradation of guanine by the action of himichrome. SIMON AND VAN VUNAKIS1,1~ had concluded that methylene blue was one of the more efficient dyes for the selective destruction of guanine derivatives; acridine orange, under similar conditions, induced little photochemical alteration of the compounds. However, more recent work from this laboratory 3 showed that the efficacy of acridine orange compared to methylene blue in causing the photodynamic inactivation of TMV-RNA is much greater than would be predicted from studies with simple model compounds. Hence, it was decided to study the effect of high concentrations of acridine orange and extensive irradiation on the photodynamic breakdown of guanosine. A b b r e v i a t i o n : TMV, t o b a c c o m o s a i c virus. * P r e s e n t a d d r e s s : B i o c h e m i s t r y Division, D e p a r t m e n t of C h e m i s t r y , O s m a n i a U n i v e r s i t y , H y d e r a b a d - 7, I n d i a . ** To w h o m r e p r i n t r e q u e s t s s h o u l d be a d d r e s s e d .

Biochim. Biophys. Acta, I29 (1966) 4 2 - 4 8

PHOTOSENSITIZED DEGRADATION OF GUANOSINE

43

MATERIALS AND METHODS

Materials Acridine orange was purified as described earlier3, i. Pure guanosine was purchased from P-L Biochemicals Inc., Milwaukee, Wisc.

Procedure /or irradiation In exploratory experiments, where the possible destruction of nucleosides b y acridine orange was investigated, the desired nucleoside was used at I mg/ml in o.05 M borate buffer (pH 9.2) and I.O ml was placed in a 2-cm petri dish. The acridine orange concentration was lO -4 M at the beginning of the irradiation, and after 5 h addition of an identical amount of acridine orange was made. Illumination for a total period of IO h was b y 5oo-W photoflood lamps positioned above to provide 12 50013 ooo-ft candies a. Residual nucleoside concentrations were determined after termination of irradiation as described in later sections. When the photoproducts of gnanosine breakdown were isolated, irradiations were repeated in a slightly different manner. A 3oo-ml batch of gnanosine (I mg/ml) was irradiated in 0.05 M NH4HCO ~ adjusted to p H 9.2 with NH4OH. Irradiations were performed in a flat rectangular glass trough with two 5oo-W lamps at a distance of 5 cm. Water filters were interposed and magnetic stirring was provided. The initial acridine orange concentration was lO -4 M, and after 7 h an identical amount of acridine orange was again added. Irradiations were carried out at 4 ° for 13 h.

Chromatographic procedure Ascending chromatography was performed throughout. Concentrates of the irradiated mixtures were chromatographed on W h a t m a n 3 MM paper in one of the following solvent systems: Isoamyl alcohol-o.2 M NH4HCO 3 ( I : I , v/v), with both phases in the t a n k (Solvent No. I). n-Butanol-acetic acid-water (4:1:5, v/v/v), top phase (solvent No. II). n-Butanol-95~/o ethanol-water (I : 4 : I, v/v/v) (Solvent No. I I I ) .

Electrophoretic procedure. Electrophoresis was performed in o.o 5 M borate buffer (pH 9.2), on W h a t m a n 3 MM paper, 15 cm wide, at 15oo V and 47-50 mA. Standards were run concurrently. Distances moved b y compounds have been calculated in all experiments relative to the distance moved b y picric acid and have been expressed at " M / ' . A negative sign denotes movement towards the cathode.

Isolation o/ photo,'products At the end of the irradiation, the entire guanosine-acridine orange mixture was lyophilized until most of the NH4HCO 3 had been removed. The fluffy residue obtained was taken up in 4.0 ml water and adjusted to p H 8.0 with dilute HC1. The mixture was then centrifuged, and the supernatant was used for isolation and identification of photoproducts. This supernatant will be termed the "crude extract". Biochim. Biophys. Acta, 129 (1966) 42-48

44

K. S. SASTRY, M. P. GORDON

The crude extract was streaked on paper and chromatographed overnight in Solvent I. Solvent I was very convenient for separating the photoproducts of guanosine from unaltered guanosine. Most of the detectable photoproducts were well separated from guanosine (RF ----0.67) and occupied a region with an R~ of 0.90. Many of the photoproducts appeared as a faintly absorbing band, RF 0.90, when observed with a low-pressure ultraviolet lamp placed beneath the paper and a fluorescent plate on top. This band was cut out and eluted with water. The eluate was again lyophilized and taken up in approx. 2.0 ml of water, clarified if necessary b y centrifugation, and used for all identification purposes. This concentrate will be simply referred to as "concentrate".

Spray reagents Ribose was detected b y an aniline phthalate reagent 5. Ureido compounds were detected b y a p-dimethylaminobenzaldehyde spray 6. Guanidine was detected b y spraying with a nitroprusside-ferricyanide reagent 7. Ribose was estimated using orcinol 8. Urea was estimated with a-isonitrosopropiophenone g. Allantoin was estimated b y the Rimini-Schryver reaction according to YOUNG AND CONWAY 10, which is based on conversion of allantoin b y alkaline hydrolysis to glyoxylate, and estimation of the latter with phenylhydrazine and KsFe(CN)6. Residual nucleoside concentrations were estimated spectrophotometrically after chromatography of irradiation mixtures in Solvent I. The ratios of the absorbances at 260 to 280 m# were used as criteria of purity.

RESULTS

Irradiation of guanosine in the presence of acridine orange at p H 7.o showed no significant destruction of the nucleoside. Since the methylene blue-sensitized reaction was much faster at p H 9.2 (refs. I, I2), all later experiments were performed at this pH. It was noted that approx. 50 % destruction of guanosine took place only in the presence of lO -4 M acridine orange and irradiation with 12 5oo-13 ooo-ft candles for IO h. In three trials, the disappearance of guanosine under the above conditions was 60, 44.4, and 46.3 %, averaging 50 %. There was negligible breakdown of uridine, cytidine and adenosine under these conditions. Attempts were made to characterize the breakdown products of guanosine, found in Solvent I, at Rp 0.9 o. The material in this region (the "concentrate") was found to possess only end absorption. Hence, the product(s) could not have had the intact pyrimidine moiety of guanosine. The possibility of the occurrence of compounds such as parabanic acid, oxaluric acid, allantoin and allantoic acid was quickly ruled out b y electrophoresis and chromatography. Parabanic acid was not expected since the mixture of products had no absorption around 260-280 In# and the alkalinity of the irradiation conditions should have resulted in the conversion of this compound to oxaluric acid. However, while checking for oxaluric acid b y electrophoresis at p H 9.2 followed b y spraying with p-dimethylaminobenzaldehyde, a strongly ureido-positive compound was found at a M p of 0.62. Allantoin was ruled out since allantoin moved at M p = 0.4o. Further, when this spot was eluted, it did not react Biochim. Biophys. Acta, x29 (1966) 42-48

PHOTOSENSITIZED

DEGRADATION

OF

GUANOSINE

45

like an a l l a n t o i n d e r i v a t i v e in t h e R i m i n i - S c h r y v e r reaction. A small a m o u n t of urea, w i t h M p = --0.38, i d e n t i c a l to t h a t of a u t h e n t i c urea, was also f o u n d in these e l e c t r o p h o r e t i c runs of t h e " c o n c e n t r a t e " . E l e c t r o p h o r e s i s of t h e c o n c e n t r a t e , followed b y s p r a y i n g w i t h aniline p h t h a l a t e showed a s t r o n g r e d - o r a n g e - p o s i t i v e r e a c t i o n a t M p -~ 0.73. This was confirmed to be free ribose. A n e x a m i n a t i o n of t h e e l e c t r o p h o r e t o g r a m in u l t r a v i o l e t fight showed t w o e n d - a b s o r b i n g regions; one at M p = 0.54 b e h i n d t h e m a i n u r e i d o - p o s i t i v e region a t M p ~ - 0 . 6 2 , a n d a n o t h e r at M p = 1.1-1.2. These results are d i a g r a m m a t i c a l l y r e p r e s e n t e d in Fig. I. T h e u r e i d o - p o s i t i v e M p 0.62 region h a d no absorption.

+1.0

i

088 o

Concentrate

Mp: 0.62compound ~

8

offer acid hydrolysis -0.38 []

picric acid uv absorption

~ ~ , 0.54 0.62 0.73

0 [] ~

ll.2

l

aniline phthalofe positive ureldo positive

Fig. i. Distribution of the products of acridine orange-sensitized photodegradation of guanosine. Electrophoretogram of "concentrate" run at pH 9.2 in o.o5 M borate buffer. Guanidine, with M p = --2.6, is not shown in the figure. For experimental details, see text.

C h r o m a t o g r a p h y of t h e " c o n c e n t r a t e " in Solvents I I a n d I I I confirmed t h e m a j o r aniline p h t h a l a t e - p o s i t i v e c o n s t i t u e n t to b e free ribose a n d also confirmed t h e presence of traces of urea. I n b o t h solvents, t h e r e was o n l y one i n t e n s e l y ureidop o s i t i v e a r e a i n d i c a t i n g t h e h o m o g e n e i t y of this m a t e r i a l . T h e R F values o b t a i n e d are given in T a b l e I.

TABLE I CHROMATOGRAPHIC CHARACTERIZATION OF SOME OF THE PRODUCTS OF ACRIDINE ORANGE-SENSITIZED BREAKDOWN OF GUANOSINE

Chromatography of the "concentrate", see text. Solvent No.

Compound tested

II

"concentrate" urea ribose "concentrate" ribose

In

RF Ureido-positive

Aniline pht~alatepositive

o.2o*; o.47"* 0.47

0.30

o. i4"

o.31 o.28 o.28

" Main product. ** ]Faint reaction. Biockim. Biophys. Acta, 129 (1966) 42-48

46

K.s.

SASTRY, M. P. GORDON

The ureido-positive compound was isolated b y electrophoresis followed b y elution. Hydrolysis was then performed for 15 min at IOO° using 0.5 M HC1. The hydrolyzed reaction mixture was taken to dryness in vacuo, and electrophoresis was performed in borate buffer at p H 9.2. Examination of the electrophoretogram with p-dimethy!aminobenzaldehyde showed the disappearance of the ureido-positive reaction at M e = 0.62 and the appearance of all the ureido reaction at M e = --o.38. Free ribose was detected at M e = 0.73 so t h a t the original ureido-positive material was postulated to be ribosylurea. A prolonged electrophoresis of the "concentrate" was then performed. The ureido compound was thereby isolated free of ribose, eluted, and hydrolyzed with acid as before. Urea and ribose were estimated in the hydrolyzate (see Table II). The agreement between the expected and observed values for ribose and urea proves that the compound is ribosylurea.

TABLE II IDENTIFICATION

OF THE

UREIDO-POSITIVE

PRODUCT

OF GUANOSINE

DEGRADATION

BY ACRIDINE

O R A N G E AS R I B O S Y L U R E A

P u r e ureido c o m p o u n d isolated b y electrophoresis, h y d r o l y z e d w i t h o. 5 M HC1, h y d r o t y z a t e m a d e to v o l u m e a n d a l i q u o t s a n a l y z e d . F o r e x p e r i m e n t a l details, see t e x t . A n a l y t i c a l v a l u e s p e r m l hydrolyzate.

Expt. No.

Ribose

Urea ]ound

Urea expected

I 2

21. 5 40.0

8. 5 16.o

8.6 16.o

Attempts were made to check for guanidine which could result from the degradation of the pyrimidine ring. The "crude extract" from guanosine irradiation was subjected to electrophoresis at p H 9.2 along with pure guanidine. The electrophoretogram was sprayed with the nitroprusside-ferricyanide reagent ~ and a positive pale-red reaction was found at M p = --2.6, which was also the mobility of pure guanidine. T~hat this material was free guanidine was confirmed b y subjecting the crude e x t r a c t and guanidine simultaneously to electrophoresis, cutting out the entire M p = --2.6 region, and chromatographing in Solvent I I I . On spraying the chromatogram both free guanidine and the guanidine-positive compound were found to have an RF ---- 0.20.

DISCUSSION

This investigation has shown t h a t acridine orange can bring about a selective photosensitized degradation of guanosine. However, the conditions required to obtain a marked destruction of guanosine with acridine orange are very severe compared to those adequate with methylene blue. This explains why SIMONAND VAN VUNAKIS1,12 did not find breakdown of guanosine by acridine orange; it is not possible to obtain any noticeable breakdown with acridine orange under their conditions. The reaction is very complex and leads undoubtedly to a mixture of several Biochim. Biophys. Acta, 129 (1966) 42-48

PHOTOSENSITIZED DEGR&DATION OF GUANOSINE

47

products. All of them could not be identified. However, the identification of four, namely, guanidine, urea, ribose and ribosylurea, is significant. This shows that the,e is a total breakdown of the guanosine molecule, and suggests at least five points of attack on the guanosine molecule (see Fig. 2). Apparently at least two modes of breakdown of guanosine or of a photochemically produced derivative are possible since both free ribose and ribosylurea were found.

oH

N,~C~c~N~, /c% H,N

N R =.ib.... -----

Points o f

/

q R

attock

Fig. 2. Some possible sites of breakdown of the guanosine molecule by the photosensitized reaction with acridine orange.

A study of photosensitized breakdown of purines has led to the identification of 1,3-dimethylallantoin from theophyllin1 and parabanic acid and guanidine from guanine~. Ribosylurea is yet another product produced from guanosine. These findings indicate that the mode of breakdown with each photosensitizing dye could be different. In other experiments11, it has been found that methylene blue also gives rise to free ribose, guanidine and ribosylurea from guanosine. Since methylene blue converts theophyllin to 1,3-dimethylallantoin, it is also evident that the ~photodegradation of related compounds by the same dye is not always by the same mechanism. In sharp contrast to the action of acridine orange and methylene blue on free guanine derivatives, the difference between the rates of destruction of the infectivity of TMV-RNA by these dyes is not very different as shown in the preceding paper 3. It is possible *_hat in the more ordered environment of TMV-RNA, the mode of attack of guanine residues by methylene blue is hindered and/or the attack on guanine residues by acridine orange is facilitated. The final consequence of either of these two possibilities would be more equal rates of destruction of the biological activity of TMV-RNA by the two dyes. A detailed study of the reactions involved is indicated and is in progress.

ACKNOWLEDGEMENT

This work was supported by funds from the U.S. Public Health Service (grant No. AI o3352-o5), the National Science Foundation (grant No. G 23oo2), and the State of Washington Initiative 171. Biochim. Biophys. Acta, 129 (1966) 42-48

48

K. S. SASTRY, M. P. GORDON

REFERENCES I 2 3 4 5 6 7 8 9 io ii 12

M. I. SIMON AND H. VAN VUNAKIS, J. Mol. Biol., 4 (1962) 488. J. S. SUSSENBACH AND W . BERENDS, Biochim. Biophys. Acta, 95 (1965) 184. K. SIVARAMA SASTR¥ AND M. P. GORDON, Biochim. Biophys. Acta, 129 (1966) 32. K. SIVARAMA SASTRY AND M. P. GORDON, Biochim. Biophys. Acta, 91 (1964) 406. S. M. PARTRIDGE, Nature, 164 (1949) 443. R. M. FINK, R. E. CLINE, C. McGAUGHEY AND K. FINK, Anal. Chem., 28 (1956) 4C. J. WEBER, J. Biol. Chem., 78 (1928) 465. H. G. ALBAUM AND W . W . UMBREIT, J. Biol. Chem., 167 (1947) 369. R. 3{. ARCHIBALD, J. Biol. Chem., 157 (1945) 507. E. G. YOUNG AND C. F. CONWA¥, J. Biol. Chem., 142 (1942) 839. L. WASKELL, K. SIVARAMA SASTRY AND M. P. GORDON, Biochim. Biophys. Acta, 129 (1966) 49. M. I. SIMON AND H. VAN VUNAKIS, Arch. Biochem. Biophys., lO5 (1964) I97.

Biochim. Biophys. Acta, 129 (1966) 42-48