v o L 31 (x959) BIOCHIMICA ET BIOPHYSICA ACTA I 9 5

ELIMINATION OF SUBSTANCES INTERFERING WITH

NUCLEIC ACIDS ESTIMATION

M. DE DEKEN-GRENSON AND R. H. DE D E K E N

Laboratoire de Physiologie animale de l' Universit$ de Bruxelles and Service de Reckerches du Centre d'Enseignement et de Recherches des Industries Alimentaires et Chimiques, Bruxelles (Belgique)

(Received May 24th, 1958)

SUMMARY

A simple method allowing quantitative purification of the ribonucleotides obtained by alcaline hydrolysis of ribonucleic acid (ScHMIDT-THANNHAUSER procedure) is presented. Nearly all the substances interfering with ultraviolet photometry and pentose estimation (orcinol) are eliminated by this procedure.

This ribonucleic acid estimation method is tested on plant and animal tissue and on several microorganisms. The method is compared with three other procedures: the SCHNEIDER technique, the OGuR-RosEN procedure and the classical SCHMIDT- THANNHAUSER method.

Extraction of deoxyribonucleic acid performed on the same tissues by the three methods is compared. Estimations made by the diphenylamine method (DIScHE) and the indol reaction (CERIOTTI) a re compared.

The results are commented and discussed.

INTRODUCTION

It seems to be preferable when performing ribo- and deoxyribonucleic acid estimations to utilize a method which separates the two kinds of nucleic acids. Until now, the SCHMIDT-THANNHAUSER procedure 1 seems to be the most specific method for the separation of ribonucleic and deoxyribonucleic acids. Unfortunately the solution of ribonucleotides obtained after the alcaiine treatment is often unsuitable for ultraviolet spectrophotometry, the mean value of the ratio O.D. at 26o mtz/O.D, at 23o m~ being x.I (see RESULTS, Part II). Moreover, the orcinol reaction is seldom in agreement with the ultraviolet photometry. Interfering substances are known for both methods: degradation products of proteins containing aromatic amino acids may give rise to erroneous interpretation of ultraviolet photometry and, on the other hand, the orcinol reaction is sensitive to protein breakdown products ~, 3 and sugars 4.

In order to obviate this situation, a simple and quantitative method of ribo- nucleotide purification has been developed and is presented here*. Ribonucleotides are sorbed on an anion exchanger, the resin is eventually washed, and the purified nucleotides are released by acid elution. Experience shows that most of the substances

* Note: A preliminary report was published e l s e w h e r e s.

Re/erences p. 207.

I96 M. D]~ DEKEN-GRENSON, R. H. DE DEKEN VOL. 31 (x959)

interfering with ultraviolet photometry and orcinol estimation are not sorbed by the resin. Ultraviolet photometry and orcinol reaction results are in very good agreement after this purification.

This improved SCHMIDT-THANNHAUSER procedure is compared with three other methods: the classical SCHMIDT-THANNHAUSER procedurO, the SCHNEIDER method 6 and the one elaborated by OGUR AND ROSEN 7. These comparisons are made for several tissues.

Deoxyribonucleic acid estimations made by four different methods, namely ultraviolet photometry, orcinol reaction s, diphenylamine reaction * and CERIOTTI reaction 1° are also compared for these different tissues.

METHODS

I. Removal o] acid-soluble compounds and delipidation

This procedure is performed as described by VOLKIN AND COHN n. Perchloric acid is preferred to trichloroacetic acid. Cell suspensions or tissue homogenates are precipitated with one volume of Io % perchloric acid at o% All acid treatments are performed in an ice bath. After delipidation, the tissue residue is washed with ether and air-dryed. All nucleic acid extractions were made on these lipid-free dry powders.

2. The SCHNEIDER procedure

Nucleic acids are extracted with 5 % perchloric acid, as proposed by SCHNEIDER et aL e. Generally 5o mg of dry delipidated tissue residue are heated for 15 min at 9 °0 with 1.5 ml of 5 % perchloric acid. After centrifugation, the insoluble residue is washed twice with 1.5 ml of 5 % perchloric acid. The combined extracts are made up to 5 ml.

3. The OGUR AND ROSEN procedural

50 mg of lipid-free tissue residue are extracted with 3 ml of IO % perchloric acid for 18 h at 4 ° for ribonucleic acid extraction. The residue is washed twice with 3 ml of cold IO % perchloric acid and the combined extracts made up to IO mh

The insoluble residue, resuspended in 1.5 ml of 5 % perchloric acid, is heated at 70 ° for 20 min. The procedure is repeated and the residue washed once with 1.5 ml of 5 % perchloric acid. The combined extracts, containing the deoxyribonucleic acid, are made up to 5 mh

4. The SCHMIDT-THANNHAUSER-SCHNEIDER procedure

This procedure is essentially performed as described by VOLKIN AND COHN 11. I00 mg of dry lipid-free tissue residue are dissolved in Io ml of I N potassium hy- droxide. The time of incubation was 2o h. After this treatment, the solution was not centrifuged, except in the case of plant material (tobacco leaves). In this case, the insoluble residue was washed twice with I N potassium hydroxide.

The ribonucleotide solution is chilled in an ice bath and neutralized with cold concentrated perchloric acid. After this, cold 70 % perchloric acid is added until a final concentration of 2.5 % is reached in order to precipitate the deoxyribonucleic acid. After centrifugation, the precipitate is washed with 2 ml Of cold 5 % perchloric acid.

The supernatants axe combined and carefully neutralized at o ° with 4o %

Relere~re~ p. 207.

voL. 31 (1959) ESTIMATION OF NUCLEIC ACIDS I97

potassium hydroxide. After centrifugation, the precipitate is washed once with 2 ml of ice cold water. The combined supernatants containing the ribonucleotides are made up to a known volume and kept at low temperature.

The deoxyribonucleic acid precipitate is heated for 15 min at 9 °0 with 2 ml of 5 % perchloric acid. The residue is washed once with I ml of 5 % perchloric acid. The combined extracts are made up to 3.5 ml.

5. Ultraviolet spectrophotomctry o/ribonudeic acid

A stock solution of purified ribonucleotides (see § 12) was diluted (either by 5 % perchloric acid or by I N hydrochloric acid) for ultraviolet spectrophotometry. In both cases, the conversion factor was 33.16, i.e. : O.D. at 260 m/z × 33.16 = t~g ribonucleic acid per ml. This factor was calculated on the basis of a 9.0 % phosphorus content for the ribonucleic acid x=. The ratio O.D. at 26o mtz/O.D, at 230 m~ was 3.41.

6. Ultraviolet spearophotomctry o[ deoxyribonucleic acid

Purified and orthophosphate-free deoxyribonucleic acid (see § 13) was treated for 2o rain at 7o ° with 5 % perchloric acid. This solution was analysed for organic phosphorus xa and diluted with 5 % perchloric acid to a suitable concentration for spectrophotometry. The conversion factor was 32.94, i.e.: O.D. at 267 mtz × 32.94 = V-g deoxyribonucleic acid per ml. This factor was calculated on the basis of a 9.22 % phosphorus content for deoxyribonucleic acid x4. The ratio: O.D. at 267 mtz/O.D, at 230 m p was 2.65.

7. Ordnol reaction/or ribonucleio acid

This reaction was performed according to DISCHE AND SCHWARZ 15 as reported by DlSCHE 4, except that the volume of the reaction mixture was reduced. Between 2o and 1oo vg, the factor was 175, i.e. : O.D. at 66o mtz × 175 ----- V-g ribonucleic acid per assay as calculated on the basis of phosphorus content.

8. Ominol reaction/or deoxyribonucleic acid 8

Experimental conditions were the same as for ribonucleic acid. Between 1oo and 500 tzg, the factor was 1,463. 9, i.e. : O.D. at 660 mt~ x 1,463.9 = v-g deoxyribonucleic acid per assay (on the basis of phosphorus content).

9. Diphenylamine reaction/or deoxyribonucleic acid

This reaction was performed according to DlSCHE*, 16. Heating t ime was 12 min. Between 50 and 250 /~g, the factor was 415, i.e.: O. D. at 6oo mt~ X 415 = deoxyribonucleic acid per assay (on the basis of phosphorus content).

IO. The CERIOTTI reaction/or deoxy~,ibonudeic acid x°

Procedure: 2 ml of 5% perchloric acid containing Io to 5o t~g of hydrolysed deoxyribonucleic acid are mixed with I ml of a 0.04 % aqueous solution of indol and I ml of concentrated hydrochloric acid (sp. gr. 1.19). After IO min heating in a boiling water bath, the reaction mixture is cooled and extracted 3 times with 4 ml of chloro- form. The aqueous layer i~ centrifuged and measured at 49 ° mr,. Between 1o and 5o vg, the factor was lO7.5, i.e.: (O.D. at 490 m/~ - - O.OLO) X Io7.5 = tzg deoxyribo- nucleic acid per assay.

Re/etences p. 207o

198 M. DE DEKEN-GRENSON, R. H. DE DEKEN VOL. 31 (1959)

I I . Ribonucleotides purification on Dowex-2

A. Preparation o/the ion-exchanger resin. Dowex-2 is alternately treated with I N sodium hydroxide and I N hydrochloric acid.

The resin (C1- form) is then washed several times with I N hydrochloric acid in the following manner: the resin is brought into suspension by mild stirring during a short time (30 sec). Immediately after sedimentation of the resin, the liquid is decanted. The aim of this operation is to discard the very fine particles of the resin which give rise to an important light diffusion at short wave lengths. If the stirring time is prolonged, grinding of the resin takes place into finer particles. The same washing procedure is performed with twice distilled water, and after filtration the resin is kept wet.

B. Sorption and elution o] the ribonucleotides. The principles of this technique are taken from COHN1L

A neutralized solution of ribonucleotides is diluted with a tris(hydroxymethyl)- aminomethane (Tris)-HC1 buffer in order to obtain a final Tris concentration of M/4o and pH 7.8. The appropriate amount of washed Dowex-2 is added, and the mixture is left at room temperature for half an hour, with occasional stirring. After cen- trifugation (conical tube) the supernatant is discarded* and the resin is transferred to a sintered glass filter (G 4, 15-2o mm ~) with Tris/HCl M/4o, pH 7.8, if washing is desired, or directly with 4 ml I N hydrochloric acid. After 20 min of contact (occasional stirring), the acid eluate is filtered with suction and collected in a gradu- ated tube. 3 ml more are added and filtered after 5 min of contact. Finally two washings are made with I ml of acid each. The filtrate (pure ribonucleotides in I N hydrochloric acid) is brought to IO ml.

Convenient quantities are 15o, 300 and 6o0 mg of Dowex-2 (dry weight) and I to 2 ~g of ribonucleotides per mg of resin. With 300 and 6o0 mg of resin, both ultra- violet photometry and orcinol reaction may be easily achieved. When interfering substances are present at very high concentrations, as it is the case with plant tissues, it is necessary to utilize more than I mg of resin per ~g nucleotide, in order to obtain quantitative results.

12. Re[erenee solution o] ribonucleic acid

Commercial yeast ribonucleic acid (Schwarz) has been purified by the method of VISCHER AND CHARGAFF TM and freed from proteins by the method of SEVAG et al. TM.

This purified ribonucleic acid was hydrolysed with I N potassium hydroxide (ScHMIDT- THANNHAUSER procedure). The ribonucleotides solution was carefully neutralized at o ° with perchloric acid. This stock solution, which was orthophosphate-free, was analysed for organic phosphorus by a modified FISKE AND SUBBAROW method TM. This solution served as a standard for ultraviolet photometry, pentose estimation and nucleotides purification by means of the ion-exchanger. All the conversion factors were calculated on the basis of a 9.0 % phosphorus content for the ribonucleic acid 1~.

13. Re]erence solution o/deoxyribonucleic acid

A sample of deoxyribonucleic acid, isolated and purified by Dr. M. ERRERA l°, served as a standard. This sample was orthophosphatefree. After digestion for 20 min

* Note: The p H of this superna tan t has to be checked. I n the presence of great quant i t ies of impurit ies, the p H falls below 6 and sorpt ion is no longer quant i ta t ive in this case.

Re#fences p. 207.

vOL. 31 (I959) ESTIMATION OF ~IYCL~IC ACIDS I99

at 7 °0 with 5 % perchloric acid, the stock solution was analysed for organic phosphorus TM. This solution served as a standard for ultraviolet photometry and colorimetric reactions. The conversion factors were calculated on the basis of a 9.22 % phosphorus content for the deoxyribonucleic acid 14.

Notes

I. Optical density measurements were made in a Zeiss spectrophotometer Model PMQ I I or in a Beckman spectrophotometer Model DU.

2. The authors were unable to achieve nucleotide purification with the ion- exchanger Amberlite IRA 400, conditions for quantitative sorption and quantitative elution being too different. Uridylic acid was very hard to desorb.

RESULTS

Part I. Purification o/ribonudeotides on Dowex-2

Ribonuc]eic acid is quantitatively converted into rihonucleotides through the alcaline hydrolysis of the SCHMIDT-THANNHAUSER procedure x, as described under METHODS. All the acid treatments are performed in an ice bath to avoid hydrolysis of the nucleotides. The neutralized solution of ribonucleotides is ready for subsequent Dowex-2 treatment.

A. Establishment o/ quantitative working conditions with pure ribonucleotides. A mixture of pure ribonucleotides was obtained as described under METHODS, § 12. The working conditions are those described under § I I . Different volumes of a solution of known O.D. at 260 m~ were treated on 15o mg of Dowex-2. The results are given in Table I.

T A B L E I

QUANTITATIVE ELUTION OF PURE RIBONUCLEOTIDES FROM DOWEK-2

Approximate ratio Measured O.D. Tkeoretical O.D. Volume o/ Over-all #g nucleotide'mg at 260 m# eluate a/ter recover~,

resin at 260 mt~ (calculated) dilution in %

O.6 0.464 O.5O6 5 ml 91.6 1.8 O.8OO o.841 io ml 95.1 2.7 0.44o o-452 25 ml 97.3 5.3 o.452 o.452 5 ° ml IOO

I t was ascertained that sorption was quantitative in these working conditions (O.D. measurement on the supernatant of sorption). One may thus conclude that quantitative elution is obtained for a ratio of 5/zg nucleotides per mg of resin with a mixture of pure ribonucleotides.

B. Establishment o/ quantitative working conditions with impure ribonudeotides. Fig. I shows the behaviour of a mixture of impure ribonucleotides obtained from green Euglena gracilis after a SCHMIDT-THANNHAUSER treatment. Substances interfering with ultraviolet photometry and orcinol reaction are found in large amounts in this material.

The lower curve of Fig. I shows the spectrum of a substance or mixture of sub- stances which are not fixed by the resin. This absorption spectrum was the same for all the tissues tested.

Table I I shows that sorption and elution of a mixture of impure nucleotides are quanti tat ive in a very wide range of the ratio/zg ribonucleotides/mg of resin.

R e l e r e n c e s p . 2 0 7 .

2 0 0 M. DE DEKEN-GRENSON, R. H. DE DEKEN voL. 31 (I959)

O.D-

0Ii 0.75 I

0.50

0.25 .

0 I i i i i | i i

220 240 260 280 300 m/a

Fig. I. Curve I : absorpt ion spec t rum of the initial solution of impure ribonucleotides. O.D. 260 m#] O.D. 23 ° m # = 0.74. Curve 2: absorp t ion spec- t r u m of the same solut ion after t r ea tmen t on Dowex-2. O.D. 260 mp]O.D. 23 ° m # = 2.36. Curve 3: absorp t ion spec t rum of the supe rna tan t liquid of sorpt ion. The sum of spectra 2 and 3 equals spec t rum I within exper imenta l errors.

T A B L E I I

PURIFICATION OF RIBONUCLEOTIDES WITH DOWEX-2

Different volumes of a solution of r ibonueleotides coming f rom Euglena gracilis (white strain), are t reated wi th different quant i t ies of Dowex-2

Approximate Vol. o/ initial Quantity o/ Vol. o/eluate alter Recovered #g nucleo- ratio pg solution in ml resin in mg O.D. at a6o m~ tk~es/ml o[ initial

nucl ling resin dilution (in ral) solution

2,o I.O 15 ° 0.908 i o 3 ° i. i i .25 i .25 300 0.454 25 300.9 i .o i .o 300 0.354 25 293.5 0.75 1.5 600 0.558 25 308.3 o.5o i .o 600 0.346 25 286.8 0.25 0. 5 600 o.188 25 311.5 o.I25 0.25 600 o.216 io 286. 4

Mean value 298. 3

T a b l e I I I s h o w s t h a t q u a n t i t a t i v e w o r k is p o s s i b l e u p t o 3 tzg of n u c l e o t i d e s

p e r m g r e s in .

T A B L E I I I

~ U A N T I T A T I V E S O R P T I O N O F I M P U R E R I B O N U C L E O T I D E S B Y D O W E X - 2

Same conditions as for Table I I . Material: Euglena gvacilis (normal green strain).

VOl. o/ eltuae Recorded # g A ~ o x i m a t ~ ratio VOl. o/ initial Qua~iOt o/ O.D. at a6o m~ alter dilation nuaeotides/ml o/ #ff mwl./mg resin solution in ml resin in mg (in ml) initial solution

0.6 0.25 I5O 0.292 IO 387.3 1.2 o.5o I5O 0.57 ° IO 378.0 2.4 0.95 xSO 0.436 25 380.4 4.8 1,9 I50 0.346 5 ° 299.3

Releren~s p. ~o7.

VOL. 3 1 (1959) E S T I M A T I O N O F N U C L E I C ACIDS 2 0 I

When interfering substances are present in very high concentration, as it is the case for most plant tissues, sorption is quantitative only below I #g of nucleotides per mg of resin, and it is necessary to wash the resin after sorption. This case is illustrated by Table IV.

TABLE IV t'URnnCATION O1' A MIXTURE O1' EIBONUCLEOTIDES COMING t'EOM TOEACCO LEAVES

(Dowex-2 treatment)

A pprozimate ratio #g Vol. o/initial Quantity o~ resin Vol. o/elute in ml nucl./mg resin solution in ml in rag

ttg Nucleotides/ml o/initial solution as measured by

spearophotomdry oreinol reaclion

1. 5 2 15o IO 84. 5 - - o .75 2 ' 3 o 0 IO 116. 4 - - o . 5o 2 45 ° IO 116 .o - - 0 . 75 6 9 0 0 IO I I 7 . 5 122. 5

Part II. Comparison o/ribonudeic acid estimations by different methods on several tissues

TABLE V TOBACCO LEAVES ESTIMATIONS OF RIBONUCLEIC ACID

Results are expressed as mg of ribonucleic acid per ioo mg oi dry delipidated tissue.

Ultraviolet pkotom~ry ExtracBon or Orcinol reaction

/ractionation procedure Ratio O.D. ~6o rap/ Restdts results O.D. z3o m#

SCHNEIDER 1 .36 3 .5 ° 1 9 . 4 4 1.31 3 .39 I 8 . 3 7

OGuR-ROSEN 2 . 7 4 2"98 5 .76 2.67 3.o7 6.23

SCHMIDT-THANNHAUSER o . 6 6 3 .6o 6 .45 o .65 3 .69 6 .52

SCHMIDT-THANNHAUSER 1.7 ° 2.51 2.65 + D o w e x - 2 t r e a t m e n t 1 .65 2 .53 2 .77

I t is clear that all the results obtained with the orcinol reaction are too high, except the one obtained after Dowex treatment. This last result is in fairly good agreement with the corresponding ultraviolet absorption data. After OGuR-RosEN extraction, the ultraviolet result is about 13 % higher (see DISCUSSION).

TABLE VI B ~ c i l ~ s ~tsgaffJ~ggt4~B ESTIMATIONS OF RIBONUCLEIC ACID

(in exponential phase of growth) Results are expressed as mg of ribonucleic acid per 1oo mg of dry delipidated tissue.

U~,avio~ photon~try E~raaion or Orc/nol reaaion

/raaiona~on procedure Ratio O.D. a6o m#/ Results results O.D. 23o rap

SCHNEIDER 2 .84 2 3 . 7 2 25 .92

O G u R - R o s E N 3.52 2 4 . 2 5 2 0 . 8 4

SCMMIDT-THANNHAUSER 2 .2 I 2 5 . 4 4 2 4 . 4 0

SCHMIDT-THANNHAUSER 2 .55 2 2 - 8 9 - - + Dowex treatment 2.52 22.58 22.69

2 . 8 6 2 2 . I 6 2 2 . 4 9

Re/erences p. ~o7.

202 M. D~ DEKEN-GRENSON, R. H. Dig DZKEN VOL. 31 (I959)

The lowest resul t is given b y the orcinol react ion af te r the OGuR-ROSEN procedure. Es t imat ions made on the deoxyr ibonucle ic acid f ract ion af ter the same OGuR-ROSEN procedure (see Table XI) show:

(I) t ha t the r ibonucleic acid ex t rac t ion was incomple te (high orcinol resu]t and m a x i m u m of u l t rav io le t absorpt ion at 260 mt~ for the deoxyr ibonucle ic acid fraction) ;

(2) t ha t pa r t of the deoxyr ibonucle ic acid was ex t r ac t ed s imul taneous ly wi th the r ibonucleic acid (d iphenylamine and CERIOTTI react ions give ve ry low values for the deoxyr ibonucle ic acid fraction).

TABLE VII

B A K E R S ~ Y E A S T E S T I M A T I O N S O F R I B O N U C L E I C A C I D

Results are expressed as mg of ribonucleic acid per IOO mg of dry delipidated tissue.

Ultraviolet pkotometry Extraction or Orcinol reaction

lractionation procedure Ratio O.D. z6o mlt/ Results results O.D. 230 ml~

SCHNEIDER 2.19 5.93 7" 13

OGUR-ROSEN 3.56 6.63 5.61

SCHMIDT-THANNHAUSER I. 16 7.52 8.13

SCHMIDT-THANNHAUSER 2.20 6.32 - - + Dowex-2 treatment 2.7o 5.98 5.96

2.52 6.14 6.04

Both u l t rav io le t pho tome t ry and orcinol d a t a ob ta ined af ter SCHMIDT-THANN- HAUSER ext rac t ion plus Dowex-2 t r ea tmen t are in good agreement wi th the photo- met r ic value ob ta ined af ter SCHNEIDER ext rac t ion . The orcinol d a t a of the OGUR- ROSEN procedure seem to indicate an incomple te ex t rac t ion of the r ibonucleic acid (7 % lower). W i t h the OCI:R-RosEN procedure, the u l t rav io le t value is about IO % higher than the orcinol results, as i t is genera l ly the case (see DISCUSSION).

TABLE VIII

Bacillus subtilis E S T I M A T I O N S O F R I B O N U C L E I C A C I D

(exponential phase of growth)

Results are expressed as mg of ribonucleic acid per ioo mg of dry delipidated tissue.

Ultraviolet pkotomet~y E xtraetion or Orcinol reaction

tractionalion procedure Ratio O.D. 260 mp/ Results results O.D. z3o rot*

SCHNEIDER 2.25 I 1.62 10.39

OGUR-ROSEN 3.41 9.59 8.91

SCHMIDT-THANNHAUSER 1.57 13-25 12.3

S C H M I D T - T H A N N H A U S E R 2 . 4 2 9.89 - - + Dowex-2 treatment 2.57 9.67 12.27

Considerable d iscrepancy is observed here between the u l t raviole t pho tome t ry and the orcinol d a t a ob ta ined af ter SCHMIDT-THANNHAUSER plus Dowex-2 t r ea tmen t .

References p. ~o 7.

V0L. 31 (1959) ESTIMATION OF NUCLEIC ACIDS 203

I t can be seen tha t the orcinol-posi t ive interfer ing substance is quan t i t a t i ve ly taken up b y the resin.

TABLE IX

RAT LIVER ESTIMATIONS OF RIBONUCLEIC ACID

Results are expressed as mg of ribonucleic acid per ioo mg of dry delipidated tissue.

Ultraviolet photomavy Extraction or Orcinol reaction

/ractionation procedure Ratio O.D. z6o m/*/ Results results O.D. a3o ms*

SCHNEIDER 1.72 4.32 3.89

OGUR-ROSEN 3.22 3.07 2.69

SCHMIDT-THANNHAUSER 0.97 4.26 3.04

SCHMIDT-THANNHAUSER 1.93 2.6 7 2.94 + Dowex-2 treatment 1.87 2.53 2.50

1.86 2.65 2.74 1.91 2.66 2.49

Very good agreement is no ted be tween bo th resul ts of the SCHMIDT-THANNHAUSER plus Dowex-2 procedure on the one hand and the orcinol resul t of the OGUR-ROSEN technique, on the o ther hand. The u l t rav io le t absorpt ion resul t given b y the OGuR-ROSEN procedure is I2 % higher t han the corresponding orcinol value (see DISCUSSION).

Part I I I . Comparison o[ deoxyribonuc.leic acid estimations by di~emnt methods on several tissues

TABLE X

TOBACCO LEAVES ESTIMATIONS OF DEOXYRIBONUCLEIC ACID

Results are expressed as mg of deoxyribonucleic acid per ioo mg of dry delipidated tissue.

Ultraviolet photometry Extraction or Diphenylamine CEslorrt

lraaionaBon procedure Ratio O.D. 267 ml~] Results reaction results reaction results O.D. 230 m#

SCHNEIDER* 0.93 O.6I

0.89 0.70

OGUR-ROsEN * 0.94 O.75 O.67 O.64 O.9I O.77 0.69 O.69

SCHMIDT-THANNHAUSER* O.7 ° O.74 O.48 O.46 0.70 o.7I 0.47 0.45

* The pairs of results are obtained on independent samples of dry powder.

Af te r OGuR-ROSEN extrac t ion , es t imat ions made b y the d ipheny lamine and CEmOTTI react ions are in good agreement ; the same value (about 0.67 %) is ob ta ined wi th the CERIOTTI react ion af ter SCHNEIDER extract ion. On the o the r hand, the SCHMIDT-THANNHAUSER procedure gives a 30 % lower resul t (about o.46 %), e i ther b y the d ipheny lamine or the CERIOTTI reaction.

Relevences p. zo7.

204 M. DE DEKEN-G~X~SON, R. H. DS DZKB~ vOL. 31 (i959)

T A B L E X I

Bacillus megather ium ESTIMATIONS OF DEOXYRIBONUCLEIC ACID

Results are expressed as m g of deoxyribonucle ic acid per i o o m g of dry del ipidated tissue.

Ultraviolet photometry Orcinol Diphenylamine CERXOTTt Extraaion or reaaion reaction reaction

/ractionation procedure Ratio O.D. ~6 7 ml~] Results results results results O.D. ~3 o mtJ

SCHNEIDER i .48 1.25

OGuR-ROSEN 1.94" 1.58* 7.42 0.47 0.39

S C H M I D T - T H A N N H A U S E R - - 1 . 2 5

M a x i m u m of absorpt ion lies at 260 m p in th is caSe.

OGug-RosEN procedure: data obtained by the diphenylamine and CERIOTTI reactions on the one hand and by the orcinol reaction on the other hand clearly show that most of the deoxyribonucleic acid has been extracted together with the ribonucleic acid and that the ribonucleic acid has not been extracted quantitatively. Fortunately, values given by the CERIOTTI reaction, after SCHNEIDER extraction, a~ well as after SCHMIDT-THANNHAUSER extraction are in full agreement. After the SCHNEIDER

procedure the diphenylamine reaction data are higher than the CERIOTTI reaction data, as it is usually the case.

T A B L E X l I

BAKERS' YEAST ESTIMATIONS OF DEOXYRIBONUCLEIC ACID

Resul ts are expressed as m g of deoxyribonucle ic acid per zoo m g of dry del ipidated tissue.

Ultraviolet photometry Orcinol Diphenylamine CEmor'r t Extraction or reaction reaction rea~ion

tractionation procedure Ratio O.D. ~6 7 mt~] Results results results results O.D. ~3o ml~

SCHNEIDER 0.29 O.2I

OGuR-ROSEN 0.69 0.26 3.95 0.27 0.27

SCHMIDT-THA.NNHAUSER 0.58 0.39 0.20 0.20

A value of about o.2o % deoxyribonucleic acid was found by three different ways: the SCHmDT-THANNHAUSER procedure (diphenylamine and CERIOrrI) and the CERIOTTI reaction after the SCHNEIDER procedure. This value is nevertheless about 26 % lower than the values given by OGuR-ROSEN technique.

T A B L E X I I I

Baci l lus 5~btili$ ESTIMATIONS OF DEOXYRIBONUCLEIC ACID

Resul ts are expressed as m g of deoxyribonucle ic acid per IOO m g of dry del ipidated t issue.

Ultraviol~ photon~ry Orcinol Dipkasylamine CxRtoxnrt Extraaion or reaaion raoxfion reaction

/ractionation procedure Ratio O.D. aM7 ml~[ O.D. ~3o ral~ Restdta results rssult$ res~J~

SCHNEIDER 2.64 2.57

OGUR-ROSEN 2.IO 2.96 3.65 2.66 2.88

SCH M IDT-THANNHAUSER I. 26 3.29 - - Z. 16 2.12

Re[evences p. .2o7,

voL. 31 (1959) ESTIMATION OF NUCLEIC ACIDS 205

As far as the diphenylamine and CERIOTTI reactions are concerned, the data obtained after the SCI-IMIDT-THANNHAUSER extraction are in good agreement but the figures are 20 % lower than with the other methods of extraction.

T A B L E X I V

RAT LIVER ESTIMATIONS OF DEOXYRIBONUCLEIC ACID

Resu l t s are expressed as m g of deoxyr ibonucle ic acid per ioo m g of d r y de l ip ida ted t i ssue .

Ultraviolet photometry Orcinol Diphenylamine CEmOTrI

Extraction or reaction reaction reaction ]ractionation procedure Ratio 0.D.267 rag [ Results resnlts results results

0"D.23o ra#

SCHNEIDER I. I O 0.93

OGuR-ROSEN 1.75 1.55 5.37 0.86 O.81

SCHMIDT-THANNHAUSER 0.72 1.20 2.07 0.75 0.78

For rat liver, the case is essentially the same as for Bacillus subtilis. The results obtained after SCHMIDT-THANNHAUSER extraction are 12 % lower than with the OGuR-ROSEN method of extraction.

DISCUSSION

A. Ribonucteic acid estimations

Beside the obvious advantages of the OGUR AND ROSEN procedure over the other methods, serious disadvantages were already pointed out by one of the authors (lack of selectivity of the separation of the two nucleic acids for certain tissues, ~1, n). Moreover, the present work shows that in all the tissues extracted (Tobacco leaves excepted) by the OGuR-ROSEN procedure the ultraviolet absorption data are higher than the corresponding pentose estimation data*. Now it is certain that the orcinol result has to be considered as a maximum value, this estimation technique being generally liable to give too high a result (interferences due to sugars, etc.). Under these conditions it is necessary to accept that the values given by ultraviolet photo- metry in the OGuR-ROSEN procedure are too high and that this situation results from the presence of interfering substances absorbing light in the 26o m/~ region. The presence of such substances might explain the strikingly high value always obtained for the ratio 0.D.26 o m~/O.D.23 o ~ (> 3) when the OGuR-ROSEN extraction is performed.

I t seems that the method developed in the present work, i.e. the SCHMIDT- THANNHAUSER procedure with added Dowex-2 treatment, gives the most valid results. I t is indeed the only method giving regularly a good agreement between the estimations made by ultraviolet photometry and the orcinol reaction. Substances which are not

" It is i m p o r t a n t to no te t h a t OGUR AND ROSEN c a m e to t h e oppos i te resul t , i . e . t h e a m o u n t of r ibonucleic acid e s t i ma t ed b y t h e orcinol reac t ion is 8 % h igher t h a n b y t h e u l t r a v i o l e t p h o t o - m e t r y for r a t l iverL Th i s d i s ag reemen t m i g h t be exp la ined in t h e following w a y : OGUR AND ROSEN h a v e adop t ed a long h e a t i n g t i m e for t h e orcinol reac t ion; t h i s m a k e s i t more sens i t ive to in ter - fe t ing s u b s t a n c e s 2. I n t h e p r e sen t work, a h e a t i n g t i m e of 3 m i n u t e s was chosen, wh ich gives a more specific reac t ion .

ReJerences p. ~o7.

206 M. DE DEKEN-GRENSON, R. H. DE DEKEN VOL. 31 (1959)

taken up by the resin and which absorb light at 270 m/~ do not seem to be nucleic acid derivatives. MAURITZEN et al. 23 already pointed out the presence in alkaline ribo- nucleic acid hydrolysate of tyrosin- and possibly tryptophane-containing material responsible for absorption at about 270 m/z.

The main advantage of the new method presented here is that the procedure may be directly applied to plant tissues as well as to animal tissues and microorganisms without additional control by an other method. Several possibilities of verification are indeed provided by the method itself.

B. Deoxyribonucleic acid estimations

If the results are considered from the point of view of agreement between the diphenylamine and CERIOTTI reactions, it is important to note the following points:

(I) SCHNEIDER extraction procedure gives rise to a disagreement between the data of the diphenylamine and CERIOa'TI reactions, the first being too high. This high result with diphenylamine already pointed out for liver tissue ~4, ~ seems thus to be a general phenomenon.

(2) After the OGvR-ROSEN extraction, the values given by the diphenylamine and CERIOTTI reactions are in good agreement.

(3) Very good agreement is obtained between the data of both reactions after the SCHMIDT-THANNHAUSER procedure.

The problem is that the values obtained by the OGuR-ROSEN procedure are nearly always higher than the results given by the SCHmDT-THANNHAOSER procedure. Two possibilities must be taken into account:

(I) The diphenylamine and CERIOTTI reactions are sensitive to impurities released by perchloric extraction.

(2) The precipitation of the deoxyribonucleic acid performed during the SCHMIDT- THANNI~AIJSER procedure is not quantitative.

The action of acids on the deoxyribonucleic acid which is known to render it alcali-labile, might be taken into account, but care was taken to avoid this.

If it is impossible under the present conditions to decide what procedure of extraction of the deoxyribonucleic acid is the most adequate, some points concerning the SCHMIDT-THANNHAUSER method may be emphasized: on the one hand deoxyribo- nucleic acid estimation by the SCHMIDT-THANNHAUSER procedure is very reproducible. This is in opposition with the hypothesis of non-quantitative precipitation of the deoxyribonucleic acid. On the other hand, it must be pointed out that perchloric acid extraction of the deoxyribonucleic acid is made on a less complex material with the SCHMIDT-THANNHAUSER procedure than with the other methods. Possibilities of occurrence of interfering substances are thus smaller.

I~IOTE ADDED IN PROOF (Received November Ist, 1958 )

I t w a s observed afterwards:

I. that the CERIOTTI and diphenylamine reactions, when performed on deoxy- ribonucleic acid hydrolysed at 9 o°, give results which are IO % lower than those obtained after a 70 ° hydrolysis. Thus, the values given in this paper for deoxy- ribonucleic acid after SCHNEIDER and SCHMIDT-THANNHAUSER extractions, must be

Re/erence$ p. 207.

VOL. 31 (I959) ESTIMATION OF NUCLEIC ACIDS 207

r e a d j u s t e d to a i o ~/o h ighe r va lue . Th is r e su l t s i n to a b e t t e r a g r e e m e n t b e t w e e n t h e

SCHMIDT-THANNHAUSER and 0GuR-ROSEN procedures; 2. t h e d e o x y r i b o s e c o n t e n t of a d e o x y r i b o n u c l e i c ac id h y d r o l y s a t e (5 % pe r -

ch lor ic acid) , dec rea se s w i t h t i m e , w h e n s t o r e d a t r o o m t e m p e r a t u r e ;

3. t h e p u r i t y of t h e h y d r o c h l o r i c ac id af fec ts s e n s i t i v i t y of t h e CERIOTTI r e a c t i o n

w i t h i n a w i d e range .

W e a re v e r y g r a t e f u l t o S. BOURGEOIS w h o d i r e c t e d ou r a t t e n t i o n u p o n t h e s e fac t s .

REFERENCES

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Vol. I, Academic Press, Inc., New York, 1955, P. 3o3 • 3 E. VOLKIN AND W. E. COHN, in D. GLICK, Methods o/Biochemical Analysis, Vol. I, Interscience

Publishers, Inc., New York, I954, p. 292. 4 Z. DISCHE, in E. CHARGh, FF AND J. N. DAVIDSON, The Nucleic Acids, Chemistry and Biology,

Vol. I, Academic Press, Inc., New York, 1955, P. 3Ol. s M . DE DEKEN-GRENSON AND R. H. DE DEKEN, Arch. int. physiol, et biochim., 66 (I958) I15. s W. C. SCHNEIDER, G. N. HOGEHOOM AND H. E. ROSS, J. Natl. Cancer Inst., 1o (I95 o) 977.

M. OGUR AND G. ROSEN, Arch. Biockem., 25 (I95O) 262. 8 W. C. SCHNEIDER, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Emymology, Vol. III ,

Academic Press, Inc., New York, I957, p. 68o. 8 Z. DISCHE, Mikrochemie, 8 (193o) 4.

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Publ. Inc., New York, 1954, p. 29o. 12 B. MAGASANIK, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Chemistry and Biology,

Vol. I, Academic Press, Inc., New York, 1955, P. 393. 18 C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (i925) 375. 14 E. CHARGAFF, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Chemistry and Biology,

Vol. I, Academic Press, Inc., New York, 1955, p. 335. xs Z. DISCHE AND K. SCHWARZ, Mihrochim. Acta, 2 (1937) I3. in Z. DlSCHX, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Chemistry and Biology,

Vol. I, Academic Press, Inc., New York, 1955, p. 287. 1~ W. E. COHN, J. Am. Chem. Soc., 72 (195 o) i47I. 18 E. VISCHER AND E. CHARGAFF, J. Biol. Chem., I76 (1948) 715 . 18 M. SEVAG, D. B. LACKMAN AND J. SMOLENS, J. Biol. Chem., i24 (I938) 425 . ~o B. TAYLOR, J. P. GREENSTEIN AND A. HOLLAENDER, Cold Spring Harbor Symposia O~uant. Biol.,

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(1952 ) 175. 8~ E. K. PATTERSON AND M. E. DACKERMAN, Arch. Biochem. Biophys., 36 (1952) 97- ~8 C. M. MAURITZEN, A. n. RoY AND E. STEDMAN, Proc. Roy. Soc. London, B I4o, (I952) 18. ~4 V. R. POTTER, R. O. RECKNAGEL AND R. B. HURLBERT, Federation Proc., IO (1951) 646. 15 W. C. SCHNEIDER, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzwmology, Vol. I I I ,

Academic Press, Inc., New York, 1957, p. 681.