A SENSITIVE METHOD FOR THE DETERMINATION OF RIBONUCLEIC ACID IN TISSUES

AND MICROORGANISMS

BY JUNIUS M. WEBB

(From the Laboratory of Infectious Diseases, National Microbiological Institute, National Institutes of Health, United States Department of Health, Education,

and Welfare, Bethesda, Maryland)

(Received for publication, December 12, 1955)

The increasing interest in the composition of nucleoproteins has em- phasized the need for more specific methods of assay of constituent nucleic acids. In a previous paper (1) a method was reported for the assay of deoxyribonucleic acid (DNA) in tissues and microorganisms based on the reaction of p-nitrophenylhydrazine with deoxyribose following hydrolysis of the biological material with 5 per cent trichloroacetic acid (TCA). The present paper reports a method for ribonucleic acid (RNA) assay with an aliquot of the same TCA hydrolysate.

The most commonly employed method for RNA assay is that of Mej- baum (a), in which orcinol is used to develop a blue-green color with fur- fural formed from ribose when the latter is heated with prescribed amounts of FeCl,-HCl solution. The results must be corrected for DNA inter- ference (3, 4). In a similar method developed by von Euler and Hahn (4) phloroglucinol is employed. DNA does not interfere, but the method is less sensitive than the orcinol procedure. In a third method, used by Davidson and Waymouth (5), the furfural formed from ribose is extracted with xylene and the xylene extract is allowed to react with aniline acetate to form a pink color. Although DNA does not interfere, the method is complicated by the necessity of removing HCl prior to color formation and the photosensitivity of the color (6). However, the extraction procedure offers certain advantages, namely (1) concentration of the furfural formed and (2) elimination or minimization of interfering substances.

In an effort to combine these advantages with a more efficient chromo- genic reagent, a method has been developed in which p-bromophenyl- hydrazine (PBPH) reacts quantitatively in acidified xylene-alcohol solu- tion with furfural formed from the liberated ribose of RNA. As seen be- low, the specific extinction coefficient of the colored product formed with the proposed reagent is about l$ times that of the colored product result- ing from the Mejbaum or&no1 procedure when optical densities are taken at their respective wave-lengths of maximal absorption. The color is stable and reproducible. Experiments performed with several carbohy-

635

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636 RNA IN TISSUES AND MICROORGANISMS

drates show that the proposed method is as specific or more so than the or- cinol procedure. The presence of DNA does not interfere with the test.

EXPERIMENTAL

Reagents- 1. Trichloroacetic acid, Merck reagent. 2. Xylene, Mallinckrodt, c.p. Some brands of xylene gave a slightly

higher blank which was not improved by using the redistilled solvent. A Caulfield Pipettor (Caulfield Safety Devices, Havertown, Pennsylvania) was used for most pipetting operations.

3. NaCl crystals, Bakers’, c.p. 4. Ethyl alcohoLHC1 solution. 2 ml. of HCl, c.p. (37 per cent), were

added to 100 ml. of 95 per cent ethyl alcohol. 5. p-Bromophenylhydrazine hydrochloride, Eastman Kodak Company,

Rochester, New York. A 2.5 per cent solution of the hydrochloride in ethyl alcohol-HCl solution was prepared fresh daily. It was found ex- pedient to decolorize the solution with charcoal (Norit A, Fisher Scientific Company, New York). About 1 gm. of charcoal per 30 ml. of volume was adequate.

6. Ribonucleic acid, Nutritional Biochemicals Corporation, Cleveland, Ohio. The material was purified, following the method of Kunitz (7), by reprecipitation from glacial acetic acid. Analysis of the RNA showed N 14.6 per cent, P 9.0 per cent.

Materials Analyzed for RNA- 1. Rat tissues. The livers, lungs, and kidneys from young Sprague-

Dawley rats were removed immediately after sacrificing the animals and were frozen at once in dry ice.

2. Yeast. Bakers’ yeast (Fleischmann), dried in 2racuo, was used. 3. Bacteria. Escherichia coli, from a 17 hour culture and Proteus am-

moniae, from 17 and 48 hour cultures grown at 37” on horse meat infusion agar, were harvested in 50 ml. of 0.85 per cent saline. The suspensions were treated with 1 ml. of 37 per cent formaldehyde and after 15 minutes the residues were recovered by centrifugation for 10 minutes at 20,000 X g in a Servall SS-1 centrifuge. The residues were washed twice with water, recovered by centrifugation, and dried in vacua.

The biological materials were prepared for analysis by acid washing with cold 10 per cent TCA, defatting with alcohol and ether, and finally convert- ing to a dry powder as described (1). For purposes of testing analytical procedures, the dry material serves as a convenient source of reproduc- ible material from one experiment to another. However, the analyti- cal procedure to be described may be applied to known amounts of homog- enized tissue or microorganism following the acid-alcohol-ether extractions, without conversion to powder.

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Analytical Procedure

Hydrolysis-In the manner previously described (l), the material to be analyzed was hydrolyzed with 5 per cent TCA for 30 minutes in a boiling water bath followed by dilution with an equal volume of 5 per cent TCA. Since, in some experiments to follow, it was desired to have sufficient aliquots for other assays, 3 ml. of 5 per cent TCA were used for hydrolysis after which an additional 3 ml. volume of 5 per cent TCA was added. In other experiments, hydrolysis was with 2 ml. of 5 per cent TCA followed by an additional 2 ml. volume. 1 ml. of the diluted hydrolysate was equiva- lent to 0.75 to 6.0 mg. of the dry powder and to 9 to 200 y of RNA.

100 ml. of a stock solution of standard hydrolyzed RNA (1 ml. = 1 mg.) were prepared in a similar manner. RNA standards of lower con- centration were prepared by appropriate dilution of this stock solution with 5 per cent TCA.

Conversion to Furfural-To 1 ml. of the diluted hydrolysate in a 12 ml. centrifuge tube was added 1 ml. of 8 N HCI followed by 1 ml. of xylene and enough NaCl crystals to saturate the mixture. 1 ml. of a standard solu- tion of hydrolyzed RNA of appropriate concentration (usually 150 y) and 1 ml. of 5 per cent TCA, constituting the blank, were treated likewise. The centrifuge tubes, the mouths of which were covered with sealed ampul bulbs, were placed in a boiling water bath for 3 hours. After cooling, 2 ml. of xylene were added to each tube and the contents were mixed and centrifuged.

Color Development-2 ml. of the xylene layers were transferred to 5 ml. volumetric flasks to which were added 2 ml. of the PBPH reagent. After mixing the contents, the flasks were stoppered with ground glass stoppers Andy placed in aconstant temperature- ronm_&37P for_ l_hour~ _ ‘The. .con, tents of the flasks were diluted to volume with ethyl alcohol-HCl solution, mixed, and transferred to matched + inch Bausch and Lomb tubes. Opti- cal densities of the solutions were measured against the blank by means of the Bausch and Lomb Spectronic 20 calorimeter set at 450 mp.

Standardization and Validity of Method

Optimal Conditions for Hydrolysis with 5 Per Cent TCA-When the pro- posed method was applied to 15 and 30 minute hydrolysates, no difference in color intensities of the final solutions was observed. However, in order to use the same hydrolysate for both RNA and DNA determinations, the 30 minute hydrolysis period recommended for DNA (1) was adopted.

Optimal Conditions for Conversion of Ribose to Furfural-Two variables were studied: time of heating in a boiling water bath (1 to 6 hours) and concentration of HCl (1 to 8 N), while the amount of hydrolyzed RNA and the volume of the final mixture were held constant. A 6 N HCl concen-

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638 RNA IN TISSUES AND MICROORGANISMS

tration with 3 hours of heating was found to be optimal. Higher concen- trations of HCl resulted in appreciable furfural destruction.

Since Foster (8) has shown that the presence of NaCl in the acid con- version medium enhances the production of furfural from pentoses with lower acid concentration, the above experiment was repeated by using final acid concentrations of from 1 to 6 N and saturating each mixture with NaCl. Under these conditions and with 3 hours of heating the results with a 4 N

HCl concentration were about 10 per cent higher than that with a 6 N

HCl concentration without NaCl. Furthermore, the time of heating was not as critical as without NaCl.

Optimal Conditions for Color Formation-The amount of PBPH reagent prescribed was found sufficient for the assay of at least 210 y of standard RNA. Doubling the amount of reagent at this concentration gave no further increase in color intensity. Color development took place slowly at room temperature, and a maximum was attained sooner (60 minutes) at 37” in an incubator room (or a slightly shorter time in a constant tem- perature bath at this temperature). The color was stable for at least 2 hours.

Although HCl was necessary to the reaction and to stabilize the reagent, its concentration was not critical. Doubling the concentration gave no i~~reas~.iir-.~~l~i.r,i~t~~;si.~y .ijj~r.-r;&t+& .th. -pi& .f*r. ~0~~~. &.+-&piriq~jp.:

Larger volumes of HCl, however, were insoluble in the xylene-alcohol mixture.

Source of Reactive Ribose-Purified yeast RNA (N 14.9 per cent, P 9.0 per cent) was analyzed by the proposed method with recrystallized n-ribose as a standard. The RNA was found to yield 22.2 per cent ribose, which was about 50 per cent of the total theoretical ribose content of RNA as calculated from the tetranucleotide formula. Since yeast RNA composi- tion has been found to correspond approximately to the tetranucleotide formula, and since purine nucleotides hydrolyze easily while pyrimidine nucleotides do not, it is likely that the purine nucleotides were the main source of reactive ribose. To demonstrate further the probability of this assumption, the purity of the RNA can be calculated, based on the theoret- ical amount of purine nucleotide ribose (23.4 per cent calculated from the tetranucleotide formula). This purity was computed to be 94.9 per cent and corresponds well with that calculated from nitrogen analysis (91.4 per cent) or from phosphorus analysis (94.7 per cent).

Xtandardixation of Test-To show that the amount of colored product formed with PBPH is directly proportional to the amount of RNA present and to determine the reproducibility of the procedure, the method was applied to various dilutions of hydrolyzed RNA standard solution. One set of such dilutions, along with a reagent blank, was run independently

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J. M. WEBB 639

eight times and optical density readings were made against distilled water. The resulting direct proportionality is shown in Fig. 1.

The reproducibility for the eight determinations at each concentration level may be shown by the relative mean deviations. At concentrations of 9.4, 18.8, 37.5, 75.0, and 150 y the mean deviations, expressed as a per cent of their respective means, were 7.4,4.0,3.9,4.5, and 4.5, respectively.

Spectral Characteristics-Spectral transmittance curves presented in Fig. 2 show the absorption characteristics of the colored product resulting from

FIG. 1. Standardization curve, showing the reIationship between optical density and amount of RNA, as obtained by the p-bromophenylhydrazine method. The amounts of RNA represent the initial amounts of hydrolyzed RNA taken. The final aliquots used for color development contained the equivalent of two-thirds of these initial amounts, as described in the assay procedure. Each point represents the mean of eight determinations. The readings were made against distilled water with matched $ inch tubes in the Bausch and Lomb Spectronic 20 calorimeter at 450 rnp.

the PBPH reaction. For comparison, the curve for the colored product resulting from the orcinol reaction is also given. These curves represent solutions of the same concentration of RNA (15 y per ml.) read against their respective blanks and illustrate the quantitative sensitivity of the two reactions. The extinction coefficients at their respective maxima (450 rnp for the PBPH product and 660 rnp for the orcinol product) show the absorption of the former to be about 14 times that of the latter.

Relationship between Amounts of Tissue Powder and Amounts of RNA Found-Fig. 3 illustrates the results of an experiment in which the method was applied to increasing amounts of liver tissue powder. It will be seen that there is a direct proportionality between optical density readings and the amounts of powder used.

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640

0.5

0.4

2- 0.3 l- 5

2 0.2

$1

I=

80 3

RNA IN TISSUES AND MICROORGANISMS

I 1 I I I I I

I I I I I I I 0 450 550 650 750

WAVE LENGTH (M/t)

FIG. 2. Absorption spectra of the products of the p-bromophenylhydrazine reac- tion and of the orcinol reaction with hydrolyzed RNA. The initial concentration was such that the final colored solution in each case was equivalent to 15 y of RNA per ml. The curves were taken from the Bausch and Lomb Spectronic 20 colorime- ter. Essentially the same curves and maxima were obtained with the Beckman quartz spectrophotometer (model DU). Matched 3 inch test-tube cells were used, and the solutions were read against their respective blanks.

7 LIVER TISSUE POWDER (MILLIGRAMS)

FIG. 3. Application of the p-bromophenylhydrazine method to increasing amounts of liver tissue powder. Each amount of tissue powder represents the initial amounts of hydrolyzed liver powder per ml. The two determinations at each level were made in-separ~~-h.ydrel-yysates.. l\FJ.in~g-ltfbefina.l~tliql!nts.used for color development contained the equivalent of two-thirds of the hydrolyzed RNA that was in the weight of tissue powder shown. The optical density readings were made as described in Fig. 1.

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J. M. WEBB 641

Recovery of RNA-A given volume of hydrolyzed RNA standard solu- tion was added to a given volume of hydrolysate of liver tissue powder so that a 1 ml. volume of the mixture was equivalent to 1 mg. of liver tissue powder and to a known amount of RNA. Three concentrations of hy- drolyzed RNA were used so that the final equivalences of added RNA, per ml., were 20, 40, and 80 y. After applying the test procedure, the average of duplicate determinations made at each level showed recoveries ranging from 98 to 100 per cent.

Specificity--The compound produced from ribose and RNA, under condi- tions of the test, gave the same absorption maxima and spectrophotometric curves with PBPH as pure furfural. It should be noted that some carbo- hydrates yield this aldehyde with similar acid treatment. However, the proposed method, in contrast to the orcinol or phloroglucinol procedures, eliminates those other possible interfering substances which do not yield furfural and are not extractable by xylene.

Under conditions of the proposed test, the assay of 1 mg. of deoxyribose anilidel gave no color, and from 1 mg. of DNA only a trace was observed. This small amount of color presumably came from an impurity, possibly RNA itself, inasmuch as present methods of preparation may not com- pletely separate the two nucleic acids.

Galacturonic acid, which gave about the same amount of color as that given by the same weight of RNA, was the most serious possible interfer- ing substance tested. Among other materials, glycogen and methyl glu- coside yielded about one-twentieth, while levulose, dextrose, and saccha- rose yielded one-tenth of the color given by the same weight of RNA. Though these interfering substances are soluble in one or both extracting solvents used in preparing the biological material for analysis, there is no assurance of their complete absence. For example, some furfural-yielding materials (polysaccharides containing uranic acid groups) are bound to proteins in the native state (9). It is conceivable that they would not be removed by the cold extractions but would be released, at least in part, by heating with 5 per cent TCA.

Application of Method and Comparison of Results

In order to determine the applicability of the proposed method, the re- sults with PBPH were compared with those with orcinol and with phos- phorus and ultraviolet absorption measurements in the following manner. The RNA content of extracted powders of several tissues and microor- ganisms was determined by the PBPH and orcinol methods (2) and the corresponding phosphorus and ultaviolet absorption values were calculated. Similarly, phosphorus and ultraviolet absorption values were calculated

1 Deoxyribose anilide may be obtained from the California Foundation for Bio- chemical Research, Los Angeles, California.

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642 RNA IN TISSUES AND MICROORGANISMS

from results of DNA assays (1). By employing the RNA phosphorus re- sults as found by the two RNA methods, two figures for total phosphorus content were calculated by adding to each RNA phosphorus value the DNA phosphorus content. These calculated results were then compared with total phosphorus content found by direct determinationP The cal- culated phosphorus contents were based on the average phosphorus con- tents found experimentally for the two nucleic acids used for standards, 9.0 per cent for RNA and 7.9 per cent for DNA.3 Calculated ultraviolet ab- sorption values were summed in a similar manner and compared with ultraviolet absorption measurements found directly. The ultraviolet values were calculated from extinction coefficients (& ,,.) of 0.224 and 0.285 found for 0.001 per cent solutions of hydrolyzed DNA and RNA standards, respectively, as determined at 260 m,u in the Beckman quartz spectrophotometer (11). The same hydrolysate was used for the different assays and quadruplicate analyses were performed on each material.

The data obtained are summarized in Tables I, II, and III. Analyses of Tissues and Microorganisms for RNA by PBPH and Orcinol

Methods and for DNA by p-Nitrophenylhydraxine Procedure-The results obtained are reported in Table I. It may be seen that, with the exception of yeast, the RNA values as found by the PBPH method, although some- what lower, were in fair agreement with RNA values as found by the orcinol procedure. The RNA values for yeast by the latter method were obviously increased by a brownish red non-specific color which contributed to the total absorption and for which no adequate correction could be made. Levy et al. (12), using the phloroglucinol procedure of von Euler and Hahn (4), experienced similar difficulty with yeast. Perhaps the con- sistently higher RNA values found here for the other materials by the or- cinol method could also be attributed to such non-specific color.

The orcinol RNA values shown have been corrected for DNA interfer- ence. Experimentally, DNA gave 12.5 per cent of the color given by the same weight of RNA at 660 rnp. This was the same amount of correction found necessary by von Euler and Hahn (4).

The reproducibility of the PBPH method is illustrated by the data

2 Phosphorus determinations were performed by the molybdenum blue reaction. The reducing agent was 0.5 per cent p-hydroxyphenylglycine in 2.5 per cent Na803 solution. After dilution of the H&S04 digests with 10 ml. of HzO, 2 ml. of (NHd)r.- Mo~O~~.~H~O (2.5 per cent in 9 N HkSOd), followed by 1 ml. of the reducing agent, were added. A stable color was developed in an approximately 2 N acid concentra- tion by heating for 30 minutes in a boiling water bath. After dilution to 15 ml., the solutions were read at 660 mp.

3 The DNA, prepared from calf thymus by the method of Hammarsten (lo), also gave an analysis of 13.7 per cent nitrogen.

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TABLE I

Analyses of Tissues and Microorganisms for RNA by p-Bromophenylhydrazine and Orcinol Methods and DNA by p-Nitrophenylhydrazine

Original material Analysis No.

- I DNA, y per mg.

extracted powder PBPH Orcinol*

- -- Rat liver 1 36.9 37.9 13.4

2, I?.~~7 1 ~$j&. 3s * 3 32.0 37.8 12.3 4 34.2 39.4 12.3

Average . 34.5 38.4 12.9

Rat lung

--

-_

--

--

15.5 16.1 37.8 13.3 12.9 40.4 13.1 14.6 39.9 15.1 17.3 38.0

Average . . . . . . 14.3 15.2 39.0

Rat kidney 1 22.0 28.1 20.6 2 20.7 24.5 21.8 3 20.4 24.7 20.6 4 20.8 24.8 20.2

Average . . . . . . . 21.0 25.5

.-

--

.-

_-

20.8

Yeast 1 75.0 124.4 2 72.5 104.6 3 66.3 113.7 4 69.5 124.4

5.1 5.0 4.5 4.8

Average 70.8 116.8 4.9

E. coli 1 2 3 4

127.5 132.1 127.5 128.2 108.5 122.3 125.9 129.4

Average .

--

--

-

58.8 58.2 52.0 53.9

122.4 128.0 55.7

RNA, y per mg. extracted powder

* Corrected for DNA interference, but not for other non-specific color (see the

text).

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644 RNA IN TISSUES AND MICROORGANISMS

TABLE II

Total Nucleic Acid Phosphorus Content Found in Tissues and Microorganisms and That Calculated from RNA and DNA Methods

DNAP = deoxyribonucleic acid phosphorus; RNAP = ribonucleic acid phos- phorus.

i Phosphorus, y per mg. extracted powder

Original material Analysis No.

Rat liver

Average . . . . . . . . . . 4.4 4.2

Rat lung

Average . . . . . . . . . . . . . 4.5 4.4 4.5

Rat kidney 1 3.7 3.6 4.2 2 3.7 3.6 3.9 3 3.8 3.5 3.9 4 4.0 3.5 3.8

Average . . . . . . . . . . . . . . . . 3.8 3.6

Yeast 1 7.2 7.2 11.6 2 7.3 6.9 9.8 3 7.3 6.3 10.6 4 7.1 6.6 11.6

Average . . . . . . . . . . 7.2 6.8 10.9

E. coli 1 14.9 16.1 16.5 2 15.2 16.1 16.1 3 14.6 13.9 15.1 4 14.9 15.6 15.9

Average . . , . . . . . . . . . . . . . . . . 14.9 15.4 15.9

Found ?NAP + RNAP

(orcinol)

4.3 4.4 4.5 4.2 4.3 3.9 4.3 4.1

4.5 4.5 4.4 4.5

4.5

4.4 4.4 4.4 4.7 4.4 4.4 4.5 4.3 4.5 4.4 4.4 4.6

4.0

Calculated (see text)

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J. M. WEBB 645

TABLE III

Total Nucleic Acid Ultraviolet Absorption Found for Tissues and Microorganisms and That Calculated from RNA and DNA Methods

Original material Analysis No.

Rat liver 1 2 3

I 4 Average . .

Rat lung

Average .

Rat kidney

Average . . . . . . . . 0.116 0.107 0.120

Yeast 1 0.188 0.225 0.366 2 .0.188 0.218 0.309 3 0.180 0.199 0.334 4 0.179 0.209 0.365

Average . . 0.184 0.213 0.344

E. coli 1 0.453 0.495 0.508 2 0.403 0.494 0.495 3 0.384 0.426 0.465 4 0.392 0.480 0.490

Average .

* 1 cm. light path at 260 mp.

- I -

--

--

--

--

--

--

-

Optical density* per 0.1 mg. extracted powder per ml. TCA

Found DNAP + RNAP

(orcinol)

0.126 0.135 0.138 0.130 0.129 0.140 0.129 0.119 0.135 0.128 0.125 0.140

0.128 0.127 0.138

0.126 0.129 0.131 0.120 0.128 0.127 0.125 0.127 0.131 0.123 0.128 0.134

0.124 0.128 0.131

0.116 0.109 0.126 0.123 0.108 0.119 0.113 0.104 0.117 0.112 0.105 0.116

0.408 0.474 -

0.490

Calculated (see text)

shown. With the five different materials on which four analyses are re- ported on each, the per cent deviation of each determination from its re- spective mean of four such determinations can be calculated. By the

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646 RNA IN TISSUES AND MICROORGANISMS

PBPH method the average of all twenty of the per cent deviations was 4.7 and the maximal single per cent deviation was about 11 per cent. By the orcinol method, the average of the deviations (omitting the yeast deter- minations) was about the same (4.6), whereas the maximal single devia- tion was about 15 per cent.

Comparison of Total Phosphorus and Ultraviolet Absorption Values Found Experimentally with Calculated Data from Nucleic Acid Assays-The results obtained are given in Tables II and III. It may be seen that calculated data for yeast, based on the RNA results as determined by the orcinol method, reflected the extraneous absorption, since significantly higher phos-

TABLE IV

Conversion of Nucleic Acid Values Found for Extracted Powders to Corresponding Values for Original Biological Material

Mg. extracted

powder per 100 mg. original

materialt

iverage RNA, mg. per 100 mg. Average

original material DNA, mg. per iop mg.

PBPH / Orcinol* orlglnal material

Average RNA, y per mg. extracted “EGA:

Tissue or organism powder Y Per mg. extracted

PBPH Orcinol’ powder ~~ ~_____

Rat liver.. 34.5 38.4 12.9 17.2 0.59 0.66 0.22 “ lung.. 14.3 15.2 39.0 13.3 0.19 0.20 0.52 LL kidney. . 21.0 25.5 20.8 14.0 0.29 0.36 0.29

Yeast. 70.8 116.8 4.9 73.2 5.18 8.55 0.36 E. coli. 122.4 128.0 55.7 69.2 8.54 8.86 3.85

* See foot-note to Table I. t Wet weight for rat tissues; dry weight for yeast and E. coli.

phorus and ultraviolet absorption values than those found by actual assay were obtained. On the contrary, calculated data for this material, based on the RNA results found by the PBPH method, agreed satisfactorily with values obtained by actual assay. For rat tissues, it is obvious that there was good agreement between phosphorus and ultraviolet absorption values calculated and those found, regardless of which RNA value was taken. For. E. coli, however, calculated data were in greater agreement with those of actual assays when RNA values as found by the PBPH reaction were taken.

There was a tendency, in general, for greater agreement between calcu- lated and found phosphorus values than between calculated and found ultraviolet absorption values, regardless of which RNA results were in- valved .

Conversion of Nucleic Acid Values of Extracted Powders to Equivalent Values for Original Material-For comparison with data in the literature,

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J. M. WEBB 647

which are often given as per cent of wet weight in the case of animal tis- sues, or per cent of dry weight in the case of microorganisms, the nucleic acid values for the extracted powders (Table I) have been converted to corresponding values for the original material. Table IV gives the values obtained for the several tissues and microorganisms when their average RNA values, as found by the two methods, and their average DNA values are multiplied by their respective conversion factors (mg. of extracted powder per 100 mg. of original material).

DISCUSSION

Since only the purine nucleotides hydrolyze readily to give pentose, in the PBPH procedure, as in the orcinol method, essentially only purine- bound ribose is measured. Hence, RNA determination based on the carbo- hydrate moiety is complicated by possible differences in the purine-pyrimi- dine nucleotide ratio from one nucleic acid to another. That considerable variations in the purine-pyrimidine ratio can occur has been demonstrated by Chargaff et al. (13-15) and also Loring (16) who found the purine con- tent of tobacco mosaic virus to be lower by 20 per cent than that required by the tetranucleotide hypothesis. DNA does not present this difficulty, as its purine-pyrimidine ratio has been shown to be relatively constant (13, 17).

In the present investigations, differences between calculated and found phosphorus values or calculated and found ultraviolet absorption values for the several materials analyzed could indicate differences in RNA com- position as compared to the standard. The case of P. ammoniae, studied in a similar manner at two stages of growth (17 and 48 hours), offers a more conclusive illustration of this point; for both phosphorus and ultraviolet calculated values were significantly higher than found values, regardless of which RNA results were taken in the calculations. It is of interest that less variation between calculated and found values was observed in the 48 hour culture of this organism than in the 17 hour culture. Since by both the orcinol and PBPH methods, the amount of RNA in the 17 hour culture was about twice that in the 48 hour culture, whereas the DNA content was approximately the same in both, one implication may be that, with increasing amounts of RNA, differences in RNA composition as compared to the yeast RNA standard become more apparent.

Besides compositional and structural differences from one nucleic acid to another, there may be present interfering substances which give a posi- tive test with both the orcinol and PBPH reagents. Thus, it is well to check nucleic acid results found by calorimetric sugar methods against to-

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648 RNA IN TISSUES AND MICROORGANISMS

tal phosphorus and ultraviolet measurements. However, possible struc- tural differences in the polynucleotide group, as suggested by Gulland (18), or small amounts of non-nucleic acid phosphorus could cause erroneous con- clusions from phosphorus determinations. Likewise, besides variations in the proportion of different bases, ultraviolet measurements may be affected by the presence of other materials which absorb in the ultraviolet for which no adequate correction can readily be made.

It should be noted that the proposed method, by virtue of the extraction before color formation, can eliminate or minimize effects of interfering sub- stances or non-specific color resulting from organic matter charred by strong acid. For example, in preliminary experiments with formalin-fixed tissue which gave non-specific color with both the orcinol and phloroglucinol methods, the PBPH method yielded results which were suggestive of its usefulness for the analysis of such material.

SUMMARY

A calorimetric procedure has been devised for determining the ribonucleic acid (RNA) content of biological material. Ribose, from hydrolyzed RNA, when heated in a 4 N HCl medium saturated with NaCl, yields furfural which is extractable with xylene. The xylene extract reacts with p-bromophenylhydrazine (PBPH) in HCl-ethanol solution to give a yellow color (maximal absorption at 450 mp) which, under standardized condi- tions, is a measure of the amount of RNA present. The developed color followed Beer’s law over the range of 9.4 y to more than 150 y of purified RNA. The PBPH method was found to be more specific than the orcinol procedure, the one most generally used for RNA assay, since deoxyribo- nucleic acid (DNA) did not interfere with the test and the xylene extrac- tion minimized the effect of interfering substances. The results were reproducible and good recoveries were obtained.

RNA values for several tissues and microorganisms were determined by the proposed method and the orcinol procedure, and thoeretical phosphorus content and ultraviolet absorption were calculated from each result. Total phosphorus content, calculated by adding RNA phosphorus values to the phosphorus content calculated from deoxyribonucleic acid assays on the same materials, was compared with total phosphorus values found by actual assay. Calculated ultraviolet absorption values were treated likewise. In general, the calculated phosphorus and ultraviolet values based on PBPH assays for RNA agreed more closely with experimental findings than did those based on the orcinol assays. Furthermore, such comparisons indicated that the proposed method may be applicable to more diverse materials.

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J. M. WEBB 649

The author wishes to thank Dr. Hilton B. Levy, National Microbiologi- cal Institute, for furnishing cultures of bacteria used in this work and for his advice during the course of this investigation. The author is also in- debted to Miss Gladys Kauffmann, National Microbiological Institute, for various suggestions in preparing this manuscript.

BIBLIOGRAPHY

1. Webb, J. M., and Levy, H. B., J. Biol. Chem., 213, 107 (1955). 2. Mejbaum, W., 2. physiol. Chem., 268, 117 (1939). 3. Schneider, W. C., I. Biol. Chem., 161, 293 (1945). 4. von Euler, H., and Hahn, L., Svensk Kern. Tidskr., 68, 251 (1946). 5. Davidson, J. N., and Waymouth, C., Biochem. J., 38, 39 (1944). 6. Reeves, J. E., and Munro, J., Ind. and Eng. Chem., Anal. Ed., 12,551 (1940). 7. Xunitz, M., J. Gen. Physiol., 24,31 (1940). 8. Foster, R. L., Ioowa State Coil. J. SC., 8, 191 (1933). 9. Pigman, W. W., and Goepp, R. M., Jr., Chemistry of the carbohydrates, New

York, 639-647 (1948). 10. Hammarsten, E., Biochem. Z., 144, 383 (1924). 11. Caspersson, T., Skand. Arch. Physiol., 73, suppl. 8 (1936). 12. Levy, H. B., Hale, M. G., Wallerstein, J. S., and Schade, A. L., Wallerstein Lab.

Communicut., 14, 43 (1951). 13. Chargaff, E., .I. Cell. and Comp. Physiol., 38, suppl. 1,41 (1951). 14. Chargaff, E., Magasanik, B., Vischer, E., Green, C., Doniger, R., and Elson, D.,

J. Biol. Chem., 186, 51 (1950). 15. Vischer, E., and Chargaff, E., J. Biol. Chem., 176, 715 (1948). 16. Loring, H. S., J. BioZ. Chem., 130, 251 (1939). 17. Chargaff, E., Ezperientia, 6, 201 (1950). 18. Gulland, J. M., Symposia Sot. Exp. Biol., 1, 8 (1947).

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Junius M. WebbMICROORGANISMS

ACID IN TISSUES ANDDETERMINATION OF RIBONUCLEIC

A SENSITIVE METHOD FOR THE

1956, 221:635-649.J. Biol. Chem. 

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