Colorimetric Determination of Ribose, Deoxyribose, and Nucleic Acid with Anthrone N. D. G A R Y
and
R. E. KLAUSMEIER
Biological Laboratories, Chemical Corps, Camp Detrick, Frederick,
Md.
deoxyribonucleate, and 400 y of sodium ribonucleate. The similarity between the respective pentose and nucleic acid curves indicates that the colors of the nucleic acid reactions are due to the pentose moiety. The relatively short heating time, 2.5 minutes, used for ribose and ribonucleic acid mas found to be optimum for maximal color development and was rigidly adhered to. The color was stable for 15 minutes after cooling to room temperature mith 96 to 99% of the ribose color (from 40 to 180 y ) and 94 to 98y0of the ribonucleic acid color (from 200 to 700 y ) remaining after 30 minutes. Hoii ever, additional heating a t 100" C. resulted in a rapid decrease in color (blue to light amber) a s indicated by the flattening of the ribose and ribonucleic acid curves in Figure 1. A heating time longer than 2.5 minutes n a s required for maximal color development IT ith deoxyribose and deoxyribonucleic acid. These periods of heating are essentially in agreement with those reported by Koehler ( 4 ) : 1.5 to 2.0 minutes for ribose and ribonucleic acid, and 6 to 10 minutes for deoxyribonucleic acid. Ten minutes vere selected not only for maximal color but also because the absorbances of ribose and ribonucleic acid reactions have decreased to a minimum a t this time. This latter factor affords an obvious advantage when assaying mixtures a s described below. The color of anthronated deoxyribose and deoxyribonucleic acid n as stable a t room temperature for several hours. The data presented in Figures 2 and 3 represent the linear relationship that exists betmeen ahsoibance and concentration of the anthronated compoundP. This relationship holds true onlj to the upper concentration reported for each compound except ribonucleic acid which was not assayed above 500 y . From the data reported in Figures 2 and 3 it is noted that the absorbance values
The anthrone reaction, under rigorously controlled conditions, can be used for quantitative determinationof D-ribose, D-2-deoxyribose,and their nucleic acids.
T
HE use of the anthrone reagent for the determination of hexoses and hexose polymers has attained widespread application. Its use for the assay of pentoses has been someTvhat limited, probably because of the transient color formed during the reaction and the lower degree of sensitivity (1, 2, 4 ) . The data presented below indicate that the anthrone reaction, under rigorously controlled conditions, can be used for the quantitative determination of D-ribose, ~-2-deoxyribose, and their respective nucleic acids. REAGENTS
The D-ribose, sodium nucleate, and deoxyribonucleate, were obtained from the Sutritional Biochemicals Corp. The anthrone reagent contained 0.2% of anthrone (melting point 154' C.)) prepared according to Vogel (j), in 95% reagent grade sulfuric acid. PROCEDURE
After varying the reaction time and temperature in a n attempt t o stabilize the anthronated ribose and ribonucleic acid color, a procedure which was found to be applicable for the assay of both pentoses and nucleic acids was adopted as follows. The samples are transferred t o 18-mm. colorimeter tubes, adjusted to a 2-ml. volume with distilled water, and cooled in an ice bath. With the tubes held in an inclined position, 4 ml. of cold anthrone reagent are permitted to drain into the tubes, forming a layer below the aqueous sample. The tubes are returned to the ice bath until all additions of the reagent have been completed. The contents of all tubes are then thoroughly mixed by shaking while in the ice bath, and the tubes are covered with marbles and transferred to a boiling water bath to develop the color. Tubes containing ribose or ribonucleic acid are heated for 2.5 minutes and those containing deoxyribose or deoxyribonucleic acid are heated for 10 minutes. After the appropriate heating period the tubes are promptly cooled to room temperature with the aid of an ice bath and the absorption is determined immediately a t the proper wave length in a colorimeter or spectrophotometer with a reagent blank (2 ml. of water and 4 ml. of anthrone reagent treated as above), set a t 100% transmittance. A Coleman spectrophotometer, Model 11, was used for these determinations. Absorption spectra were determined with 3ml. aliquots of the reaction mixtures in 1-em. cuvettes in a Beckman DU spectrophotometer.
I. D-2-deoxyribose, IO min 2. DNA,IOmin. 3. D-ribose, 2.5 min. 4. RNA, 2.5min. D-ribose, I O m i n . ,RNA. io min.
RESULTS A N D DISCUSSIOR
From the absorption spectra presented in Figure 1, a maximum a t 620 to 625 mp (blue color) is noted for ribose and ribonucleic acid, and maxima at 565 and 530 mp (red color) for deoxyribose and deoxyribonucleic acid. Koehler ( 3 ) reported onlj maxima a t 550 to 560 mp for both 2-deoxyribose and 2-deoxyxylose in contrast to a maximum at 520 mp for 2-deoxyglucose, thus suggesting the possible contamination with deoxyglucose of the samples of deoxyribose and deoxyribonucleic acid used in this investigation. Hox-ever, the close similarity of the absorption spectra and the 530/565 mp absorbance ratios for both the synthetic (deoxyribose) and biological (deoxyribonucleic acid) compounds make this unlikely. The absorption curves shown in Figure 1 were obtained ~ i t h the following amounts of test material in a final reaction volume of 6 ml.: 150 y of ribose, 150 y of deoxyribose, 300 y of sodium
Figure 1. Absorption Spectra of Anthronated %ribose, D-2-Deoxyribose, Ribonucleic Acid, and Deoxyribonucleic Acid
1958
V O L U M E 2 6 , NO. 1 2 , D E C E M B E R 1 9 5 4
1959
same (620 to 625 mp), and the sensitivity is g r e a t e r f o r hexoses. Absorbance T o determine whether one 620 mp, 2 5 Minutes 565 mp, 10.0 Minutes nucleic acid can be determined Rcaled. *D in the presence of the other, Assay R + D Ddetd. Rcalcd. Rdetd. Rdrtd. R -t D Rdetd. D e a l c d . Ddetd. Ddetd solutions containing mixtures Pentose 150 y R 0.367 0.109 0.256 0 . 2 6 2 0.977 0.678 0.071 0.807 0 . 6 2 9 0.965 of ribonucleic (RKA) and de200 y D oxyribonucleic ( D S A ) acids Kucleate 250 y RNA 0,131 0.040 0,091 0.093 0.978 0.282 0.041 0 . 2 4 1 0.244 0,988 (concentrations unknoxn to 230 y DNA investigator performing assay) a Measured with Beckman DU spectrophotometer. R = ribose, RKA. D = deoxyribose, DNA. were prepared and assayed. R + D = anthrone reaction of mixed compounds. Rdetd., D d e t d . = anthrone reaction determined with individual compounds. Solutions containing several Rcslcd , D c s l c d . = calculated: mixed minus individual reactions k n o n n q u a n t i t i e s of each nucleic acid were also assayed. The quantities present in the unkn0m-n mixture were calculated as follows: The absorbance of the 10-minute reaction a t 565 mp wave length was assumed to n' be due t o deoxyribonucleic acid only, as represented in Equation l : 0.5 DNA = D565 (1)
Table I. Determination of D-Ribose and D-2-Deoxyribose, or Ribonucleic and Ueoxyribonucleic Acids in the Presence of One Anothera
-
The absorbance of the 2.5-minute reaction a t 620 mp wave length is the sum of the reactions of ribonucleic and deoxyribonucleic acids. By subtracting the absorbance due to deoxyribonucleic acid, ribonucleic acid could then be determined as in Equation 2: RNA = ( R D)620 DeZo (2)
0.4
w
0
z a
+
-
0.3
0 v)
m U
0.2
0.5
0.1
0.4 W
u z
a
50
100 150 CONCENTRATION, p g
200
Figure 2. Relationship between Absorbance of Anthrone Reaction and Concentration of D-Ribose at 620 mp and D-2-Deoxyribose at 565 mp
for ribose and ribonucleic acid average 43.8 and 41.0%, respectively, of the absorbance values for equal weights of deoxyribose and deoxyribonucleic acid. On a molar basis the absorbance of the ribose reaction is approximately 50% of that observed for deoxyribose. The possible interference of ribo- and deoxyribo- compounds during the reaction with anthrone was examined. Johanson ( 2 ) reported that the presence of pentoses interfered with the determination of hexoses by causing a shift in the absorption maximum from 625 to 680 mp and increasing the extinction beyond the additive absorbance levels of the two compounds when anthronated separately. From the data presented in Figure 4 (using 200 y of deoxyribose and 150 y of ribose) it is evident that no shift in the absorption spectra occurs when ribose and deoxyribose are anthronated together. It is apparent by comparison of curves 3 and 4 of Figure 4 and from the results recorded in Table I that the absorbance values for mixtures a t 565 and 620 mp are essentially additive. The values calculated from the mixtures are equal to 96 to 99% of the values obtained from the assay of the individual compound. These findings suggest that the pentoses or nucleic acids can be determined in the presence of one another. The presence of hexoses would probably interfere with the assay of ribose or ribonucleic acid since the absorption maximum is the
m a 0.3 0 v)
m
a
0.2
0.I
100 200 300 400 CONCENTRATION,pg sodium salt
Figure 3. Relationship between Absorbance of Anthrone Reaction and Concentration of Ribonucleic Acid at 620 mp and DeoxyribonucleicAcid at 565 mp
The amount of deoxyribonucleic acid color present in the mixture a t 620 mp can be calculated by multiplying the value, measured a t 565 mp, by a conversion factor, K (Equation 3 ) : D620 = KD585 (3 ) where K is equal to the ratio of the absorbance values obtained with standard solutions of deoxyribonucleic acid a t 620 and 565 mp, after reaction periods of 2.5 and of 10 minutes, respectively (Equation 4):
K = -D N A620 D N A666
(4)
ANALYTICAL CHEMISTRY
1960 By sulmtituting the values for h-and De% in Equations 2 and 3, ribonucleic acid in the mixture can be calculated. The results of the experiment are recorded in Table 11. For equivalent levels of ribonucleic and deoxyribonucleic acids the recoveries were fair. Hon ever, the recoveries of ribonucleic acid (102%) in the presence of lower quantities of deoxyribonucleic acid and for deoxyribonucleic acid (95.601,) in the presence of lower quantities of ribonucleic acid are well within experimental limits. The situation of one nucleic acid's occurring in greater concentration than the other is most likely to be encountered in saniples separated and purified from biological materials.
3
I D-2-deoxyribose
z
D-ribose
I
2
08
07
06
0 5 W
Table 11. *
Recovery of Ribonucleic Acid and Deou)-ribonucleic Acid from Mixtures" Known, yb Determined, y h Recovery, Yo
Sample
RNA
DNA
RNA
1
250.0 400.0 100.0
250.0 100.0 400.0
279.5 406.5 128 8
2 3 a
b
u z a
DN-4 286.0 165.8 382 2
RNA
DSA
112.0 102.0 129.0
114.0 166.0 95,6
m [L
0
04
cn m
a
03
lleasured with Coleman Model 11 spectrophotometer. Concentrations expressed as sodium salts.
0 2
01
CONCLUSIONS
The determination of pentose and pentose nucleic acid by the procedure described presents certain advantages and disadvantagre. .4e a qualitative test the compounds can be readily differentiated by the differences in reaction color and stability. Quantitatively the procedure is rapid and relatively simple, with one procedure serving for the determination of both types of compounds. An obvious disadvantage ie the expected interference by hexoses which must therefore be eliminated from the assay mixture. Assay of mixtureE of nucleic acids (ribo- and deoxyribonucleic acid) indicate that one type can be determined in the presence of small quantities of the other. ACK3OWLEDGMENT
The fiample of D-2-deoxyribose u as kindly furnished tiy John C. Sowden, Washington University 7 , St. Louis, 310.
2
550 600 WAVELENGTH, rnp
650
Figure 4. Absorption Spectra of DRibose and D-2-Deoxl-ribose Anthronated (2.5 Minutes), Separately and Together LITERATURE CITED
(1) Bridges, R. R.. ~ A L CHEM., . 24, 2004 (1952). (2) Johanson, R., Vature, 171, 176 (1953). (3) Koehler, L.H., .kbstracts, 124th Xketing Of -4YERICAN CHEMICIL
SOCIETY, p. 14D, 1953. (4) Koehler, L. H., ANAL.CHEM..24, 1.576 (1952). (5) Vogel, A. I., "A Text-Book of Practical Organic Cheinistry," p. 704, London, Longmans, Green and Co., 1948. R E C E I V Efor D review April 20, 1954. Acceiited August 1 1 , 1954.
Determination of Methylhydrazine HERBERT MCKENNIS, JR., and ALLAN S. YARD Department o f Pharmacology, M e d i c a l College o f Virginia, Richmond, V a .
The reaction of methylhydrazine with p-diniethylaminobenzaldehyde in aqueous sulfuric acid has been studied. Solutions of these reagents are highly colored. The similarity between the absorption spectra of these solutions and those obtained from hydrazine suggests the presence of chromophores of analogous structure. Colorimetric determination of methylhydrazine is subject to interference by amines known to interfere with hj-drazine as well as, by hydrazine itself. The oxidation of methylhydrazine by acidic iodate solution follows the stoichiometry of hydrazine oxidation. This similarity points to methanol as an end product in the oxidation of methylhydrazine.
T
HE analytical chemistry of hydrazine and its derivatives has in recent years, with the advent of economical production and multiplicity of uses for hydrazine (4, 5, I d ) , been subjected to a number of investigations ( I O , I S , 16). There are a number of
reports dealing with the analysis of hydrazine derivatives. The recent employment of acyl derivatives of hydrazine in the chemotherapy of tuberculosis (4,5, 11) and the agricultural use of maleic hydrazide ( 1 6 ) have increased interest in the determination and detection of small amounts of hydrazine derivativeb. For the most part (6 1 6 ) the analytical methods are dependent upon the regeneration of hydrazine by hydrolysis and colorimetric determination of the freed hydrazine. During an investigation of the biological activity of hydrazine derivatives in this laboratory (14), need arose for determination of methylhydrazine concentrations. AIethods for the determination of methylhydrazine do not appear in the literature. For determination of small amounts of methylhydrazine the colorimetric method of Pesez and Petit (10) for hydrazine was selected for initial consideration. Detailed procedures for hydrazine determination which employ the reaction of p-dimethylaminobenaaldehpde in acidic solution with hydrazine ( 1 0 ) have been given by Watt and Chrisp (13)and more recently by Wood (16). Pesez and Petit (IO) considered that the acid salt of p -
