ANALYTICAL CHEMISTRY
1914 transfer the residue from the crucible into a 250-m1. beaker containing the melt. Dilute to approximately 80 ml. with water and heat the solution until the salt has decomposed. Chill the solution to 5 ” C. for 1 hour and filter through a sintered porcelain crucible. Wash the insoluble hydroxides with several 10-nil. portions of ice water. Pass the filtrate through 5 grams of the perchlorate form of Amberlite IRA400 in an ion exchange column, a t a r:,te of 10 to 20 ml per minute. Rinse the column with 100 ml. of water a t a rate of 1 to 2 ml. per minute. Remove the adsorbed molybdenum with 300 nil. of 1X potassium oxalate solution a t a rate of 2 ml. per minute. Rinse the column with 50 ml. of water to remove any excess oxalate ion. Pass 200 ml. of 1dP perchloric acid through the column collecting the effluent in a 250-ml. volumetric flask. Dilute the effluent to exactly 250 ml., take J iuitahle aliquot and determine the
rhenium concentration, using the method employing a-furildioxime (4,5 ) . LITERATURE CITED
(1) Atteberry, 11. W., and Boyd, G. E.. J . A m . Chem. Soc.. 72, 4805 (1950). (2) Ephraim, Fritz, “Inorganic Chemistry,” pp. 496-510, Yew York, h’ordeman Publishing Co.. Inc., 1943. (3) Fisher, S. A , , Ph.D. thesis, University of Wisconsin, 1949. (4) Fisher, S. b.,and AIeloche, V. W., AS.AL.CHEX, 24, 1100 (1952) ( 5 ) Larson, W.J., Ph.D. thesis, University of Wisconsin, 1950. (6) Xlalouf, E. E., and White, A I . G., ANAL.CHEM.,23, 497 (1951). (7) Tompkins, E. R.. and lIayer, S. W.,J . Am. Chem. Soc., 69, 2889 (1947). R E C E I V E for D review August 20. 1954. Accepted Septeiiibcr 20, 1054,
Reaction of Deoxy Sugars with Anthrone LEONORE HOLLANDER KOEHLERI The Institute for Cancer Research and The Lankenao Hospital Research Insfifute, Fox Chase, Philadelphia
In this work, 2-deoxj -D-glucose, %deoxy-D-ribose, and 2-deoxy-D-xylose were observed to produce red colors with anthronesulfuric acid, in contrast to the bluegreen colors with absorption maxima near 620 mM shown by fully hydroxylated sugars. The deoxypentoses showed greatest absorbance at 550 and 560 mfi, deoxyglucose at 520 n a p . By use of different heating times and concentrations, characteristic reaction rates and color intensities were obtained and studied to explore the possibility of quantitative application. After 10 minutes of heating with anthrone in a boiling water bath, deoxyribose gate 45% and deoxjxylose 36% of the color gilen by deoxyglucose at 540 mp. Quantitative use might be feasible with controlled heating time and appropriate standards. A study of mixtures showed si:nple addithe effects at most wave lengths in optimum concentration range, making possible the detection of the two tjpes of sugars or their derivatives (nucleic acids) in the presence of each other.
THE
importance of deoxyribose in nucleic a -id chemistry prompted a detailed inquiry a s to the value of the anthrone reaction for the detection and estimation of deoxy s u q r s . I n a previous investigation ( 4 ), the color produced by deoxypentoee nucleic acid was observed to be red; a negative reaction had been reported by Sattler and Zerban (6) for 2-deoxyribose When synthetic 2-deoxy-~-ribose, 2-deoxy-D-xylose, and 2-deoxv-~glucose became available, through the courtesy of Francis B. Cramer ( I ) , a comparative study was made of the absorption spectra, rates of development] and intensities of their anthrone colors, The absorption spectra of the fully hydroxylated analogs were also observed in order to evaluate the behavior of mixtures such as might be found in nature in nucleic acid fractions. The common hexoses and pentoses are known to react with anthrone a t different rates and with different color intensities (4), and all show absorption maxima near 620 mp ( 7 , 8). Hydrowymethylfurfural and furfural have been reported to give similar colors ( 5 ) . Other organic compounds have been noted to produce a red, violet, o r brownish color (6, 7, 8); in the course of this investigation, tryptophan wis found to give a faint pink color, but tyrosine gave 110 color. In view of the recent work of Graff, McElroy, and IIonnev ( g \ and of Holechek and Collins (3) on 1
Present address, St Luke’s Hospital, Bethlehem, Pa.
7 1, Pa.
differential anthrone colors of steroids, it is likely that the reaction may come to be applicd in ne\y ways. METHOD
The materials and technique6 here employed have been described (4);absorption spectra TTere measured by means of a Coleman universal spectrophotometer. Ribose and xylose give anthrone colors which fade, thus necessitating rapid readings. Comparisons between s:nyle sugars and mixtures were made on tubes treated and read concur1ently. Satisfactory readings were obtained when 100 y of deosyglucose or 200 y of deoxypentose were used in a 5-ml. reference sample; it was found necessary to hold to the optimum concentration range for accuracy, not exceeding an absorbance of 0.500. I n the study of mixtures, the sugar solutions were combined before the reaction with anthrone. RESULTS A N D DISCUSSION
The findings illustrated in Figures I and 2 are comparable with those in the author’s previou? work (4). Klett colorimeter readings are plotted against heating time in Figure 1, and against concentration in Figure 2, using filter No. 54 (540 mp), Deoxyribose reacted rapidly to give piactically maximum color in 3 minutes, a t a color density (anthrone factor) which might be expressed as 0.45, to parallel that of ribose of 0.42 a t 620 mp. After 10 minutes of heating, the color density produced by deoxyglucose was 0.95 on the same scale, compared with 1.55 for glucose However, no “plateau” or maximum was observed;
-150
)D
0
DEOXYGLUCOSE
DEOXYRIBOSE
2
Figure 1.
5
e IO HEATING T I M E , MIN
15
20
2
Reaction Rates of Some Deoxy Sugars K l e t t filter No. 54
1915
V O L U M E 2 6 , NO. 1 2 , D E C E M B E R 1 9 5 4
DEOXYGLUCOSE
.+
.i
I3O0
-0-
HE ATE D 3 O M I N I' 10 "
.L'
,;.
DE OX Y X Y LOS E
E 0 J
--.--
r'+ ,
CONCENTRATICU,
Figure 2.
I
PER 5 ML ALIQUOT
Concentration Curves Klett filter No. 54
nature of the major component. The absorption curves show that the deoxypentoses have maxima a t 550 mp, and deoxyglucose has a masimum a t 520 mp, in contrast to maxima a t 615 mp for the pentoses and a t 625 mp for glucose. The Klett filter No. 54 is intermediate between the absorption maxima, for deoxyglucose and the deoxypentoses. The results observed on heating mivtures (Jf ribose and deoxyribose, in different proportions, with arithroiie-sulfuric acid, are shown in Figure 3 and in Figure 4. The upper, brohen lines, which represent a t each wave length the sum of the corresponding separate values for the two sugars, approximate tLe position of the points indicating found values foi mixtures. The agreement is close despite the difficulty of timing and reading the ribose color accurately a t 2 minutes; this coloi fadrs a t lesrst 10% I er hour a t room temperature, Each component appeared to develop its characteristic color independently of the other, and could thus be detected in the composite absorption spectrum. After 15 minutes of heating, the green color due to ribose had faded to a nonspecific brown, adding its expected increment to the curve i n the case of the miyture.
IOOE RI BO SE
the value continuously rope to 1.86 in 40 minutes of heating. Deoxyxylose, not shown in Figure 1, maintained a pyactically constant level a t 0.35 after 5 minutes of heating. A somewhat higher maximum, sometimes observed between 1 and 2 minutes' heating, was not reproducible under the conditions used. These characteristics of the reaction rates of deoxy sugars are not favorable to quantitative application; nevertheless, a s seen i n Figure 2, approximately linear concentration curves are produced when heating time is rigorously controlled. Satisfactory results could be expected if standards were always run simultaneously with unknowns. The sharp difl'erence in color between the deosy sugars and the fully hydroxylated ones, when treated with anthrone, is illustrated in Figures 3 to 8. Qualitatively, it provides a distinction which should be very useful, as the test is rapid, simple, and sensitive. To the eye, the reaction mixtures were observed t o be clear red or green, respectively. Mixtures of the two types appeared gray, with red or green predominating according to the
50-50, CALC
x FOUND
,":*
t
CALC
----
a 0 K
m 4
550
500 WAVE LENGTH
600
,mr
Figure 4. Absorption Spectra of Ribose, Deoxyribose, and RIixture at 15 Minutes of Heating
IO08 X Y L O S E
+
I
10011 D E O X Y X Y L O S E
2 M I N HEATING t
o
I
500
550 WAVE L E N G T H
I
I
600
650
I
Figure 5 . Absorption Spectra of Xylose, Deoxyxylose, and Mixture at 2 iMinutes of Heating
I
500
550 600 W A V E LENGTH.^^
650
Figure 3. Absorption Spectra of Ribose, Deoxyribose, and Mixtures at 2 Minutes of Heating
Similar results were obtained with xj1oi.e and deoxyxylose, aa illustrated in Figure 5. The xylose color after 2 minutes of heating is much more intense than that produced by deoxyxylose. After 10 minutes of heating, the relationship is reversed (Figure 6). These effects are reflected in the shapes of the curves obtained from mixtures, although the observed values aere loner than those calculated in the 10-minute series. Glucose and deoxyglucose, studied in the same wa) after being heated in anthrone-sulfuric arid for 10 and for 40 minutes, yielded the results s h o w in Figures 7 and 8 Here again, the green color of the fully hydroxylated sugar v-ab obliterated in the longer heating time, while the red color of deouyglucoee was greatly
ANALYTICAL CHEMISTRY
1916 Table I.
1.600 -BOO
Anthrone Factors of Deoxy Sugars Timeo a t 100 , blin. 10 2
Substance 2-Deoxy-D-glucose 2-Deoxy-D-ribose
Anthrone Factor, No. 54a Klett Filter 0.95 0.40 0.43 0.54 0.13 0.35,0.23 0.35,0.44
10
i5 10 1.5 10
2-Deorypentose niicleic acid 2-Deoxy-~-xyloee
IO08 GLUCOSE
,,./"-',,, ,!"-,'
j' x
x\,i, x\,i.
XFOUND
,.' x \:
x
-~
\
-.400
14 0.00
