Differentiation of Carbohydrates by Anthrone React ion Rate and Color Intensity LEONORE HOLLANDER KOEHLER Institute f o r Cancer Research and Lankenau Hospital Research Institute, Philadelphia 11, Pa. The aim of this investigation was to adapt the anthrone-sulfuric acid carbohydrate color reaction to the study of native polysaccharide complexes. The behavior of known carbohydrates was first established as the basis of comparison. Known sugar types showed distinctive reaction rates and intensities; aldohexoses gave readings of 110 to 165 Klett units per 100 micrograms at their characteristic maximum of 5 to 10 minutes of heating at 98’; lietohexoses, 220 to 270 units per 100 micrograms at their maximum, 1 to 2 minutes at 98”. Pentoses produced color readings of 40 to 70 units
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OR the qualitative and quantitative determination of polysaccharides, Dreywood’s anthrone color test ( 3 ) has the advantages of sensitivity and simplicity; it does not require previous hydrolysis. First adapted for quantitative use by Morris ( l a ) ,it has been applied for the determination of mannosidostreptomycin by Koxald and MacCormack (IO); of blood glucose by Durham, Bloom, Lewis, and hfandel ( 4 ) ; and of liver glycogen by Seifter, Dayton, Novic, and Muntwyler (16). Among other applications have been assays of blood and urine amylase described by Kibrick, Rogers, and Skupp (8), and dextran, by Bloom and Wilcox ( 2 ) . In the present work, a comparison of several well-known carbohydrates made it possible to develop standards for applying the anthrone method to natural polysaccharide mixtures. MATERIALS
Anthrone reagent \\-as prepared in the manner employed by the authors above cited, and recommended in the Eastman Organic Chemical Bulletin ( 5 ) , from reagent grade sulfuric acid, and anthrone made as described in “Organic Syntheses” (11). Although the required amount of reagent for each day was usually made fresh, it was found that the same reagent could be used for as long as a week, if it was stored in the refrigerator and blanks were run daily. Polysaccharides from Serratia marcescens \?-ere prepared by following the general procedure of Perrault and Shear (13). The dextrin was that described in a previous work of the author (9). The amylose was A-fraction potato starch, furnished by T. J. Schoch, Corn Products Refining Go. This company also furnished n-glucurono--~-lactone. D-Fructose, inulin, and octaacetjl sucrose were prepared a t the Eastern Regional Research Laboratories and furnished by Elias Yanovsky. Guaran, xylan, and acetyl xylan were obtained from R. L. Whistler, Purdue University, and the preparations of n-sorbose, D-arabinose, and n-xylose were obtained from the National Institutes of Health. The other materials were of commercial origin: D-glucose, Merck reagent. Glycogen, Pfanstiehl, = a t 200”. D-Mannose and glucosamine (2-amino-2-desoxy-n-g~ucose), Eastman Kodak Co. Sucrose and o-galactose, Difco. D-Ribose, Schwarz Laboratories. Sucleic acid (ribose nucleic acic), Nutritional Biochemical Laboratories. Desoxyribonucleic acid (desoxypentose nucleic acid), Krishell Laboratories. Analytical samples of carbohydrates were prepared as follows: For soluble sugars, solutions were made up to contain 200 micrograms of hexose or 400 micrograms of pentose per ml., in saturated benzoic acid solution. Of the former, 0.25 ml., containing 50 micrograms, and of the latter, 0.3 ml., containing 120 microg r a m , were used in each 2.5-mI. aliquot. Glycogen and inulin were dissolved in water by heating; the dextrin re uired 1 minute of boiling. Amylose was dissolved by gradual ajdition of the dry powder to rapidly stirred boiling
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per 100 micrograms at a maximum of 1 to 2 minutes at 98’. Polymers reacted like the constituent sugars; derivatives developed the decreased amount of color expected from the content of inert component. Bacterial polysaccharides often could b e differentiated and partially characterized. This procedure can be used to indicate the probable identity or nonidentity of various polysaccharjdes, or, to estimate the percentage of the carbohydrate portion of a mixture or complex using known standards. In some cases evidence can be deduced about the nature of the carbohydrate components of a complex.
water, and 5 minutes of boiling. Xylan, after 1minute of boiling in relatively concentrated solution, was filtered through glass
wool to avoid introducing paper fibers, and diluted so that aliquots containing 100 micrograms could be conveniently measured. Guaran, homogenized with water in a Waring Blendor, was heated for 1 hour on a steam bath, filtered, and suitably diluted. All the polymers except xylan were used in the same concentration as the hexoses. Recrystallized sucrose octaacetate R as dissolved in ethyl alcohol and diluted to volume with an approximately equal volume of water and used in aliquots containing 55 micrograms each. Acetyl xylan was dried a t 100” C. after reprecipitation with alcohol from a glacial acetic acid solution filtered through sintered glass. The purified product, dissolved in concentrated acetic acid, was used in 120-microgram aliquots. Ribose nucleic acid and desoxypentose nucleic acid, dissolved in very dilute sodium hydroxide, were used in aliquots of 815 and 555 micrograms, respectively, per 2.5-m1. sample. (The product commercially known as “nucleic acid,” being prepared from yeast, is known to be largely ribose nucleic acid, That known as “desoxyribonucleic acid” should more accurately be called desoxypentose nucleic acid, as its carbohydrate moiety has not been adequately identified.) The samples of lyophilized Serratia marcescens polysaccharide were suspended in water, the lumps Rere carefully dispersed, and the mixture was shaken on a mechanical vibrator until no more particles could be seen. For each determination, 200 to 400 micrograms were used. PROCEDURE
To each 2.5-mI. test sample in a borosilicate glass 25-mm. bore test tube, 5 ml. of a 0.2y0 solution of anthrone in 95% sulfuric acid were added from a buret free of stopcock grease, During the addition of reagent, the tube was immersed in cold, but not iced, water and its contents were shaken vigorously. It was then closed with a glass bottle stopper and heated in a boiling water bath for an accurately measured time interval. Bfter cooling in a cold, but not iced, water bath, the contents were found to have a constant color intensity for a t least 4 hours at room temperature. For time curve analyses, identical reaction mixtures were heated for various lengths of time. The blank consisted of 5 ml. of reagent and 2.5 ml. of water. A Klett-Summerson photoelectric colorimeter was used for measuring the color intensities, the reaction mixture having been transferred to standard uncalibrated Klett tubes. I n the optimum range of accuracy, giving net readings between 100 and 250 Klett scale units, the color was a clear grass green. Red filter KO.62 was employed for all readings except on desoxypentose nucleic acid. RESULTS AND DISCUSSION
The anthrone reaction rate could be followed by plotting the colorimeter readings, in Klett scale units per 100 micrograms of
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1577 there must be the possibility of dehydration in the 2-3 position, resulting in a furfural derivative. Segative anthrone reactions, other than those listed by Sattler and Zerban, were given in this study by glucosamine and glucuronolactone. A negative reaction with sorbitol showed that the carbonyl group is also necessary. The difference in position of the carbonyl group is responsible for the striking divergence between glucose and fructose. The differencesin behavior of the related sugars, arabinose on the one hand, and glucose and mannose on the other, illustrate the effect of chain length. Possibly the ease of formation of a furfural derivative is the regulating factor in the rate of development of color, and the nature of that derivative determines relative color intensity.
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Figure 1.
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..inthrone Reaction Rate Curves, Aldohexose Type
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RIBOSE RIBOSE NUCLEIC ACID DESOXYPENTOSE NUCLEIC ACID
-. FRUCTOSE ,,...,,.,
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INULIN SORBOSE SUCROSE OCTAACETYLSUCROSE
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Figure 3.
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Figure 2.
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:inthrone Reaction Hate Curves, Ketohexose Type 0
carbohydrate, against heating time in minutes. Distinctive curves were obtained for the different sugars; those for polymers in each case closely resembled the one produced by the corresponding simple sugar. Three classes of curves could be distinguished. The aldohexose type, illustrated by Figure I, \Tas exemplified by glucose, dextrin, glycogen, amylose, galactose, mannose, and guaran. The aldohexoses reacted relatively slowly, giving a maximum color after 5 to 10 minutes' heating and showing no significant fading in 12 minutes. The ketohexose type (Figure 2)-namely, fructose, its polymer inulin, and sorboseproduced a very deep color per unit n-eight after 0.5 to 2 minutes' heating. Although it faded gradually, the color was still slightly more intense after 10 minutes' heating than that of glucose after the same heating time. Finally, as seen in Figures 3 and 4, the pentoses, ribose, arabinose, and xylose, together with xylan, gave the least color per unit weight, reaching their maxima in 1 to 2 minutes and fading very significantly after 4 to 5 minutes' heating. The results show that carbohydrates react with anthronesulfuric acid in accordance with the configuration of the sugar involved. As pointed out by Sattler and Zerban (14, 15),
Figure 4.
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Anthrone Reaction Rate Curves, Pentose Type
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XYLOSE XYLAN ACETYLXYLAN
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Anthrone Reaction Rate Curves, Pentose Type
The work of Morris (12) indicated that a difference exists between sugars in the depth of the color they produce; Seifter, Dayton, Novic, and Muntwyler (16) pointed out the desirability of controlling heating time. Since most investigators utilized heat of mixing to produce color, results are not always comparable-for instance, the finding of Morris ( I @ , that "equal amounts of glucose and fructose give identical color," was duplicated in these experiments only a t the stage where both reaction mixtures had been heated for 12 minutes. I n making comparisons, therefore, i t is necessary to consider not only the observed color but the rate a t which it develops. Studies of this kind, using orcinol, were employed by Albaum and Umbreit (1) on pentose derivatives, and by Kapp (Y), using naphthylresorcinol, on glycuronic acids. I n the present investigation, the possibility is indicated that anthrone reaction rate may sometimes furnish a basis for distinguishing between sugars even in mixtures, derivatives, and polymers.
A K E. L Y T IC A L C H E M IS T R Y
1578 I t was of importance to establish that noncarbohydrate components in admixture or combination have no effect other than t o reduce the amount of color developed per unit total weight. In agreement with Morris (12) and Kowald and MacC‘orniack (IO), i t was found in this work that the diminution in color corresponds quantitatively to the amount of inert constituent. Polymers would be expected t o give about 11% more color than monomers because of their lower water content. In accord with this expectation, xylan was found to give 11% more color than xylose (Figure 4), and glycogen and 18-glucose dextrin, 6% more than glucose (Figure l ) , a t the respective time maxima. Derivatives, as exemplified by octaacetyl sucrose and acetyl xylan (Figures 2 to 4), showed time curves similar to those of the unsubstituted carhohydrates, but \+ith maxima 47 and 30% lower, respectively. This, in turn, could be explained by the fact that the content of acetyl groups in octaacetyl sucrose, calculated on the basis of molecular weights, bhould be 50%; monoacetyl uylari should contain 25% acetyl groups.
Table I.
Tirne, Uin.
Anthrone Color Intensity Readings for GlucoseFructose Mixtures and Sucrose Klett Scale Reading per 100 y Calcd. for 50 y glucose f o u n d . using Calcd. for 50 fructose mixture 100 y siicrose
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Found, 100 Sucrose
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Table 11. Anthrone Color Readings for Xylan and Guaran Tinie. 1Iin.
Klett Scale Readings per 100 y 50 y Xylan 90 y Xylan 10 y Xylan 7 10 y Guaran 90 y Guaran 50 y Guaran Calcd. Found Calcd. Founcl Calcd. Found
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The green anthrone color produced by ribose nucleic acid was only 24% as deep as that of ribose (Figure 3) assuming a nucleotide unit weight of about 300, 2770 might be expected. The time curve followed that of ribose, reaching the maximum in 2 minutes of heating, and falling off again in 5 minutes. I n contrast, the sample of desoxypentosenucleic acid which was studied gave a red color under the same conditions, which was estimated usiiig green filter S o . 54. The reaction rate was slower than that of ribosenucleic acid, and the color maximum was reached in 5 minutes and maintained during a t least 10 minutes (see Figures 3 and 4). The implications of this difference will be made the subject of another investigation. The results of the study of carbohydrate mixtures are summarized in Tables I and 11. Sucrose behaved almost like the corresponding glucose-fructose mixture, in that the color intensities read a t stated times were very close to those calculated b,~, adding the expected values based on data obtained from the separate sugars. For mixtures of the polymers xylan and guaran, the results were compared with calculated values obtained from those observed for the two polymers separately (Tahle 11). Agreement was good a t 2 and 3 minutes of heating, though the observed values were in general lower than those calculated at 1.5 minutes and higher at 4 to 6 minutes. Each component is seen to contribute its approximately proportional share to the resultant total color. The assumption seems valid that, in appi’opriate cases, reaction-rate studies using the anthrone-sulfiiiic* acid reagent may permit the detection of one type of Fugar, evt’ii in compounds or polymers, in the presence of another. ~
Cases in which anthrone time-curve analyses codd he useful are those in whic,h one type of sugar is present and is to lie identified, and those in which information is sought as to the components of a mixture. A4fact permitting such deductions is the finding that aldohexoses developed less than 30% of their niaximum color in 1.5 minutes of heating. An unknonm showing this type of curve would not be expected to contain an appreciable amount of pentose or ketohexose. For example, guaran gave 16.5% of its maximum color in 1.5 minutes, while 90% guaran with 10% sylan gave 32%, and 50% xylan with 50% guaran, 62% of its maximum. Conversely, a substance yielding 75% or more of its maximum color in 1.5 minutes would be expected to consist predominantly of pentose or ketohexose; if aldohexose were present a t all, i t would be to the extent of less than half of a mixture. Another fact helpful in distinguishing sugars is the rapid fading of the color obtained from pentoses. If an experimental mixture had less than 40% of its maximum anthrone color left after 5 minutes of heating, hexoses could be presumed to be practically absent. Thus, as little as 10% guaran in 90% xylan maintained the color reading a t 50% of the maximum after 10 minutes of heating, while for xylan alone only 16% of the color was left after 4 minutes of heating. On the other hand, the deep, rapidly appearing, long sustained color characteristic of ketoses ~ o u l dbe expected to mask all b u t predominant amounts of aldohexoses or pentoses. A numerical value which mag be called “anthrone factor” served to compare the color intensities produced by the various carbohydrates a t their respective time maxima. This conversion factor Klett scale reading A.F. = y of carbohydrate per 5 ml. of solution
represents the slope of the concentration curve of each carbohydrate a t its optimum heating time. Provided the readings were kept in the range 100 to 250 on the (logarithmic) N e t t scale, the color intensity was found to bear a linear relation t o the concentration. The amount of bacterial polysaccharide in unkrio sn solutions could he estimated accurately, if a standard rontaining a known \wiglit of the same type of pol?saccharide \vas used. The anthrone fartors of the carbohydrates here studied, together with the heating times a t which maximal color was observed, are listed in Table 111. Each value r e p resents the average of two or more determinations: differences were within &2% of the mean. I n comparing anthrone factors, a difference greater than 0.05 may be considered significant. Although 2.5 ml. of sample plus 5 ml. of reagent was here found sufficient for t’he determinations, the calculations are based on 5-ml. samples plus 10 ml. of reagent to facilitate comparison with the work of other investigators.
Table 111.
Anthrone Factors of Carbohydrates
Carbohydrate or Derivative D-Glucose Glycogen 18-Glucose dextrin Aniylose D-Galactose D-Mannose Guaran D-Fruc t ose Inulin L-Sorbose Sucrose Octaacetyl ;‘ucro+e D-Arabinose D-Rihosr D-SiIOSe Xylan Acetyl xylan Ribose nucleic acid Desoxspentose nucleic acid Bacterial polysaccharide 31P2 hIP5
Time of .\faximum C o l y , Min. a t 100 8-1 1 8-1 1 8-1 1 8-1 1 6 4 6- Y fi-O
1.5-2 1.5 1.5. 2.13 1
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Anthrone Factor, No. 62 N e t t Filter
(Filter 54)
2.68 2.58 2.00 1.92 1 02 0.48 0.42 0.70 0.79 0.57 0 10 0.13 0 3.1 0 59
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PREPARATION M P 2 PREPARATION (CRUDE) M P 5 HEXANE-TREATED M P 5 REPRECIPITATED M P I , FIRST RUN REPRECIPITATED M P5, SECOND RUN
minor components, as well as noncarbohydrate constituents, undoubtedly vary appreciably in the preparations obtained from different bacteria, different cultures of the same or different bacterial strains, or even different fractions obtained from the same culture. The use of anthrone factors and anthrone reaction rate curves appears useful in detecting variations of thi. kind and aid- in the study of natural polysaccharide mixtures. ACKNOWLEDGMENT
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Figuie 3. .knthrone Reaction Rate Curves, Bacterial Polysaccharides (Serratia marcescens)
To illustrate the application of anthrone time-curve analysis to bacterial polysaccharides, results are shown in Figure 5 for two preparations, MP2 and MP5, of a polysaccharide-lipide complex from Serratza marcescens. These were processed from bacterial paste, kindly supplied by Lferck & Co., Inc., and obtained from r,ultures of different strains of the organism. Trypsin-digested hIP5 gave appreciably less color per unit weight-Le., a lower anthrone factor-than the two runs of the product after reprecipitation by n-propyl alcohol. The latter gave essentially the same reaction curve a8 material treated with hexane by the method of Perrault and Shear (IS). Reprecipitation with n-propyl alcohol thus appears t o eliminate some components inert to the anthrone reagent. Comparison of the curves obtained from the two different preparations MP2 and hIP5, hoxever, reveals a significant difference in the color depth per unit aeight and also in the reaction rate. At a heating time of 1.5 minutes, only 18% of the maximum color was developed by XIP2, as against 50% produced by the 11P5 preparations a t the corresponding time. Besides indicating nonidentity of these two preparations, the anthrone time curve difference shows the probability that MP2 is lacking in the component of MP5, which causes it to react more rapidly with anthrone than aldohexoses do, since the curve for MP2 more closely resembles those of the aldohexose~. Meth~-Ipentoseand glucosamine were demonstrated by Hartwell. Shear, Adams, and Perrault (6) to be present among the hydrolysis products of this polysaccharide along M ith the predominant carbohydrate, an aldohexose shown not to be galactose 01 111:i11no=e3 and "pi.ovisionally regarded a3 glucose." These
The counsel and encouragement of Hugh J. Creech are gratefully acknowledged. The author is indebted to R. L. Whistler of Purdue Cniversity, to Elias Tanovsky of the Eastern Regional Research Laboratories, and to T. J. Schoch of Corn Products Refining c'o., who very kindly furnished materials. LITERATURE CITED
(1) Albaum, H. G., and L-mbreit, IT. W,, J . Biol. Chem., 167, 389 (1947). (2) Bloom, IT.L., and Wilcox, XI. L., Proc. Soc. Erptl. Bid. M e d . , 76,3-4 (1951). (3) Dreywood, R., ISD. Esc. CHEM.,- 1 s ~ED., ~ . 18, 499 (1946). (4) Durham, W.F., Bloom, W ,L., Lewis, G. T., and Mandel, E. E., L-. S . Public Health Service, Pub. Health Repts., 65, 670 (1950). (5) Eastnian Kodak Co., Orguriic Cherii. BUZZ.,23 ( l ) ,6 (1951). (6) Hartwell. J. L., Shear, AI. J., -idanis, J. R., and Perrault, A , , J . T a t l . CanccrInst., 4, 107 (1943). (7) Kapp, E., J . Biol. Chein., 134, 143 (1940). ( ~ 8 Kibrick, ) A. C., Rosers, H. E., aud Skupp. S., Ihid., 190, 10; ( 1951). (9) Koehler, L. H., Proc. Peim. d c n d . Sci.. 23, 196 (1949). (10) Kowald, J. -I., and IIacCormark, R. B., ABAL.CHEM.,21, 1383 (1919). (11) AIeyer, K. H . , "Organic Syntheses," Coll. Vol. 1, p. 52, S e n Tork, John TTiley & Sons, 1932. (12) Morris, D. L., Science, 107, 254-5 (1948). (13) Perrault, -1.. and Shear, M.J., Cancer Research. 7, 714 (1947). (14) Sattler, L., and Zerban, F. W.,J . Am. Chem. Soc., 72, 3815 (1950). (15) Sattler, L., and Zerban, F. W., Scierzcr. 108, 207 (1948). (16) Seifter, S., Dayton, S., S o r i c , B., and Muntwyler, E., r l ~ c h . Biochtm., 25,191 (1950). RECEIVED for review February 7 , 1952. Accepted July 23, 1952. M-ork supported in part hs a grant-in-aid to Hugh J. Creech from the American Cancer Society upon recoinmendation by the Committee on Growth of the Srttional Research Council.
X-Ray Diffract ion Patterns for Some Tet razole Derivatives LOHR A. BURKARDT AND DONALD W. MOORE Chemistry Division, c'. S . .Vat.al Ordnance Test Station, China Lake, Calij. Where x-ray diffraction powder patterns of known materials are available, they offer a means of identifying the components of a crystalline material without separation of such components. This feature of x-ray analysis was utilized to assist another group of investigators in this laboratory engaged in a study of reactions producing tetrazole derivatives. A group of x-ray diffraction patterns of various tetrazole derivatives from know-n materials has been accumulated. These patterns are made available for anal>-ticalpurposes to other investigators of tetrazole derivatives.
T
HE use of x-ray diffraction methods as an aid in the study of
high-nitrogen compounds being carried out by others in this laboratoi y hac resulted in the accumulation of powder data on a number of tetrazole derivatives. X-ray spectrometer patterns are relatively insensitive to impurities and will permit the identification of the results of reactions with a minimum of effort spent on purification of reaction products. Compounds which have poor melting point characteristics or which derompose before melting are readily identified.
These data are presented here in a form similar to that used by Hanawalt (1) for the presentation of x-ray powder data for analytical purposes. It should be borne in mind that these compounds may exist in several crystalline forms and hence different x-ray diffraction patterns would be obtained, depending on the form present. The intensities observed on x-ray spectrometer patterns exhibit marked variations with differences in crystal habit and size. These variations arise from the presence of preferred orientation in the samples. Efforts to avoid this
