QUALITATIVE AND QUANTITATIVE COLORIMETRIC DETERMINATION OF HEPTOSES*

BY ZACHARIAS DISCHE

(From the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York)

(Received for publication, March 26, 1953)

The possibility that heptoses and their esters may be intermediates in various metabolic processes in animal and plant tissues suggested that sensitive color reactions should be developed for their detection and micro- determination. The present report deals with such reactions and their application to quantitative analysis.

EXPERIMENTAL

General Reactions of Heptose

Reactions of Heptose with Orcinol

All heptoses produce with orcinol in dilute HCl two characteristic colored compounds which differ greatly in their absorption spectra. Three differ- ent modifications of this reaction were found useful.

Procedure i--To 2 cc. of a solution containing 20 to 100 y per cc. of ketoheptose or 5 times as much of an aldoheptose is added 0.4 cc. of con- centrated HCl, sp. gr. 1.19, in a test-tube with a ground glass stopper, and the mixture is immersed for 1 hour in a boiling water bath. Then 0.4 cc. of a solution of 13 mg. of FeC13.6HzO in 100 cc. of 2 N HCl and 0.15 cc. of a 6 per cent solution of orcinol in ethanol are added to the sample. A blank containing water instead of the unknown is run simultaneously. The tube is submerged for exactly 3 minutes in a boiling water bath. A bluish purple color appears in the heptose solution. The solution is then diluted with an equal amount of distilled water. The color becomes a reddish purple.

Procedure g-The bluish purple reaction mixture obtained after 3 min- utes heating with orcinol is diluted with double its volume of either ethanol or glacial acetic acid. In this case the bluish purple color changes to green- ish blue. If the mixture diluted with glacial acetic acid is further heated for 15 minutes in a boiling water bath, the intensity of the bluish green color increases.

Procedure Z-The heptose solution (2 cc.) is heated and mixed with FeC13 as in Procedure 1; 4 cc. of glacial acetic acid and 0.15 cc. of 6

* This work was supported by a grant of the Rockefeller Foundation.

983

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984 COLORIMETRIC DETERMINATION OF HEPTOSES

per cent orcinol in ethanol are then added, and the mixture is heated for 15 minutes in a boiling water bath. The same greenish blue color as in Procedure 2 appears.

Specificity of Three &cinol Reactionsl-D-Gala-L-gala-octose, D-ghCO-L-

gala-octose, T-desoxy-L-manno-L-gala-heptose, hexose, pentose, and tetrose nor triose have been found in Procedures 1 and 2 to give either the purple or the greenish blue color yielded by heptose. Ketohexoses in concentra- tions exceeding 100 y per cc. produce a red color which does not change after addition of either ethanol or glacial acetic acid. As other sugars give a weak and uncharacteristic brown color, there will, in general, be no inter- ference from other sugars when the orcinol reaction is carried out according to Procedure 1 or 2. On the other hand, Procedure 3, which has the ad- vantage of producing a higher intensity of color, is much more influenced by other sugars. Pentoses readily give the green color, with an absorption maximum at 670 m,u, and hexoses, especially ketohexoses, produce an in- tense brown color. In this case the detection of heptose is possible only by examination of the absorption curve.

Absorption Spectra-The purple color obtained after 3 minutes heating before and after addition of water shows an absorption maximum at 560 to 565 rnp (Fig. 1). The maximum shifts to 620 rnF after addition of ethanol and is found at 610 to 615 rnp after addition of glacial acetic acid. The red color obtained with ketohexoses has an absorption maximum at 530 w.

E$ect of Alkali on Orcinol Reaction of Heptose---The product which is formed during 1 hour’s heating in 2 N HCl alone, and which produces in Procedure 2 the greenish blue color with glacial acetic acid, is alkali-labile. If, therefore, the heated acid solution is made alkaline (0.1 N), heated for 3 minutes at lOO”, then acidified again (2 N) with HCl, it yields in Procedure 2 only 50 to 60 per cent (depending on the nature of the heptose) of the color obtained without treatment with alkali. The determination of this char- acteristic property can serve to confirm a tentative identification of heptose.

Reaction with Sulfuric Acid. Procedure-To 1 cc. of solution, containing 20 to 100 y per cc. of heptose, are added, with cooling in ice water, 4.5 cc. of a mixture of 6 parts of H&J04 and 1 part of water. After 1 minute the mixture is shaken and held for successive 3 minute periods at O”, at 20-25”, and at 100”. It is then cooled to room temperature. A weak brownish color appears.

1 The author is greatly indebted to Dr. C. S. Hudson and Dr. Nelson K. Richtmyer of the National Institutes of Health, Washington, D. C., for samples of all heptoses used in this investigation, and to Dr. Melvin Calvin of the University of California for a sample of sedoheptulose.

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Z. DISCHE 985

Absorption spectra-The brown compound produced by the action of H,S04 alone has an absorption maximum between 400 and 405 rnp and a minimum at 350 rnp (Fig. 2). With 25 y per cc. of sedoheptulosan the

.60

1.

V&U’%00 5.0 540 560 580 600 6 20 MO 460 6$0?00

FIG. 1. Absorption spectra in the orcinol reaction of sedoheptulosan (0.01 per cent) in dilute HCl. Curve I, after dilution of the reaction mixture with water; Curve II, after dilution with glacial acetic acid.

optical density at 400 rnp is 0.38 (light pathway 1 cm.) (Fig. 2). As can be seen in Fig. 2, aldoheptoses as well as ketoheptoses give absorp- tion curves with the same maximum, although the curves do not always completely coincide at lower and higher wave-lengths. The optical den- sities at 400 rnp from equimolar solutions of various heptuloses differ by no more than about 50 per cent. Aldoheptoses in general show weaker ab- sorption and wider differences.

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986 COLORIMETRIC DETERMINATION OF IIEPTOSES

Reaction with Cyst&e and Xulfwic Rcid2. Procedure--To the react’ion mixture obtained on heating heptose with sulfuric acid as described above, 0.1 cc. of a 3 per cent solution of cysteine hydrochloride is added and the solution vigorously shaken. An intense orange color appears, which slowly changes to a pink of increasing intensity.

.55

.50

A6

.YO

%U~050 36037038039o 400 410 420 430 440

FIG. 2. Absorption curves of the breakdown products of various heptoses in 82 per cent H,SOd. Curve I, idoheptulosan 0.005 per cent; Curve II, sedoheptulosan 0.002 per cent; Curve III, guloheptulosan 0.002 per cent; Curve IV, a-glucoheptose 0.0025 per cent.

Absorption Spectra-After addition of cysteine, L)UM begins to decrease and a second absorption maximum at 430 to 440 rnp soon appears (Fig. 3). This maximum corresponds to the orange reaction product. When the

2 At the time this work was planned and prepared, the author was informed by Dr. M. Jesaitis and Dr. W. F. Goebel of the Rockefeller Institute, that, using the cysteine-HzS04 reaction for the determinat)ion of hexose on a bacterial polysac- charide preparation, they observed a purple color with an absorption maximum at 510 rnp. They suggested that this colored compound may be produced by heptoses. The author is greatly indebted to Dr. Jesaitis and Dr. Goebel for this information.

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8. DISCHE 987

subsequent transformation of this product into the pink compound is com- plete, the absorption curve shows a maximum at 506 to 510 rnp which re- mains unchanged for days.

X~eciJicity of Reaction-It was previously reported (1) that pentoses, hexoses, and methylpentose, when heated with H&04 under conditions of the reaction for heptose, produce furfural or its corresponding homologues which are stable at room temperature in the HZO-H&04 mixture. These compounds show absorption maxima between 305 and 330 m,u. Their

.70

FIG. 3. Absorption spectra of various heptoses in the reaction with cyst&e and &SO,. Curve I, idoheptulosan 0.005 per cent; Curve II, sedoheptulosan 0.0025 per cent; Curve III, a-glucoheptose 0.0025 per cent.

absorption at 400 rnp, where the breakdown product of heptose (probably hydroxyethylfurfural or a product of its polymerization or condensation) has its maximum, is negligible. After addition of cysteine, new reaction products with absorption maxima between 390 and 415 rnp appear. They seem to be analogous to the orange compound formed from heptose, having absorption maximum around 430 rnp. The products from pentose and hexose are unstable and, when the reaction mixtures stand at room temperature, they are transformed into purple (in the case of pentose) and blue (in the case of hexose) substances with absorption maxima at 540 and 600 rnp, respectively. These transformations apparently are analogous to the reaction involved in the shift of the absorption maximum from 430 to

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988 COLORIMETRIC DETERMINATION OF HEPTOSES

510 rnp observed with solutions of heptose. Similar changes occur with 7-desoxyheptose and octoses. The secondary reaction products in these cases, however, show only weak yellow colors.

Reaction of Heptose with Diphenylamine and HCI. Procedure--To 1 cc’. of the solution containing 20 to 100 y per cc. of a heptulose or 50 to 250 y per cc. of aldoheptose are added 2 cc. of a rea,gent prepared by mixing 10 cc. of a 10 per cent solution of diphenylamine (twice recrystallized from 70 per cent ethanol) in absolute ethanol with 90 cc. of glacial acetic acid (Baker’s Reagent) and 100 cc. of concentrated HCl (c.p.). The mixt,ure is heat’ed for 10 minutes in a boiling water bath. A purple color appears, which is stable for days. A blank prepared with 1 cc. of Hz0 instead of heptose solut’ion gives only a faint greenish tint.

Xpeci$city of Reaction and Absorption spectra--All carbohydrates pro- duce colored compounds with the diphenylamine reagent (2), although of very unequal intensity. Hexoses produce a blue color, with two absorp- tion maxima at 635 and 520 rnp and a minimum at 560 mp. Pentoses and tetroses give a greenish yellow color, trioses a yellow, and glycolic aldehyde a green color. Hexuronic acids produce a brown-red color, with an absorp- tion maximum at 520 to 522 rnp and a second absorption maximum in the violet part of the spectrum. The red component fades slowly at room tem- perature and the final pure brown color is obviously different from the bluish purple given by heptose, which shows a sharp maximum at 560 mp. 2-Desoxypentose in desoxyribonucleic acid produces a blue color, with two absorption maxima at 600 and 520 rnp. As can be seen from the last column in Table V, the shape of the absorption curve from heptose varies somewhat with the concentration of the sugar, and hence the ratio II,,, :LIes5 is lower with more dilute solutions.

Color Reactions of Heptose with Carbazole and H&Or-All carbohydrates are known to react with carbazole and sulfuric acid (3), and it has been shown that not only can this reagent differentiate between various types of saccharides but serve for quantitative determinations. The modification of the reaction described by Gurin and Hood (4), widely used for tentative identification of hexoses, gives a very characteristic orange color for aldo- and ketoheptoses, with an absorption maximum at 490 rnp. Although the color is very intense, the usefulness of this reaction is probably restricted to highly purified heptoses because of strong interference from practically all other carbohydrates.

Differentiating Color Reactions for Ketoheptose and Aldoheptose

Color Reaction o;f Ketoheptose with Phloroglucinol-A mixture of 2 cc. of a solution of ketoheptose containing 50 to 200 y per cc. with 0.4 cc. of concentrated HCl (sp. gr. 1.19) is heated in a test-tube with a ground glass

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Z. DISCHE 989

stopper for 1 hour in a boiling water bath and cooled to room temperature in tap water. 4.8 cc. of glacial acetic acid and 0.2 cc. of a 5 per cent solu- tion of phloroglucinol in ethanol are then added and the reaction mixture is heated for exactly 60 seconds in a boiling water bath and then cooled to room temperature. A blank containing water instead of the heptulose solution is run simultaneously. The heptulose solution shows a green color, having an absorption maximum at 635 rnp, which gains in intensity after standing at room temperature and later fades very slowly. It is still clearly visible after 16 hours.

Specificity of Reaction-Aldoheptoses in a concentration of 200 y per cc. sh’ow a brown color, with a maximum at 635 rnp and another in the blue region of the spectrum. Methylpentoses, aldo- and ketohexoses, and pen- toses show uncharacteristic yellow to brown colors, with absorption maxima in the blue region. These colors fade more rapidly than the green color derived from heptulose. The latter therefore can still be easily recog- nized, even when twice as much fructose for example is present in solution, especially if the reaction mixture has stood overnight at room temperature.

Digerentiation between Aldo- and Ketoheptoses by Orcinol Reaction of Bial, According to Procedure of Dische and Xchwarx (5)-1.5 cc. of a solution of nldo- or ketoheptose containing 10 to 25 y per cc. of the sugar are mixed with 3 cc. of a mixture of 100 cc. of concentrated hydrochloric acid and 0.5 cc. of a 10 per cent solution of FeC1,.6HsO and 0.2 of a freshly prepared 6 per cent solution of orcinol (Eastman, twice recrystallized from benzene). A blank containing water is run simultaneously. The reaction mixture is immersed for exactly 3 minutes in a boiling water bath and then cooled to room temperature. The solutions of aldo- and ketoheptoses show a dirty green color. The absorption curves, measured against t.he blank, all have maxima between 585 and 595 mp. The samples, after being read on the spectrophotometer, are again immersed for 17.5 minutes in a boiling water bath and cooled t)o room temperature, when the solutions from heptu- loses appear purplish, whereas the solutions from aldoheptoses show various shades of green. The changes in the absorption maxima of the solutions as compared with those observed after 3 minutes heating are shown in Fig. 4. With heptuloses the absorption maximum shifts to 560 to 570 rnp; with aldoheptoses shifts occur towards the longer wave-lengths but the absorption maxima for various aldoheptoses are not identical. o(-Gluco-, ar-manno-, and oc-guloheptose now show absorption maxima at 660 to 665 rnp, oc-galaheptose maxima at 610 and 665 rnp, and &glucohep- tose at 600 rnp (Fig. 4). In the rase of ,&glucoheptose the slope of the curve is slight between 600 and 665 rnp and the curve is horizontal between 650 and 665 rnp. The differences in position of the maxima from aldo- heptoses are probably due to the formation of two different colored com-

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990 COLORIMETRIC DETERMIKATION OF HEPTOSES

pounds, one with a maximum around 660 rnp and the other at a shorter wave-length, the relative amounts of these products apparently varying with the nature of the aldoheptose.

.65

-+----- l -$

,;%&O 5jO-rsO 600 6i0 bj,o 630 640 640 &0 6jO 6bO -1

FIG. 4. Sbsorption spectra of various heptoses in the Bial reaction aft*er 20 min- utes heating. Concentration of all sugars, 0.005 per cent. Curve I, a-mannohep- tose; Curve II, mannoheptulose; Curve III, cu.galaheptose; Curve IV, galaheptulose; Curve V, wguloheptose; Curve VI, sedoheptulosan; Curve VII, a-glucoheptose; Curve VIII, fi-glucoheptosc.

Di$‘erentiation among Various Heptoses

As can be seen in Tables I to V, some of the heptoses differ considerably from others in the color reactions described here. In the reaction with cysteine the differences in the extinction coefficients between heptuloses are not very marked, the greatest being 50 per cent between ido- and sedo- or guloheptulose. Among the aldoheptoses, however, a-mannoheptose and a-galaheptose have much lower extinction coefficients than does a-gluco- heptose, which does not differ significantly from that for glucoheptulose. This difference between a-mannoheptose and a-galaheptose and ar-gluco- heptose is also observed in the diphenylamine and orcinol reactions in dilute acid. In the Bial reaction after 20 minutes heating, on the other hand, in spite of differences in the form of the absorption curve, the opti- cal densities of equimolar solutions at 665 mp do not show differences com- parable with those in the other reactions.

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8. DISCHE 991

a-Gala- and a-mannoheptose can therefore be differentiated from cy-glu- coheptose by the ratio of optical densities from equimolar amounts in the Bial and in the cysteine reactions. Among heptuloses there are also marked differences in the ratios of the extinction coefficients in the orcinol and cysteine reactions, respectively. For example, galaheptulose has a higher extinction coefficient than glucoheptulose in the orcinol reaction and a lower one in the cysteine reaction, while idoheptulose shows one-third as high an absorption maximum as glucoheptulose in the orcinol reaction according to Procedure 2, but only 20 per cent less in the cysteine reaction. These differences can be used not only for differentiations of these heptuloses, but for the quantitat#ive estimation of two of them simultaneously. The differ- ences between the other three heptuloscs hitherto investigated are less pronounced, and no difference in reactivity has been established between

TABLE I

Comparison of Optical Densities of Solutions of Various Heptoses in Orcinol Reaction

a-Glucoheptose.. Glucoheptulose. Mannoheptulose. Galaheptulose Sedoheptulosan Guloheptulosan Idoheptulosan.

1 tion, mg. per

100 cc.

~ 4 4 4 4 4

I 4 / 10

Density after 3 min.*

610 mfi 564 m,., i 655 ms

72 46 ! 46 190 121 / 121 163 100 I 231 138 / 108 140 134 84 84 166 105 106 116 76 71

Density after 18 min.*

610 m,, 564 mfi 65.5 nlp

196 124 122 370 241 235 302 194 186

327 204 201 359 234 227

258 169 162 j

* All values of optical densities multiplied by 1000

gulo- and sedohcptuloses. These two heptuloses, however, can be easily differentiated by paper chromatography according to the method of Bevenue and Williams (6).

Quantitative Determination of Heptose in Presence of Other Sugars

Of all the reactions described in this report only three, namely the orcinol reaction according to Procedure 2, the cysteine reaction, and the diphenyl- amine reaction, promise to be useful for the quantitative determination of heptose in presence of other sugars.

The orcinol reaction, according to Procedure 1, appears less useful be- cause of the considerable deviation from proportionality. The same is true of the Bial reaction, in which the absorption curve of heptose is dis- torted in the presence of other sugars. The orcinol reaction according to Procedure 3 and the carbazole reaction have the disadvantage that other classes of saccharides yield colors of considerable intensity, although differ- ent in quality from those produced by heptoses.

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992 COLORIMETRIC DETERMISATIOS OF HEPTOSES

Determination by Or&no1 Reaction-The difference in the optical densities at 610 and 530 rnp should be used as a measure of the concentration to

TABLE II

In$uence of Concentralion and of Presence of Other Sugars on Orcinol Reaction of Sedoheptulose after Healing 3 and 18 Minutes

-

C

P

--

-

3

DSIO X 1000 :oncentra tion, mg. er 100 cc.

5.4 10.8

2.0 40 8.0 4.0

15.0 15.0 30.0

Experiment No Sugar

min. 1 8 min .: 1 min. 8 min

I 194 80 358 137

min. ‘1

114 221

II 61 117 230

IIIa IIIb IIIC

Sedoheptulosan “ “ “ <‘ “

Fructose “ + adenosine-5-

phosphate

134 270 151

16 24

165 322 633 382

56 110

49

14 22

78 160 102

(a + b) 169 67 102 (a + 0) 182 78 104

8 min.

105 205 403

TABLE III

Comparison of Optical Densities of Solutions of Various Heptoses in H2S04 and in

- HZSOl

reaction, ‘402 x 1001

Cyst&e reaction Heptose (2.5 mg. per 100 cc.) ho - Dsro Reaction

x 1000 time ID ‘610 x lOO(

-___ ‘640 x 1ooc

626 301 325 694 332 362 413 200 213 470 225 255 456 206 250 149 73 76

97 49 48 540 240 300 431 195 236 539 239 300 562 256 306

hrs.

24 24 24 24 20 20 20 20 20 20 20

‘G --

Sedoheptulosan Guloheptulosan Idoheptulosan a-Glucoheptose

<‘

a-Mannoheptose wGalaheptose Glucoheptulose Mannoheptulose Galaheptulose Sedoheptulosan

384 412 264 294

achieve greater accuracy of the determinations and to eliminate interference from other sugars. As can be seen from Table II, the Del0 - Dbso of solutions of various concentrations lies on a straight line which intersects the ordinate at a point corresponding to an optical density of no more than

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Z. DISCHE 993

0.01. This small deviation can be easily corrected without significant in- crease in the accuracy of the determinations by subtracting 0.01 from values for optical densities of the unknown and the standard. Hexose and pen- tose in twice as high concentration as that of heptose show very small ab- sorption at 610 and 530 rnp. De10 - D630 for these sugars is negligible, and the presence of such amounts of these saccharides does not influence the optical density produced by heptoses.

TABLE IV

Injluence of Concentration and of Presence of Other Sugars on Cysteine Reaction of Heptose

Experi- ment NO.

I

II

IIIa IIIb

IIIC IIId IIIe

IVa IVb IVC

IVd IVe

Sugar

Sedoheptulosan “ “ “ ‘I “ I‘

+ glucose Glucose Sedoheptulosan

I‘

+ glucose I‘

Fructose Sedoheptulosan

+ fructose Yeast adenylic acid

“ ‘< “

+ sedoheptulosan

tl

1.25 2.5 5.0 1.25 2.5 2.5 2.5 2.5 2.5 1.25 1.25 2.5 2.5 2.5 2.5 2.5 5.0 2.5

1 3608 x 1000

294 140 154 570 271 299

1116 524 592 292 141 151 551 259 292 521 247 274

520 265 256 46 40 6

272 127 145 276 143 133

545 271 274 30 27 3

554 290 264 4 6 -2

563 288 275

540 x 1ooc il.

‘i- ‘608 - 0640

x 1000

Determination by CyAfeine Reaction-The reaction with cysteine is more sensitive than is the orcinol reaction. The difference in the optical den- sities between 510 and 540 rnp should be used for quantitative determina- tions, as this difference is negligible for hexoses and pentoses in concentra- tions twice that of heptose. As can be seen in Table III, the optical densities corresponding to various concentrations lie on a straight line which intersects the ordinate at a point corresponding to an optical den- sity of 0.008. This value therefore has to be subtracted from the optical densities of the unknown and standard. Although the Ds~O - Db30 pro- duced by hexoses is negligible, the presence of these sugars somewhat de- presses the differential value due to heptose. It can be seen from Table

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994 COLORIMBTRIC DETERMINATION OF HEPTOSES

IV, however, that this depression in per cent of the total density is the same for various concentrations of heptose. The disturbance due to the presence of hexose, therefore, can be completely eliminated by the use of an internal standard.

Determination by Diphenylamine Readion-----In the diphenylamine reac- tion the opt]ical density at 560 rnp can be used only for approximate esti- mation of heptose because, for unknown reasons, it seems impossible to

TABLE V Optical Densities in Diphenulamine Beaction of Various Heptoses and Their

Expei$ent

I

II

IIIa IIIb (a + b) IV

V

Dependence on Concentration ( Sugar

Concentra- tion, y per cc.

Dsso x 1000

a-Glucoheptose 200 a-Mannoheptose 200 cu-Galaheptose 200 Glucoheptulose 200 Mannoheptulose 200 Gslaheptulose ~ 200 Sedoheptulosan 200

382 200 1.9 48 35 1.4

111 77 1.44 445 233 1.84 638 298 2.14 842 419 2.00

1005 475 2.1 550 298 1.85 714 430 1.66 200 129 1.56 910 447 2.04 209 359 0.58

1103 796 1.38 137 80 1 71 283 154 1.84 398 220 1 80 536 283 1.91 443 234 1.80 849 427 1.99

“

Guloheptulosan Idoheptulosan Sedoheptulosan I~ructose

100 100 100 133

30

Sedoheptulosan 25 “ 50 I‘ 75 I‘ 100 “ 83 “ 166

h x 1000 if; x 1000

obtain agreement between duplicate samples better than ~t3 per cent, for Dseo. Consequently, although the reaction seems to show proportionality between concentration of the sugar and D 56o values obtained as averages from a great number of determinations, the accuracy of individual deter- minations by comparison of the unknown with a standard solution is much lower than in t’he two other reactions (see Experiments IV and V, of Table

VI. DISCUSSION

The color reactions described in this report promise to be useful for the determination of heptoses which occur in living cells, either as permanent

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8. DISCHE 995

constituents or as intermediates in cellular metabolism. Their use for these purposes seems subject to three limitations, which have to be taken into consideration in every particular case.

1. An accurate determination of heptose is possible only when the hep- tose fractions in the material analyzed consist \vholly or predominately of one heptose, the nature of which must be established.

2. Heptose may be present in the material analyzed in the form of esters. The extinction coefficients of some esters in t,he reactions described here may differ considerably from those of free sugars. If, therefore, the nature of the ester present in the material is unknown, it mill be necessary to es- tablish whether, and to what extent, hydrolysis of appropriate duration changes the reactivity of the ester in some or all of these color reactions.

3. Tissue extracts may contain organic substances which when heated with I12S04 will yield products absorbing in the violet and near ultraviolet. To account for this absorption, when carrying out the cysteine or carbazole reaction, it is necessary to run with the experimental sample a blank which contains the unknown solution and H&Oh but not cysteine or carbazole, and to subtract the optical density of the latter from that of the sample containing the organic developer. As heptoses yield with HzS04 alone products which show a considerable absorption between 380 and 415 my and as this absorption decreases after addition of cysteine, they will inter- fere with the determination of hexoses by making it impossible to account for non-sugar substances which react with H,SOb. This difficulty will not be encountered in those cases in which disappearance or formation of heptose from substrates added to tissues is studied. The optical density in the cysteine reaction of the tissue extract to which no substrate was added subtracted from the optical density of the sample with substrate gives then the optical density due to the substrate and the products of its transformation by the tissues. No blanks without cysteine are necessary in this case.

The choice of the reaction most suitable for the quantitative determina- tion of the heptose will depend on circumstances of the particular case and the purpose of the determination. The diphenylamine reaction, which is the least sensitive and accurate of the three, has the advantage of simplicity and of being particularly suitable for orienting rapid estimation of keto- hexose and ketoheptose in the same sample of the unknown. The ratio of Db6,, and D,j35 rnp when compared with that of standards of ketohexose and heptose will immediately reveal the presence and approximate amount of either of these sugars in the solution.

The second modification of the orcinol reaction and the cysteine reaction appeared to be about equal in their usefulness, although the cysteine re- action is somewhat more sensitive. Accurate determinations with the cys-

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996 COLORIMETRIC DETERMINATION OF HEPTOSES

teine reaction, however, require the use of an internal standard, which in general will not be necessary with the orcinol reaction. On the other hand, in the determimations of heptose in trichloroacetic acid filtrates, it is neces- sary to accourlt for the acidity of the filtrate. This can be done by adding an appropriate amount of trichloroacetic acid to the standard solution or using an internal standard.

The reactions described in this paper promise to be useful for tentative identification of certain heptoses. In the case of aldoheptoses, this will not be possible until more than five of the sixteen possible heptoses are investigated. However, it seems possible at least to exclude certain eventualities by using the differences in the absorption spectrum of the Bial reaction in which oc-galaheptose and P-glucoheptose differ significantly from the three other aldoheptoses so far investigated. The case is considerably more favorable as far as heptuloses are concerned. This is particularly true when the color reactions are combined with paper chromatography according to Bevenue and Williams (6). Among the heptuloses we can immediately differentiate two groups, one belonging to the altro-allo and gulo-ido series, and the others from which they differ by the characteristic property of forming 2,7-anhydrides when heated with diluted acid (7-9). These anhydrides remain in equilibrium with the sugar itself and can be distinguished from the latter by their resistance to heating with alkali. Among this group of anhydride-forming heptuloses, idoheptulosan differs in a very characteristic way in the intensity of the orcinol reaction from gulo- and sedoheptulosans, and thus can be easily distinguished from the latter two. Guloheptulosan and sedoheptulosan show almost identical be- havior in all the color reactions described here, but they can be differenti- ated by paper chromatography after being heated with acid. When heated with 1 per cent HCl on a steam bath for 30 minutes, sedoheptulosan shows three different spots, one of which has a much higher RF than the others, while guloheptulosan in comparable concentrations appears only in two spots, the position of which does not differ very much from those produced by other heptuloses.3 In this sedo- and guloheptulose can be easily differentiated.4

Finally, it will be noted that gala-, manno-, and glucoheptuloses, which

3 After this manuscript was finished, G. R. Noggle (10) reported that sedoheptu- lossn can be differentiated from gulo- and idoheptulosan, after heating with 0.2 N

HCl, by paper chromatography with saturated phenol as solvent in so far as it pro- duces four different spots, while gulo- and idoheptulosan produce only two spots, corresponding to the sugar itself and its anhydride. The faster spot produced in our procedure does not correspond either to sedoheptulose or sedoheptulosan and appears to be identical with the fastest spot (RF 0.9) produced by acid-heated sedo- heptulosan in the procedure of Noggle.

4These experiments were carried out in collaboration with E. Pollaczek.

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Z. DISCHE 997

do not easily form 2,7-anhydrides in diluted acid and therefore are de- stroyed completely by heating with alkali, show lesser differences, and in a different sense, in the intensity of all three modifications of the orcinol reaction than in the cysteine reaction. The combination of these two types of reactions therefore in general will allow differentiation of these saccharides. It should be noted, however, that the testing of the alkali resistance of heptulose heated with acid can safely be applied only to the free sugars, as substitution of the sugars may prevent the formation of 2 ,7-anhydride.

SUMMARY

1. A series of characteristic color reactions of heptose is described. 2. The application of these methods to the differentiation of aldo- and

ketoheptoses and to tentative identification of some of these sugars, as well as to their quantitative determination, is discussed.

BIBLIOGRAPHY

1. Dische, Z., Shettles, L. B., and Osnos, M., Arch. B&hem., 22, 169 (1949). Dische, Z., J. Biol. Chem., 181, 379 (1949).

2. Dische, Z., Mikrochemie, 1, 33 (1929). 3. Dische, Z., and Robbins, S. S., Riochem. Z., 274, 345 (1934). 4. Gurin, S., and Hood, D. B., J. Biol. Chem., 131, 211 (1939). 5. Dische, Z., and Schwarz, K., Mikrochim. acfa, 2, 13 (1937). 6. Bevenue, A., and Williams, K. T., Arch. Biochem. and Biophys., 34, 225 (1951). 7. Haskins, W. T., Hann, It. M., and Hudson, C. S., J. Am. Chem. Sot., 74, 2198

(1952). 8. Stewart, 1,. C., Richtmyer, N. K., and Hudson, C. S., J. Am. Chem. Sot., 74,

2206 (1952). 9. Pratt, J. W., Richtmyer, N. K., and Hudson, C. S., J. Am. Chem. Sot., 74, 2210

(1952). 10. Noggle, G. R., Arch. Biochem. and Biophys., 43, 238 (1953).

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Zacharias DischeHEPTOSES

OFCOLORIMETRIC DETERMINATION QUALITATIVE AND QUANTITATIVE

1953, 204:983-998.J. Biol. Chem. 

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