10% practically - Plant Physiology suchreagentsas carbonates, sulphates, oxalates of sodiumor...

THE SUGARS OF THE ROOTS OF DAUCUS' CAROTA

A. G. GAWADI

Received February 15, 1947

It is well known that some reducing substances, resembling true sugarsin their behavior, occur almost invariably in plant extracts and may intro-duce great errors into the estimation of those sugars. Since the solublesugars commonly present in plants (glucose, fructose, sucrose and maltose)are easily fermented by yeast, this phenomenon suggests itself as a conveni-ent and practicable method for the fractionation of the total reducing sub-stances in any plant extract into fermentable and non-fermentable fractions.

Methods

TRIALS ON THE FERMENTATION OF PURE SUGARS BY ORDINARY

BAKER'S YEAST

After trials with various brands of baker's yeast, a commercial brand,known in England by the name "Eureka" was selected as being mostsuitable.

Considerable work was done to determine optimum conditions for fer-mentation of pure sugars, with the selected yeast in the shortest time possible,in order to evade the action of bacteria. The conditions dealt with involvedthe study of the different hydrogen ion concentrations in the yeast media, theamount of yeast necessary for the complete exhaustion of a given amount ofa fermentable sugar, the different sugars the selected yeast can digest, thetime required for the completion of the fermentation process, and some othertechnical points.

The following technique was finally adopted: 10 cc. of 10% washed yeast,2 cc. acetate buffer (pH 4.7), 2 cc. 0.2 M KH2PO4 and 25 cc. sugar solutionof known strength are mixed in a 50-ce. measuring flask and incubated at350 C. for three hours. At the expiration of this period the mixture isneutralized to phenol red with N/10 NaOH, 4 cc. alumina cream are addedwith subsequent shaking of the flask, the volume is then completed up to50 cc., and the contents filtered dry. A few drops of toluene are added tothe clear filtrate which is set aside for estimation.

Very much smaller concentrations of yeast were found adequate for thecomplete removal of glucose, fructose, and sucrose in the time stated; maltosefermentation, on the other hand, was-much slower. Using the standard con-centration it was found that of 50 mg. maltose supplied, 90% had beenremoved in three hours; while practically complete removal in the sameperiod was achieved when 5 cc. of 5% Taka-diastase solution were added tothe yeast suspension.

Trials with galactose and the pentose sugars (xylose and arabinose)under the standard conditions showed that of 35 mg. supplied 96%0, 98%o,

The variety used in this work is known in England by ''Early Market."438

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GAWADI: SUGARS IN DAUCUS

and 95( of the original amounts of these sugars were recovered, respectively.The small losses are presumably due to the development of bacteria underthe conditions of the incubation, since estimations after twenty-four hoursincubation gave very much higher losses; a result which makes HEINzE andMURNEEK'S (5) practice of 48 hours incubation and that of RYGG (11) of18 hours incubation for fractionating yeast fermentable and non-fermentableparts of plant extracts rather questionable.

Any loss of reducing power due to the absorption, by yeast, of some of thesugars under investigation can be neglected, since experiments in which thefresh yeast was precipitated with alumina cream, immediately after mixingwith the sugar solutions, gave satisfactory percentage recovery: maltose,99%o; galactose, 100%; arabinose, 100.5%; and xylose, 99%o. Glucose, how-ever, under the same treatment gave a recovery of only 967o, a result whichis ascribed to its rapid fermentation, for with boiled yeast treated in thesame way the recovery was 101%.

Thuts the technique adopted ensures the removal of glucose, fructose, andsucrose, leaving galactose and the pentose sugars practically untouched.

APPLICATION OF THE TECHNIQUE ADOPTED FOR THE REMOVAL OF THE

FERMENTABLE SUGARS FROM CARROT TISSUE EXTRACT

Time curves for complete removal of fermentable sugars in carrot tissueextract were studied on many occasions. Because of the presence of somenon-fermentable reducing substances which are continually attacked by bac-teria presenit in the yeast suspension, no constant value for these substancescould be attained at any point on the time curve. For this reason, the methodwhich was finally adopted to ensure the complete digestion of the reducingfermentable sugars in the tissue extract was as follows.

A sample of the extract (about 20 cc.) was treated with yeast under thestandard conditions, and to another aliquot sample a known amount of glu-cose was added; in both cases the reducing value of the clear digest wa-s thesame. This implies: firstly, that fermentable sugars in the extract are totallyremoved; and secondly, that nothing is present in the extract to hamper inany measurable way the action of yeast.

To rule out any possible error which might arise from the application ofyeast, a blank fermentation containing all of the reagents with distilled waterin place of the experimental extract, was always carried out. It is interest-ing to note that the titration value of this yeast blank and that of pure dis-tilled water, were always practically the same, especially when the coppermethod was being employed for the estimation of reducing values.

The amount of yeast used was decreased or increased according to thereducing value of the material to be fermented, and a corresponding increaseor decrease was made in the amount of alumina cream used for precipitation.

EXTRACTION AND CLARIFICATIONFor the extraction of carbohydrates from carrot root tissue, the 807o-

alcohol technique was employed. Extraction was carried out in a simplified

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PLANT PHYSIOLOGY

soxhlet-like apparatus until no color appeared in the drippings. This wasconsidered the end point of extraction, according to previous work done inthis laboratory. The simpler carbohydrates, such as the hexoses and thedisaccharides, were sought in the alcohol extract, the more complex forms inthe alcohol insoluble residue. From the extract, the alcohol was removed bydistillation under reduced pressure at 50-55° C. to avoid the possibility ofany sucrose inversion, especially towards the end of distillation when thebulk was reduced to a few cc. of a syrupy fluid. This latter was then takenup in water by washing it several times, with a little warm water and cleansand each time. The washings thus collected were finally clarified.

Among the many clarifying agents used by earlier investigators (1),basic lead acetate was most extensively applied, followed by one of a numberof such reagents as carbonates, sulphates, oxalates of sodium or potassium toremove the excess lead. Loss of reducing sugars, especially of fructose pres-ent in the extract, was noticed under such treatment by many workers, andwas attributed to a destructive action of the lead salt on fructose (7).ENGLIs and TSANG (2) had experimentally shown that this loss in reducingsugars from tissue extracts, clarified with basic lead acetate, was due to thenature of the deleading agents used, and not to the lead salt. In view of thefacts hitherto mentioned, many trials on clarifying carrot tissue extract werecarried out, using some of the reagents most commonly used for this purpose.Two precipitants, namely basic and neutral lead acetate, were chosen. Eachone of the two reagents was tried on two aliquot samples of the same extract.

TABLE IREDUCING POWER OF THE CLEAR PLANT EXTRACT EXPRESSED AS GRAMS "GLUCOSE" PER 100

GRAMS FRESH WEIGHT OF CARROT TISSUE

TOTAL REDUCING EXTRACT AFTER R.V. OF THECLARIFYING REAGENTS EMPLOYED VALUE OF THE XFERMAENTA- PORTION OF

EXTRACT ~~TION THE EXTRACT

THE FERRICYANIDE METHOD

(1) Basic lead acetate + disodiumhydrogen phosphate .................. 3.468 0.387 3.081

(2) Basic lead actate + potassiumoxalate ... 3.287 0.387 2.900

(3) Neutral lead acetate + di-sodium hydrogen phosphate 3.761 0.376 3.385

(4) Neutral lead acetate + potas-sium oxalate 3.525 0.382 3.143

THE COPPER METHOD

(1) Basic lead acetate + disodiumhydrogen phosphate ................ - 3.294 0.340 2.954

(2) Basic lead acetate + potassiumoxalate ............................ 3.220 0.336 2.884

(3) Neutral lead acetate + di-sodium hydrogen phosphate 3.519 0.323 3.196

(4) Neutral lead acetate + potas-sium oxalate 3.229 0.312 2.917

440




The excess of lead was removed from each member of the two pairs of ali-quots with one or the other of the two deleading agents chosen; namely,disodium hydrogen phosphate and potassium oxalate. The reducing powerof the aliquots was then estimated before and after fermenting the clarifiedextract with yeast. The estimation was carried out with both the ferri-cyanide (3) and the copper (12) methods, elaborated and modified in thislaboratory for measuring the reducing power of plant extracts.

The results presented in table I show that the application of lead acetate,either basic or neutral, followed by disodium hydrogen phosphate consist-ently resulted in higher reducing values than when potassium oxalate wasused in place of phosphate; especially for the fermentable fraction of theextract which includes all the reducing sugars. These results are in accordwith the conclusion of ENGLIS and TSANG (2).

Accordingly the treatment with basic lead acetate as a precipitant, fol-lowed by disodium hydrogen phosphate as a deleading agent, was adoptedfor the work on carrot tissue extracts.

ESTIMATION OF INDIVIDUAL CARBOHYDRATES IN CARROT TISSUE]EXTRACT AND THE METHODS EMPLOYED

Two methods, previously modified and elaborated in this laboratory, forthe estimation of reducing substances in plant extract, were used.

THE FERRICYANIDE METHOD.-This is a modification of HAGEDORN andJENSEN's (3) method which was first introduced and modified in this labora-tory by Hanes, and finally standardized by HULME and NARAIN (6) for theestimation of pure reducing sugars in solution.

THE COPPER METHOD.-This is a modification of SHAFFER and HARTMAN 's(12) method which was later standardized by MASKELL and NARAIN (workhitherto unpublished). The principal modification consists in the use of aless alkaline solution and the removal of-oxygen from the mixture of extractand copper reagent before boiling, by means of a stream of nitrogen gas.Under these conditions there is no re-oxidation of reduced copper duringboiling, and the thiosulphate titration value is proportional to the amountof sugar present, up to a maximum of 1.5 mg. of the reducing substance(hexose) to be estimated.

Each of the two methods proved to have some characteristic merits. Theferricyanide has an advantage over the copper method in the fact that it isnot affected by any oxygen that may be present in the experimental solution.Also larger amounts of reducing substances can be estimated by the ferri-cyanide than by the copper method; the upper limits being 3.0 and 1.5 mg.,respectively. On the other hand, the copper method, relative to monosac-charides, is less sensitive to reducing disaccharides than is-the ferricyanidemethod. Consequently, with the copper method, the rise in the reducingvalue after the hydrolysis of disaccharides, will be relatively greater andhence more accurately estimated, than with the ferricyanide method.

In view of the differences between the two methods and the advantages

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PLANT PHYSIOLOGY

each one has over the other in some respects, both were simultanleously usedduring the early part of this work. When the identity of the individualmembers of the carbohydrate group present in carrot tissue became wellestablished, the copper method was chosen, being more suited to carry onwith the rest of the estimations for this work (9). However, the ferricyaanidemethod was called on whenever the necessity arose, to check results obtainiedby the copper method.

ESTIMATION OF INDIVIDUAL CARBOHYDRATES IN CARROTTISSUE EXTRACT

THE ALDOSES AND KETOSES OR APPARENT HEXOSES.-Among the methodsused for the estimation of aldose sugars is the iodometric one, elaborated byROMIJN (10) and based on the oxidation of aldoses by iodine ill alkalinesolution. MASKELL and NARAIN (unpublished data) found this method togive correct results for mixtures of pure sugars; for leaf extracts, however,the figures for aldose sugars, indicated by the iodine used up, were invariablymuch too high.

In the present work, the method was tested oni carrot tissue extraet. Theresidual iodine in the experimental samples was titrated with N/10 sodiumsulphite and the difference between the values thus obtained and a blankestimation, gave values in terms of Na2SO3 equivalent to the I2 reduced by theexperimental solutions. Table II contains a few sets of results which repre-sent values for the aldoses estimated by the iodometric, ferricyanide, and thecopper methods. In using the two latter methods for the estimation of thealdoses, samples of the extract were treated with iodine as mentioned above,merely to remove the aldoses; then the ketoses, presumably left ulntouched,were estimated. The difference between the reducing value of the extractboth before and after the iodine treatment gave the value for the aldoses.The extract used for this investigation was previously hydrolyzed with N/2HCI for increasing lengths of time at 600 C. during the course of elaboratingtechniques for sucrose estimation. The figures obtained and presented intable II are in accord with NARAIN'S (8) results working on pure sugars andivy leaf extracts.

In view of the excessively high estimates obtained for apparent glucose(aldoses) with the iodometric method, the use of this method for accurateestimation of these sugars was ruled out. It not only falsifies the figures foraldoses, but it also leads to fictitious results for ketoses which are to be ob-tained by subtracting the aldose value from the total reducing value of thetwo, estimated jointly by either the ferricyanide or the copper method.Despite the limitations of the iodometric method and in view of the fact thatketoses are hardly oxidized by iodine under these conditions, the iodometricmethod was made use of merely as a means of getting the aldoses present inthe extract inactive by oxidation, leaving the residual ketoses to be estimatedby either of the standard methods. The coupling of the iodometric methodwith the standard reduction methods, proved very valuable in obtaining

442




reliable and accurate results. Trials for recovering pure sugars (glucose,fructose, and sucrose) added to the tissue extract proved satisfactory.

THE TRUE HEXOSES-GLUCOSE AND FRUCTOSE.-Some reducing substancesthat may be mistaken for glucose and fructose proved to be always presentin carrot tissue extract; since these substances proved to be completely re-sistant to yeast fermentation, the yeast fermentation technique was applied,as described previously, to fractionate the reducing substances in the extractinto non-fermentable and fermentable fractions. This latter was taken torepresent the joint value of true glucose and true fructose. Practically inevery case where the yeast fermentation technique was applied, and beforeapplying the estimates of the non-fermentable substance to the interpretationof experimental results, a test was carried out to ensure the complete removalof the fermentable sugars, without prolonging the standard fermentation

TABLE IITHE EXPERIMENTAL MATERIAL WAS HYDROLYZED WITH N/2 HCI AT 600 C. FOR VARIED

LENGTHS OF TIME BEFORE OXIDATION WITH IODINE

GRAMS APPARENT GLUCOSE PER 100 GRAMSMATERIAL USED F. WT. ESTIMATED BY THE:

IODOMETRIC FERRICYANIDE COPPER

20-cC. extract hydrolyzed for 4 ininlutes 3.735 2.617 2.7208 it 2.123 2.991

16 " 2.820 &.955 2.22030 " 3.100 2.800 2.63260 " 4.230 2.515 2.861

Results of another set from a duplicateextract

20-cC. extract hydrolyzed for 4 minutes 5.072 2.158 3.3658 (C 4.795 2.656 3.487

16 " 5.640 3.578 3.13930 it 5.215 3.559 3.34760 " 4.650 3.325 3.295

period. This involved the fermentation of an aliquot sample of the extractto which a known amount of pure glucose or pure fructose was added. Inalmost all cases, the results obtained after fermentilng the entire and mixedextracts, agreed fairly well. In cases when such an agreement was notattained, however, the quantity of yeast employed was increased to effectsuch agreement.

Although the joint value for true glucose and true fructose present incarrot tissue extract could be obtained according to the technique so fardescribed, no accurate separate values for the two hexoses could possibly havebeen obtained without firstly ascertaining whether this non-fermentable frac-tion belonged to the aldo or to the keto type of reducing substances, orwhether it was a mixture of the two; and secondly, estimating the value ofeach component of a mixture.

ALDO AND KETO NON-FERMENTABLE SUBSTANCES.-To separate the non-fer-mentable substances into aldo and keto fractions, 20- to 30-cc. samples of the

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PLANT PHYSIOLOGY

extract were fermented under the conditions of the standard method, andthe reducing value of the fermented extract was estimated. Samples of thefermented extract were then oxidized with iodine and the reducing value ofthe oxidized solution was again measured. This latter value was taken torepresent the keto non-fermentable substance, and the difference betweenthis and the total value (before oxidation but after fermentation) to repre-sent the value for the aldo non-fermentable substance. By subtracting thesetwo figures from those representing the total ketoses and total aldoses (appar-ent hexoses, respectively) the values for true fructose and true glucose wereobtained. This procedure with a slight modification, was duplicated for con-firmation. The same steps were performed, only with their sequence re-versed; namely, oxidation preceding fermentation. With both sequencesfairly good agreements were obtained. The results obtained at this stageand throughout the present work, indicated that the non-fermentable matterpresent in carrot root extract is composed of two different types; i.e., a ketoand an aldo, and that the keto type constituted the major part; at times thealdo type was entirely absent. Nothing like a constant ratio between therelative values of these two substances did exist at any time. This realiza-tion, coupled with the fact that the value of the non-fermentable fraction inthe extract was by no means too low to be ignored, rising at times to morethan 10% of the value for true hexoses, necessitated going through the tedi-ous processes of their fractionation whenever the estimation of true sugarswas required.

Before passing to the estimation of other members of the carbohydrategroup present in carrot tissue extract, a few trials were carried out to checkthe reliability of the figures so obtained in representing the true hexoses inthat extract. Recovery of pure fructose added to the extract was tried, bothbefore and after treating the mixture with iodine. Within the limits of ex-perimental error, 100%0 recovery was obtained, suggesting that nothing inthe carrot tissue extract, nor the treatment with iodine, affects the recoveryof pure sugar added to the extract in any measurable degree, and hence thegenuineness of the figures for glucose and fructose present in this extractmay be accepted.

THEI DISACCHARIDES-SUCROSE.-The method adopted in this laboratoryand applied to pure sucrose and leaf extracts was based on hydrolyzing thematerial to be analyzed for sucrose, with half normal hydrochloric acid at600 C., and measuring the reducing value after hydrolysis.

Since the value for sucrose in carrot tissue extract was to be obtainedfrom the increase in the reducing value of the extract after hydrolysis withN/2 HC1 at 600 C., the accuracy of such a value would necessarily depend on,firstly, whether the non-fermentable reducing substances in the extract wouldundergo any substantial change under the conditions of hydrolysis; and,secondly, whether any disaccharide, other than sucrose, may be present inthe extract to augment the reducing value of that extract on hydrolysis.A few trials were carried out to test the behavior of the non-fermentable

AAA




TABLE III

REDUCING VALUES AS MGS. GLUCOSEIN 20 CC. TISSUE EXqTRACT

-MATERIAL ESTIMATED BY:

THE FERRI- THE COPPERCYNIDE METHOD 'METHOD

mg. mng.

20-ce. extract fermented ... 15.80 13.6

20-ce. extract hydrolyzed and fermented .... 10.00 37* 9.4 31*

* Percentage loss.

reducing substances under these conditions. Samples of the extract werefermented and other aliquot samples were hydrolyzed and then fermented.Results obtained and recorded in table III clearly show a consistent loss ofthe non-fermeintable material; a loss which suggests that the material inquestion had either undergone partial destruction under the prevailing con-ditions, or had been partially hydrolyzed into some substance fermentableby the yeast used. To clear up this point, samples of the extract were fer-mented, and a part of the fermented extract hydrolyzed with N/2 HCI at60° C. The result thus obtained and recorded in table IV indicates that thenon-fermentable reducing matter in carrot tissue extract undergoes partialdestruction under the conditions of hydrolysis with N/2 HC1. Although theloss in the non-fermentable substance amounted to over 30%o at times, thetotal value of this substance relative to the total reducing power of the carrottissue extract (after hydrolysis) is quite small, the allowance for this destruc-tion can be safely ignored without appreciably affecting the results forsucrose.

To find out whether sucrose was presenit and if so, whether it was the onlydisaccharide that occurs in carrot tissue, a study of the hydrolysis time curvewas carried out. Such a curve would also indicate the period necessary forhydrolysis to attain completion. For this purpose two comparable tissuesamples A and B were separately extracted and clarified. From the clear

TABLE IV

REDUCING VALUES AS MGS. GLUCOSEIN 20 CC. TISSUE EXTRACT

MATERIAL ESTIMATED BY:

THE FERRI- THE COPPERCYANIDE METHOD 'METHOD

_~~~~~~~~~~y..

20-ce. extract fermented ..... 15.2 12.6

20-ce. extract hydrolyzed and fermented .... 12.2 20* 10.0 20*

* Percentage loss.

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PLANT PHYSIOLOGY

extracts, two sets of samples, one set for each extract, were hydrolyzed withN/2 HCl at 600 C. for varied lengths of time, and their reducing valuesestimated both before and after hydrolysis with both the two standardmethods. The results obtained are presented in table V. It is evident fromthese results, that the total reducing value of the extract rose rapidly withtime, until a level was reached after 16 minutes, beyond which time nofurther increase or decrease took place.

TABLE VANALYSIS OF TWO DUPLICATE EEXTRACTS A AND B

RESULTS ARE EXPRESSED AS GRAMS APPARENT HEXOSES PER 100 GRAMS FRESH WEIGHT

TOTAL R.V.METHOD AS APPAR- APPARENT APPARENTOF ESTI- MATERIAL USED ENT GLU- FRUCTOSE GLUCOSEMATION COSE

I__________I____________________ (1) (2) (3),~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The cop-per

The ferri-cyanide

Unhydrolyzed extract

Hydrolyzed at 600 C. for:4 minutes

8 "

16 "

30 "

60 "

Unhydrolyzed extract

Hydrolyzed at 600 C.4 minutes8 "

16 "

30 "

60 "

A.B.

A.B.A.B.A.B.A.B.A.B.

A.B.

for:A.B.A.B.A.B.A.B.A.B.

gm.3.780}37833.785

5.375 5.6986.020 f6.160; 65.9601006.355 1 6.3386.320 f6.250 1 6.3386.4256.310

6.3656.420

3.995 4)1644.332

5.880 l 6.4206.960

6.930 16 981

7.032

7.465)7417.365 7.415

7.135 7.2887.440 -7.185 ) 7 378

7.570 J

gm.

1870}1 775

2.788 1 2 7592.725 J3.022 3 1163.2103.140 3.0743.0743.125 3-1603.1952.808 ' 2 9793.150

2.180 2.2622.343

3.338 ~31943.0503.562 3.5563.5503.575 35053.4353.587 35993.6073.475 l3 3883.300

gm.2.070 t20081.945

2.587) 29413.295 J943.138 2.9442.7503.215 3 2643.3133.125 3 1783.230 *3.502 )33863.270

1.815 1h9021.989 1

2.542 3.2263.9103.368 3 4253.482 v3.890. 3 9103.9303.548 l 3 6913.8333.710 3.9904.270

To identify and fractionate the hydrolysis products, the reducing valuesof the extract, before and after hydrolysis, were resolved into ketose andaldose fractions, and the results obtained were again recorded in table Vunder columns 2 and 3, respectively.

Since the maximum reducing value of the hydrolysis products wasattained after 16 minutes and remained constant beyond that time, the valuesobtained for each set (A and B) in 16, 30, and 60 minutes were treated ascomparable estimates of the hydrolysis products, and accordingly their mean

446




for each set, as well as for the two sets combined, was taken to give a moreaccurate estimate for 100% hydrolysis. In table VI, column 2, the means ofthe three last estimates for the hydrolysis products, differentiated intoaldoses and ketoses are presented. In the same table, column 1, are alsotabulated the aldoses and ketoses initially present in the extract. Since nioallowance for destruction in the non-fermentable matter was involved at thisstage, the difference between the corresponding values in columns 1 and 2was taken to represent true glucose and true fructose that resulted from thehydrolysis of the substance or substances under investigation. These valuesfor true glucose and fructose are shown in column 3.

The results obtained by the copper method (table VI) suggest that theproducts of hydrolysis, within the limits of experimental error, are composedof equal quantities of true glucose and true fructose. By the ferricyanide

TABLE VIREDUCING VALUES AS GRAMS HEXOSE PER 100 GRAMS FRESH WEIGHT

APPARENT HEXOSE INCREASE IN TRUE

METHOD INITIAL AFTER HYDROL- HEXOSES DUE TOOF ESTI- MATERIAL SISMATION (1) (2) (3)

KETOSES IALDOSES KETOSES IALDOSES FRUCTOSE IGLUCOSEgm. gm. gm. gm. gm. gmn.

Copper Set A 1.71 2.07 3.02 3.28 1.31 1.21Set B 1.84 1.95 3.12 3.27 1.28 1.32Mean (A&B) 1.775 2.01 3.07 3.275 1.295 1.265

Ferri- Set A 2.18 1.82 3.55 3.72 1.37 1.90cyanide Set B 2.34 1.99 3.45 4.01 1.11 2.02

Mean (A & B) 2.26 1.905 3.50 3.865 1.24 1.96

method, however, glucose is consistently higher than fructose; a discrepancywhich is quite inexplicable and must be ascribed to some failing in thatmethod rather than to a substance in the extract responsible for this excessof glucose. This assumption is substantiated by the fact, as -mentionedearlier, that the copper method relative to hexose is less sensitive to disac-charides than is the ferricyanide method; hence it follows that in case of thepresence of a substance of this category in the extract, the copper methodshould give-after hydrolysis-higher reducing values relative to the initialreducing power of the extract, than would the ferricyanide method. In noinstance was this the case; on the contrary, the ferricyanide method invari-ably gave higher results. Nevertheless, an attempt was made to find a causefor this disparity between the two components (glucose and fructose) of thehydrolysis products, as estimated by the ferricyanide method; a cause whichwas thought to lie probably in the oxidation reagents and their possible inter-ference with the estimation of ketoses-and consequently the aldoses. Aftermany fruitless efforts, through which it was well established that the oxida-

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PLANT PHYSIOLOGY

tion reagents did in no way interfere with the estimation of the reducingpower of carrot extracts no such cause was discovered to account for thebehavior of the ferricyanide method in this respect.

Since the rate of hydrolysis of any material under any given set of con-ditions is a fairly good index to its identification, the rate of hydrolysis ofcarrot root extract under these conditions was calculated, in the hope ofshedding some light on the nature of the substance in question. The resultsof calculation are shown in table VII. The mean value of the last three esti-

TABLE VIIPERCENTAGE AND RATE OF HYDROLYSIS

METHOD OF ESTIMATION

TIME OF THE POPPER METHOD THE FERRICYANIDEHYDROL-HYRLSSHDOSIYSIS INCREASE HYRLSS INRCEASE HDOYI

IN R.V. PERCENT- IN R.V. PERCENT-GLU. + PR. E RATE* GLU. + FR. RAGEGLU.FR. AGE AGE

4 minutes 1.915 74.7 0.37 2.257 70.6 0.318 " 2.278 88.9 " per 2.818 88.2 " per

16 " 2.555 99.7 minute" 3.252 101.7 minute"30a ' 2.555 99.7 3.124 97.860 " 2.583 100.7 3.214 100.6

Mean of 16, 2.564 100.0 3.197 100.030, and60 min-utes

* Rate of hydrolysis was worked out according to the equation (4)

K = t log -t a-xWhere K is rate of hydrolysis in t time: a the amount of cane-sugar present at the start,and x the amount of cane-sugar that is hydrolyzed.

mates for the hydrolysis products (after 16, 30, and 60 minutes) was takento represent 100%o. Accordingly, percentage hydrolysis after varied lengthsof time, and also the rate of hydrolysis [after HARVEY (4) ] were calculated.

It is evident that the results obtained for the products of hydrolysis at600 C. with N/2 HCI, show that:

1. The hydrolysis time curve attained a maximum value after 16 minutes,beyond which time it kept practically level.

2. The. hydrolysis products are composed entirely of glucose and fruc-tose, as measured by the copper method.

3. The rate of hydrolysis of the extract is of the same order as that forpure sucrose under the same conditions.

Such conclusions strongly suggest that sucrose is the only disaccharidepresent in carrot tissue extract. Accordingly, the technique adopted for theestimation of sucrose in this work, was based on treating the extract withN/2 HCI at 60° C. for 30 minutes.

448




MALTOSE.-The method which has been used in this laboratory by MAS-KELL and NARAIN (unpublished data) for the estimation of maltose, basedon the hydrolysis of this sugar with N HCl at 1000 C. and allowing for thedestruction of the hydrolysis products at the rate of 1%o per hour, was triedon carrot tissue extract; also hydrolyzing the extract with 0.5% Taka-diastase was attempted. Results obtained are recorded in table VIII, to-gether with the reducing value of the same extract after N/2 HC1 hydrolysisat 600 C. The increase due to hydrolysis after both the treatment with N/2HCI at 600 C. and with 0.5% Taka-diastase is practically the same. The lowresults obtained after hydrolysis with N HCI at 1000 C. might be due to agreater rate of destruction than has been allowed for. These results furtherconfirm the conclusion, already arrived at; namely, that sucrose is the onlydisaccharide present in carrot root tissue.

TABLE VIIIREDUCING VALUES OF CARROT lISSUE EXTRACT AS GRAMS TRUE HEXOSES PER 100 GRAMS

FRESH WEIGHT BEFORE AND AFTER DIFFERENT TREATMENTS*

HYDROLYZING AGENT:

REDuCING VALUES |N/2HCI AT | N HIl AT 0.5% TAKA-

60C 100C. DIASTASE600C. ~~~~~~AT350 C.

T.R.V. beforeT.R.V. afterIncrease "

hydrolysis ........................

gm.4.1657.3653.200

gm.4.1556.9062.751

gm.4.2007.4033.203

* Figures shown in this table are means of values obtained for the duplicate extractsA and B by the ferricyanide method.

To conclude: here follows a short account of the steps followed for a com-plete analysis sheet of individual carbohydrates, present in the 80%o alcoholextract of carrot tissue, according to the technique hitherto discussed:

STEPS TREATMENTA. Extract directly estimatedB. Extract fermented with yeast and

estimatedC. Fermented extract oxidized with

iodineD. Extract oxidized with iodineE. Extract hydrolyzed with N/2 HCI

at 600 C.F. Hydrolyzed extract oxidized with

iodine

RESULTSDirect total reducing valueTotal non-fermentable reducing matter

Keto non-fermentable reducing matter

Total direct reducing ketosesTotal reducing value after hydrolysis

Total reducing ketoses after hydrolysis

Thus

A-BB-CA-D

= Total fermentable reducing matter, or true hexoses= Aldo non-fermentable reducing matter= Total direct reducing aldoses

449



PLANT PHYSIOLOGY

A-D-(B-C) =Fermentable direct reducing aldoses or true glucoseD-C = Fermentable direct reducing ketoses or true fructoseE-A = Total increase in fermentable reducing matter or the in-

crease in glucose and fructose due to inversionE-F = Total reducing aldoses after inversionF-D = Increase in fructose due to inversionE-F-(A-D) = Increase in glucose due to inversion

All the results obtained according to the above scheme represent reducingvalues in terms of glucose, and accordingly (D-C) and (F-D) in order torepresent' fructose, must be corrected by adding lO; fructose being thatamount lower in its reducing power than glucose, as' estimated by the coppermethod. Hence it follows:

(D-C ) x 10097 = Initial fructose

(Ft-D) x 100 = Increase in fructose after

inversion(F-D) x 100

+ E-F-(A-D) = Sucrose as invert sugar

360 9)7 + E-F- (A-D) =Sucroseassuch

Summary1. Carrot tissue :was extracted with 80%o boiling alcohol. The aqueous

extract was cleared with basic lead acetate and disodium phosphate.2. Using the two reduction methods (the copper and ferricyanide) glu-

cose, fructose, and sucrose were found and estimated in the clear extract.Maltose was entirely absent.

3. Trials for fermenting various sugars with ordinary baker's yeast wereattempted. Glucose, fructose, and sucrose were completely fermented inless than three hours. Maltose was more resistant than the three mentionedsugars. It was almost equally rapidly fermented when a solution of Taka-diastase was added to the yeast suspension. The pentoses, arabinose and.xylose, were not attacked by the yeast used.

4. The yeast technique was applied to carrot tissue extract for separatingthe non-fermentable reducing matter which was generally taken for reducingsugars.

This work was carried out at the Botany School, Cambridge, England.The author wishes to express his indebtedness to the late DR. F. F. BLACK-MAN, F.R.S., for his kind encouragement and to DR. E. J. MASKELL, F.R.S.,for his intense interest in the work and his continual advice and criticism.The author further wishes to thank MR. C. SEWELL for his help with theanalyses.

BROOKLYN BOTANIC GARDENBROOKLYN, NEW YORK

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LITERATURE CITED1. COMMITTEE ON CHEMICAL METHODS. Determination of carbohydrates.

Plant Physiol. 10: 387-391. 1935.2. ENGLIS, D. T., and TSANG, T. The clarification of solutions containing

reducing sugars by basic lead acetate. The effect of different de-leading agents. Jour. Amer. Chem. Soc. 44: 865-867. 1922.

3. HAGEDORN, H. C., and JENSEN, B. N. Zur Mikrobestimmung des Blut-zuckers mittels Ferricyanid. Biochem. Zeitsch. 135: 46-58. 1923.

4. HARVEY, R. B. Plant Physiological Chemistry. The Century Co.New York. 1930.

5. HEINZE, P. H., and MURNEEK, A. E. Comparative accuracy and effi-ciency in determination of carbohydrates in plant material. Univ.Missouri Agri. Exp. Sta. Res. Bull. 314. 1940.

6. HULME, A. C., and NARAIN, R. The ferricyanide method for the deter-mination of reducing sugars. Biochem. Jour. 25: 1051-1061. 1931.

7. LooMis, W. E. A study of the clearing of alcoholic plant extracts.Plant Physiol. 1: 179-189. 1926.

8. NARAIN, R. Carbohydrate metabolism in respiring leaves of Hederahelix. Thesis, Cambridge University. 1932.

9. PH ILIPS, T. G. The determination of sugars in plant extracts. Jour.Biol. Chem. 95: 735-742. 1932.

10. ROMIJN, F. tVber eine jodometrische Zucherbestimmung. Zeit. Anal.Chem. 18: 349. 1897.

11. RYGG, G. L. Sugars in the roots of the carrots. Plant Physiol. 20: 47.1945.

12. SHAFFER, P. A., and HARTMAN, A. F. The iodometric determination ofcopper and its use in sugar analysis. Jour. Biol. Chem. 45: 365-390. 1920-21.

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