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THE MECHANISM OF THE OXIDATION OF GLUCOSE

BY BROMINE.

BY H. H. BUNZEL.

(From the Laboratory of Bioch em istry of the University of Chicag o.)

(Received for pu blica tion, Decem ber 8, rgq.)

I.

INTRODUCTION.

In order to understand metabolism better the behavior of the

different fuelstuffs under various conditions outside the body

must first be investigated. A series of studies in this direction

was accordingly started, particularly on the physico-chemical

behavior of the sugars (1-7).

In a preceding investigation by Mathews and the writer (7),

the following conclusions were drawn regarding the oxidation of

glucose by bromine:

(a) Glucose behaves both as a weak acid and as a weak base.

In an alkaline or neutral solution it dissociates into metal or

Hf ions and C,H,,O,- ions; in an acid solution it forms a salt of

the acid by addit ion, yielding C,H,,O,+ ions and the anion of the

corresponding acid.

(b) Both of the glucose ions are oxidized by bromine side by

side, the oxidation of the first being depressed by the addition of

H+ ions, while the other remains unaffected.

Velocity equations were worked out on this basis, taking into

consideration the two kinds of glucose ions, the concentration

of the H+ and OH- ions and of the bromine and the hypothesis

was tested under very diverse conditions by measuring the rate

of oxidation in the presence of various acids in different concen-

trations and varying amounts of bromine. The hypothesis was

found to agree with al l the facts observed.

In the experiments just cited the concentration of the sugar

was purposely taken

SO

large that its change of concentration

was negligible. It seemed necessary, therefore, to vary also the

I.57

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158

Oxidation of Glucose by Bromine

concentration of the sugar, to observe its rate of disappearance and

to ascertain whether the equations alluded to in the above article

hold true under these conditions. It was desirable also, to

ascertain especially whether the oxidation of the positive glucose

ions leads to the formation of gluconic acid exclusively.

The methods were the same as those in the previous investi-

gation. The acidity developed during the reaction was deter-

mined in some of the experiments by titration with

0.1 N

sodium

hydroxide using phenolphthalein as the indicator, and by sub-

tracting the amount of hydrobromic acid formed, the amount

of gluconic acid was determined.

I I . DEVELOPMENT OF EQUATIONS.

It was shown in the previous paper on the subject that both

the positive and negative ion are oxidizable and that in the

presence of sufficiently high concentrations of H ions the acid

ionization of the glucose is so depressed that only one of these

two reactions needs to be considered, i.e. the oxidation of the

positive glucose ion. It was shown by experiment that concen-

trations of H ions above 0.1 N practically completely suppress

the oxidation of the negative glucose ion and in such conditions

we have to deal altogether with the reaction:

C,HL0,

+ Cl- + Br, + HOH = C,H,,O, + HCl + 2 HBr

In all of the following experiments only this reaction

IVLLS

studied, an amount of sulphuric acid being added to make the

solution o. I s as regards H,SO,.

The differential equation representing this reaction is

ax

- =K.C&& X CoH- X Cnrz(free) _. _.

dt >

which means that the rate of the reaction will depend on three

factors: (a) the concentration of the positive glucose ion, C,H,,O,:

(b) the concentration of the hydroxylions in the solution; (c)

the concentration of the free bromine in solution.

(a) The formation and quantity of the positive glucose ion

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H. H. Bunzel

I.59

is determined by the amount of the acid present, as shown for

the case of hydrochloric acid in the following equation:

C,H,,O, + H++ Cl- s C,H,,+o, + Cl-

CC6H1206+ = K’CC~ H~~O ~ x c+

H

Inasmuch as the ionic dissociation of the glucose is very small,

the amount of C,H,,O,+ ions will be practically directly propor-

tional to the concentration of the glucose solution and also

directly proportional to the concentration of the H ions.

(11) The hydroxylions are of course all derived from the water

present. Their quantity is determined by the equation :

K CHzO = ‘H, x COH -

from which it is evident that COH- is inversely proportional to

the concentration of the H ions in solution.

(c) The amount of free bromine may be computed as follows:’

CJ+~ X CB~- = o.oj CB~~-

CBr (Total) = CBrz + CBr,-

CBr - (Total) = ZCBr Consumed

CBr- + CBr, = 2 CBr Consumed

.oj Br,

BrT - Br,

+ Br, = 2 Br Consumed

% = f - 2 Br Total Br Consumed +

‘05 + Br T otal + 2 Br consumed

a

_____.~

2

>

.=‘.5 + B r Total + 2 Br Consumed

+ .___~_

2

By subtracting this value of Br, from the total bromine titrat-

able with sodium thiosulphate solution, one obtains the value for

the free Br,.

1 See literature 7, I 0, I 1, 12 at the end of the paper.

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160

Oxidation of Glucose by Bromine

By examining (a) and (b), it becomes evident at once that,

provided suffic ient acid is added to suppress completely the

oxidation of the negative ion, the H ions hasten the reaction

according to (a) exactly to the same extent as they retard it

according to (b). This allowed us to neglect the concentration

of the H ions provided it was over o.

I N.

The velocity of the reaction is accordingly proportional, under

these conditions, to two variables only; the concentration of the

glucose and the concentration of the free bromine, and is repre-

sented by the equation

= K (a - x) (b - X)

On integration this becomes

K= I

log nat

b(a - x)

t (a - b) a (b - x)

where K is the velocity constant, t the time elapsed since the

beginning of the exlzeriment, a and b the concentration respec-

tively of the active bromine and sugar at the start, and x the

amount of bromine and sugar consumed.

In a part of the experiments a known amount of sodium bro-

mide solution was added, so that the concentration of the Br-

ions in the final mixture was 0.3

N.

As the Br, in these experi-

ments was only 0.01 N at the start, the slight increase on the con-

centration of the Br- ions during the reaction due to the forma-

tion of HBr, was negligible. In the remainder of the experi-

ments, however, where the concentration of the free bromide at

the start was larger or where the reactions were so slow that it

seemed undesirable to depress their rate still more by the addi-

tion of bromide the value for Br, had to be calculated separately

for each determination. By taking the constantly changing

value for Br- in consideration we obtained a differential eqa-

tion, which we were unable to integrate. For that reason the

values for K were calculated for short time intervals, always

using the time of the last preceding determination as a starting

point. To approximate the concentration of the Br the writer

added to the concentration of the HBr present at the preceding

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H. H. Bunzel

determination (including that which is present as Br,) one

half of the HBr formed during the time interval considered and

subtracted the concentration of the bromine present in the form

of Br,.

I

t _ .--------..

-- ~~ --.- -~m~~~m-(a - b)

H Brt, - H Brt,

05 + Br-(,t &tart)+ -~ ~-~--

2

- %

The formula used in the experiment where the concentration

of the Br- ion was made .30 at the start, was

1

K=

t %(a - b)

In b G-Ei . .[III]

I I I .

EXPERIMENTAL

A. To detervnine the comtancy of K when the acidity, glucoseand

bromine

are

all varyitzg.

The figures given in Tables I and II indicate by the constancy

of the values for K when the glucose and Br, are varied within

wide limits, that the course of the reaction is actually controlled

by those factors and in the manner stated in our hypothesis at

the beginning of this paper.

B.

Is gluconic acid the sole product of the oxidation of the posi-

tive glucose on?

In all the experiments just quoted the change in the concentra-

tion of the bromine only was determined.

The change in con-

centration of the sugar was assumed to be one molecule of sugar

oxidized for each molecule of Br, used up. This assumption

gave a constant value for K. To test the hypothesis further

that glucose went thus into gluconic acid, it was necessary to

measure at least in one experiment the degree of acidity of the

mixture at varying time intervals and thus to determine the

amount of gluconic acid formed. It should of course be found

then that for each molecule of bromine which disappeared from

the solution, one molecule of glucose has been used up and the

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162

Oxidation of Glucose by Bromine

TABLE I .

Concentration of H,SO, 0.37~; Con centration of Na Br 0.30 N.

2.OOM*

1.00

0.50

0.25

0.10

0.05

-t.-

0.00500

0.00520

0.00516

0.00496

0.00515

11.60

10.68

9.70

8.98

7.53

5.80

4.91

10.01

9.25

8.82

7.89

7.05

6.48

3.28

10.40

9.29

8.41

7.25

5.30

10.32

10.00

9.74

9.29

8.20

6.83

5.31

9.92

6.31

5.44

1.93

10.30

8.37

7.64

5.85

:

-

I (MINUTES).

K (log n&t).

__ .--

0

10

20

30

50

82

100

0

15

25

50

75

100

260

0

50

100

165

300

0

30

50

100

200

0.0290

0.0312

0.0298

0.0301

0.0295

0.0301

0.0342

0.0338

0.0330

/

I

I

0.0310

0.0304

~

0.0300 0.03'21

0.0276

0.0298

I

0.0306 1

0.0314 i 0.0299

0.0293 )

0.0312 :

0.0291 I

0.0322 /

YE*?4

K (log nat)

0 0300

350 0.0330 I

570

/

I 0.0328 , 0 0314

0

1060

13%

3825

0

970

1185

2350

0.0301

0.0298

0. 0334

0.0303

0.0357

0.0343

0.0310

0.0x35

* nr refers to a molecular solution

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TABLE I I .

No NaBr added . C, = 0. N H, SO,

0.77

0.55

0.275

0.110

0.55

Calculated

ineach

determin-

ation from

thebegin-

ning of

the experi-

ment.

0.100

. l.Ol

8.88

5.78

4.20

3.19

2.67

11.02

8.82

6.50

4.04

1.94

.Sl

11.71

10.40

9.04

7.14

4.85

3.05

10.95

9.38

8.05

6.72

11.41

9.59

8.59

x.19

45.40

36.45

31.50

20.30

17.00

14.98

T.

‘Tot. Br.

0

10

30

45

60

70

00

15

35

65

120

185

0

15

35

65

120

185

0

50

100

160

0

3.00550

3.00444

3.00289

3.00210

0.00160

0.00134

0.00551

0.00441

0.00325

0.00202

0.00097

0.00041

0.00586

0.00520

0.00452

0.00357

0.00243

0.00153

0.0054s

0.00469

0.00403

0.00336

0.00571

100

170

200

0.00480 0.00015

0.00430 0.00020

0.00410 0.00023

0 0.090s

100

0.0729

200 0.0630

775 0.0406

150 0.0340

480 0.0300

I

I

I-

(

(

(

(

(

(

(

(

I

I

I

I

I

I

I

,

/

/

--

).00017

j.00026

I.00024

).00021

).00020

).00017

1.00024

3.00024

3.00015

3.00006

3.00010

0.00022

0.00029

0.00028

0.00022

0.00010

0.00021

0.00025

0.0066

0.02185

0.0263

0.0233

0.0240

C

c

C

C

C

(

(

(

(

(

(

(

(

(

(

(

(

(

(

I I

, I

’

CB*-

(log nat)

-_

).

00089

j.00342

I.00578

j.00710

j.00788

I.0283

I.0299

I.0308

I.0272

I.0269

). 00093

j.00312

I.00575

).00788

I.00959

I.0274

I.0290

J.0322

1.0282

I. 0297

I.00055

I.00177

I.00333

I.00544

I.00776

K

(

(

(

(

(

(

(

(

(

I

(

I

I

I I

I ’

I

I

1

I. 0290

3.0265

3.0306

0.0285

3.0304

I.00069

1.00203

1.00332

0.0288

Cl.0290

0.0299

3.00076 0.0315

3.00121 0.0310

3.00161 0.0313

0.0113 0.0300

0.0238 0.0282

0.0517 0.027C

0.0837 0.027E

0.0936 0.026t

IEAN K.

3.0286

D .02X3

0.0290

0.0292

0.0313

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164

Oxidation of Glucose by Bromine

TABLE II-Continued

lK(log nst)

MEAN K.

I

I-

7.49 100 0.03745 / 0.01255 0.0253

4.30 320 0.0215 0.0110 0.0300 0.0313

0.0283

0.05 31.12 0 0.1556

28.50 100 0.1425 0.0131 0.0258

24.62 320 0.1231 0.0296

Meanvalue of K (log nat).

I

).0302*

1

*The cause of the divergence of this constant fro m the one found in the previous paper by

Mathe ws and Bunael, i. e., 0.0169 is twofo ld. The re the suga r wau always present in the sam e

concentration, 0.5 M, which wits regarded iw unity; moreo ver it was though t then that the glucose

used conkdined one molecule of wate r of crystallization, which w as proved to be incorrect by

polarimetric mea surem ents carried on later. If the previous value of the constant is trans-

forme d on this basis, we obtain as mea n value for K (log nat) fro m our form er paper 0.0308,

which agrees with the one found above.

corresponding amount of gluconic acid formed. Furthermore

there ought to be for each atom of bromine transformed into

hydrobromic acid, an acidity produced which is one and a half

that of the HBr calculated, one-third of this acid being due to

the gluconic acid formed and two-thirds of it to the HBr. The

experiment was performed under the usual conditions. As the

temperature was not absolutely uniform in this experiment but

oscillated between 25’ and 25.5O, no attempt was made to calcu-

late the velocity constants in this experiment and only the paral-

lelism between the changes of concentration of the two compo-

nents named were studied.

For this purpose at various time

intervals two portions of the reaction mixture were removed

from the reaction flask. One of these portions of

IO cc.

volume

was run into KI solution and titrated with 0.1

N

sodium

thiosulphate solution to determine the Br, used up. The other

portion of 50 cc. had air blown through it for 15 minutes at room

temperature to remove the Br,. Then

IO

cc. of this portion was

titrated with

0.10

sodium hydrate and then with

0.1 N

silver

nitrate using a few drops of a saturated potassium chromate

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H. H. Bunzel

1%

solution as indicator to determine respectively the total acidity

and the bromine in the form of bromide.

To make certain that none of the hydrobromic acid is lost dur-

ing the passage of air, the following experiment was carried out: .4

solution of hydrobromic acid was made and air drawn through it.

Titer of 10 cc.

Tim e. of HBr solution

3,3 j........ ........ ........ ..... j.ZSCC. 0.1 NNaoHsol

3.55

5.30

(‘ “ “ “

4.10 . . . . . . . . . .._. ._. _..__ j Z “ “ “

From this experiment it was clear that there was no appreciable

loss of hydrobromic acid.

A vigorous current of air was blown through the bromine-con-

taining solutions in tall cylinders and by separate experiments it

was determined that a complete removal of the bromine was

affected.

Table III gives the results of the experiment. The strength

of the sodium hydrate solution used was o.

I

o

I 2

N, the silver nitrate

solution was 0.09703 N, the sodium thiosulphate solution was

0.1000 N.

From the figures in Table III the following relationships become

evident :

(I)

The total acidity at any time in the course of the reaction

determined by titration with

0.1 N

sodium hydrate solution after

removal of the free bromine, is, \yithin experimental errors, equal

to the acidity calculated from the amount of bromine which has

disappeared from the solution assuming that each molecule of

Br, thus used up has oxidized one nolecule of glucose to form one

molecule of gluconic acid and two molecules of hydrobromic acid.

(2)

Two-thirds of the acidity at any particular time is due to

the hydrobromic acid formed and one-third to the gluconic acid

produced.

(3) The amount of bromide found in the solution at varying

times agrees closely with the amount calculated from the acidity.

(4) The amount of glucose oxidized calculated from the glu-

conic acid produced agrees closely with the amount calculated

from the assumption that one molecule of Br, oxidizes one mole-

cule of glucose to gluconic acid.

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Oxidation of Glucose by Bromine

0

0

0

666666

+ I I I I I

0 0 0 0 0 0

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H. H. Bunzel

IV. DETERMINATION OF THE TEMPERATURE COEFFICIENT OF THE

REACTION.

Cklucose 0.5 M; CH, so1 0.3 N; CN~B~ 0.30 N. Temperature 0’.

12.66 j 0' 0.00633

11.50 I 1000 0.00575

10.01 2430 0.00500

7.40

I

5410 0.00369

5.45 8200 0.00273

&lean K oog nat).

0.00134

0.00136

0.00140

0.00144

I

0.00139

K, clog natI. .... .... .... .... .... .... .... .... .. o. OVJOZ

K, clognatj .... .... .... .... .... .... .... .... .. .o.oo~gg

Temp. Coeff

..................................

.j.sz for Id’

v. CONCLUSIONS.

The results of the present investigation have shown: (I) that

the amount of acid and of bromide produced and of glucose and

bromine disappearing in the oxidation of glucose by bromine in

acid solution is equal to that calculated, on the hypothesis that

one molecule of bromine oxidizes one positive glucose ion to

gluconic acid ; and (2) that the velocity of the reaction is correctly

represented by equation

I,

(p. 154) as shown by the constancy of

the velocity constant, K, under widely varying conditions of

acidity and of different concentrations of bromine and glucose.

These results strongly support the conclusions of our former

paper, that glucose, like other alcohols and aldehydes, forms two

series of salts; the first in which glucose behaves as an acid, the

salt dissociating into metal and negative glucose ions; the second,

in which glucose behaves as a base, forming an oxonium salt

dissociating into C,H,,O,+ ions and the negative acid radicle.

Both of these ions are more easily oxidized than the neutral

glucose molecule and they give rise on oxidation to different acids.

The I)ositive ion, as this research sho*,vs, goes quantitatively into

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Oxidation of Glucose by Bromine

gluconic acid. The gluconic acid thus formed is presumably

converted into lactone and in the course of time would be still

farther oxidized by the bromine. This reaction is, however, a

slow one compared to that which leads to the formation of the

gluconic acid. It probably accounts for the slight tendency of

the constant K to rise toward the end of the experiment, in long

experiments, and in those cases where Br, is present in large

amounts and the glucose in small amounts.

Whether gluconic acid is produced in small quantities by the

oxidation of the negative glucose ion, or whether it is produced

by slow oxidation also of the unionized molecule, if there is such

an oxidation, cannot be said without further investigation.

The negative glucose ion when oxidized by bromine probably

gives rise to the large variety of acids, described by many ob-

servers and studied particularly by Nef (13)

in his splendid inves-

tigation of the transformation of the sugar molecules.

The fact that glucose oxidizes both as an oxonium salt and the

salt of a metal explains the dependence of the rate and character

of the oxidation on the reaction acid or alkaline of the medium.

Glucose being a weak acid (14-15) dissociates into H+ ions and

negatively charged C,H,,O,- ions and in the presenceof acidsinto

positive C,H,,O,+ ions and the negative acid radical. The first

kind of dissociation is the principal one in the presenceof alkalies.

The effect of the alkali, therefore, is to increase the number of

the active negative sugar particles in solution. Acids, on the

other hand, by means of the large number of H ions they furnish,

greatly depress the dissociation of so weak an acid as glucose,

and at the same time they form salts with the positive ion.

In

a neutral solution both of the oxidations of both positive and

negative glucose ions go on side by side at their respective veloci-

ties, which have been determined in a former paper 7). The

addition of small amounts of acid will decrease the rate of oxi-

dation of the glucose, because, for the reason just given, acids

diminish the extent of the “negative ion oxidation” which is

rapid and leave unaltered the extent of the slower “positive ion

oxidation, ” Increasing quantities of acid will slow the rate

of oxidation farther and cause more and more a predominance

of the latter reaction; but this decrease in rate due to increasing

acidity holds true only up to a certain point i.e., to the point

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H. H. Bunzel

where the negative ion oxidation is entirely suppressed (appr. .02

NH+

ions); beyond that additional amounts of all acids tried,

except HCl and HBr have no noticeable depressing ef fect . The

particular action of these acids was discussed in the first com-

munication.

The writer wishes to express his gratitude to Prof. A. P.

Mathews, for the theoretical formulation of the reaction and for

his uninterrupted interest in the execution of this work.

LITERATURE.

I. Mathews and McGuigan: Am erican Journ al of Phys iology, xix,

P. 1997 1907.

2. McGuigan, H.: Am erican

Journ al of Phys iology, xix, p. 175,

1907.

3. Mathews and Walker: Journal of Bio logica l Chemistry, vi, p. 2 I.

‘909.

4. Mathews and Walker: Journal of Bio logica l Chemistry, vi, p. 29,

‘909.

5. Mathews: Journal of Bio logic al Chemistry, vi, p. 3, 1909.

6. Bunz el, H. H.: Am erican Journ al of Phys iology, xxi,

p. 23,

1908.

7. Bunzel and Mathews: Journal of the American Chem ical Society,

xxxi, p. 464, 1 909.

8. Mathews and Walker: Journal of Bio logica l Chemistry, vi, p. 289,

‘909.

9. Mathews and Walker: Journal of Biolo gica l Chemistry, vi, P. 299,

1909.

IO. Richards and Stull : Zeitschrift ftir physikaliscke Chemie, xli, p.

544, 1902.

I I. Roloff : Zeitsckrijt fiir physikalische Chemie, xiii, p. 353, 1894.

12. Worley : Journal of the American Chem ical Society, lxxxvii, p. I 107,

‘905.

13. Nef: Annalen der Ckemie, cc& ii, p. 214, 1907.

14. Cohen : Zeitsckrift fzir pkysikaliscke Ckemie, xxxvii, p. 69, 1909.

15. Osaka: Z eitsckrijt ftir pkysikaliscke Ckemnir, xxxv, p. 661, 1900.