James A. Hawkins and Donald D.

Van Slyke
J. Biol. Chem. 1929, 84:243-247.

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ARTICLE:
COMPARISON OF RATES OF SUGAR
DISAPPEARANCE AND CARBON
DIOXIDE FORMATION DURING
FERMENTATION OF GLUCOSE

COMPARISON
OF RATES OF SUGAR DISAPPEARANCE
AND CARBON DIOXIDE
FORMATION
DURING
FERMENTATION
OF GLUCOSE.
BY JAMES
(From

A. HAWKISS

the Hospital

AND

DONALD

of The Rockejeller
Institute
New York.)

(Received

for publication,

August

D. VAN
for

SLYKE.

Medical

Research,

5, 1929.)

243

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In an investigation
of the amount
of sugar in urine (Van Slyke
and Hawkins, 1929) we noticed that it was impossible to detect
any glucose by the reduction
method after the glucose-yeast
mixture had stood for 15 minutes, whereas it was necessary to
allow the mixture
to stand 1 hour in order to get 80 per cent of
the theoretical yield of carbon dioxide from the glucose.
This
suggested that the combination
or absorption
of glucose by the
yeast is a rapid reaction, while the breaking down of glucose into
alcohol and carbon dioxide is a much slower one.
Slator (1906) explains the mechanism of fermentation
on the
hypothesis that sugar diffuses into the yeast cell and combines
with the enzyme, and that this compound
decomposes either
directly or indirectly into alcohol and carbon dioxide with regeneration of enzyme and immediate formation
of more compound
with sugar. In this series of reactions, he states that the decomposition of the compound is the one which proceeds slowest and
is therefore
the most important
in determining
the velocity
reaction.
He found that when the concentration
of the sugar
was above 1 per cent it had no influence on the velocity reaction,
but that the concentration
of the sugar would have an influence
if the concentrations
of sugar and yeast were such that an appreciable amount of enzyme was left uncombined.
We have measured simultaneously,
by observing the disappearance of reducing material from solution, the rate at which glucose
combines with the yeast and also, by observing the rate of CO,
formation
in the yeast-sugar solution mixture, the rate at which
the sugar breaks down into alcohol and carbon dioxide.

244

CO, Lag in Fermentation

EXPERIMENTAL.

Determination

of Carbon Dioxide.

3 cc. samples of the alkali-treated

suspension of yeast in glucose
solution were transferred
by means of a rubber-tipped,
stop-cock
pipette to the chamber of Van Slyke and Neills manometric
apparatus and analyzed for COZ as described by the& (1924).
The CO2 factors of Van Slyke and Sendroy for the manometric
blood gas apparatus
(1927) were used to calculate the COZ.
Controls were run at the same time by substituting
distilled water
for the glucose solution in order to correct for the COZ present
in t.he yeast itself.
Determination

of Glucose by Ferricyanide

Reduction.

5 cc. portions of the alkali-treated

yeast-glucose solutions were
mixed with 10 cc. of distilled water, 5 cc. of 0.2 N H2SOe, and 1.5
gm. of Lloyds reagent.
The mixture was shaken for 1 minute
and filtered through a dry filter paper in order to obtain a clear
filtrate.
The sugar remaining
in the filtrate was then determined by the Van Slyke-Hawkins
gasometric
blood
sugar
method (1928).
Control analyses in which the sugar solutions were replaced by
water were run at the same time to determine p. readings.
Amount

of Glucose Removed from Solution Estimated from Decrease

in Reducing Power and From Increase in COz.

The results of these determinations

are given in Table I. They
indicate that t.he initial reaction rate for the removal of glucose

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50 cc. of a standard glucose solution were mixed with 50 cc. of

yeast suspension and 15 cc. portions of this mixture were run
rapidly into 20 cc. tubes filled with mercury and connected at
the bottom with mercury leveling bulbs.
The tubes used were
of the type marked J in Fig. 3 of Austin, Cullen, et al. (1922).
Each tube was sealed by clamping the rubber inlet tube at the
top with a screw clamp.
At various time intervals
after the
mixing of the yeast and glucose, 1 cc. of a 4 per cent solution of
sodium fluoride in 8 per cent sodium hydroxide was run into each
tube in order to kill the yeast and prevent the escape of COz.
The tubes were shaken vigorously to insure thorough mixing.

and 10

and 5

Mixture
2.
125 mg. glucose
gm. yeast.

Mixture
3.
125 mg. glucose
gm. yeast.

yeast

5
10
15
30
60

5
10
15
30
60

5
10
15
30
60

min.

Removed from

and 10

of glucose
and
100 cc. suspension.

of Glucose

Mixture
1.
250 mg. glucose
gm. yeast.

Amounts
in

Amount

55
83
102
125
125

69
119
124
125
125

120
203
250
250
250

pei%nt

Solution

i-

10.2
25.4
36.6
46.9
48.4

24.445.6
47.4.
48.4
49.3

18.6
64.5
84.0
90.8
95.8

23
52
75
96
99

50
93
97
99
101

38
132
172
186
196

ml.
%?.
er ceni ! 1Per cent

Series

Estimated

TABLE

0.42
0.63
0.73
0.77
0.79

I.

-7
-

36
42
51
98
125

56
107
121
125
125

250
250

98

2.

4.4
10.3
16.1
33.2
44.0

18.6
36.2
43.5
45.4
49.8

13.2
38.6
57.7
88.9
99.7

9
21
33
68
90

38
74
89
93
102

27
79
118
182
204

0.25
0.50
0.65
0.69
0.72

0.67
0.69
0.73
0.74
0.82

0.73
0.82

0.28

Power

ml.
mg.
1er Cc%, IP er cell

Series

in Reducing

ml.
pm cent

Decrease

0.72
0.78
0.78
0.79
0.81

0.32
0.65
0.69
0.74
0.77

from

--

3.

45
90
92
96
98

22.0
44.0
44.9
46.9
47.9
7.8
15.6
22.9
40.1
44.5

63
118
125
125
125
36
51
70
114
125

16
32
47
82
91

82
178
203
226
245

40.0
87.0
99.2
10.4
19.6

0.44
0.63
0.67
0.72
0.73

0.71
0.76
0.74
0.77
0.78

0.79
0.83
0.90
0.98

of COZ.

ml.
mg.
wr ceni tP er cm

a
6
g

Series

Formation

226
245
250
250

and from

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246

CO, Lag in Fermentation

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from solution by yeast is much more rapid than the initial reaction
rate for the decomposition
of glucose to alcohol and CO,. The
rate during the first 5 minutes at which glucose is decomposed
is in Mixtures 1 and 3 only from 0.25 to 0.44 as fast as the rate
at which glucose disappears from solution.
In Mixture 2 the ratio of yeast to glucose is evidently so high
that the initial lag in COz formation
is more nearly overcome
in the first 5 minute period.
In this mixture the glucose decomposition during the first 5 minutes is 0.67 to 0.72 as much as the
glucose removal.
Further evidence of the initial lag in COz formation
is seen in
the fact that in Mixtures 1 and 3 actually less COz was formed
in the first 5 minutes of the reaction than in the second 5 minutes.
From comparison of the results from Mixtures 1 and 2 it is
evident that, with a constant amount of yeast present, the rate
of glucose disappearance
during the first 5 minutes is nearly
proportional
to the glucose concentration.
CO, formation,
however, does not show in its initial rate any proportionality
to glucose
concentration.
From comparison of Mixtures 1 and 3 it is evident that, with a
constant initial glucose concentration,
the initial rates of both
glucose disappearance and COz formation
increase with the concentration of yeast.
Our method of COz determination
measured the total CO2
content of the yeast-solution
mixture, cells as well as fluid.
It is
consequently impossible to ascribe the observed initial lag in CO2
formaticn
to retention of COz in the yeast cells.
Mixtures
containing
glucose, water, and yeast in the same
proportions
gave in the different series of experiments
varying
ates of both glucose disappearance
and CO2 formation.
This
variation
is attributable
to inconstancy
in the activity
of the
yeast used, Different
Fleischmann
yeast cakes were used for
each days experiments, and the activity
of the yeast in them
varied.
With all of them, however, the same lag in initial COz
formation
behind glucose disappearance
was observed.
In Table I the amounts of glucose corresponding to the observed
amounts of COz formed have been calculated in accordance with
the equation
CeHlzO~ = 2CzH50H + 2C0,.
This equation is
not quite exact, as small amounts of other products are formed,

J. A. Hawkins

and D. D. Van Slyke

SUMMARY.

The rate of removal of glucose from solution by yeast and the

rate at which glucose decomposes, as indicated by CO2 formation,
have been measured.
During the initial. stage glucose removal
takes place more than twice as rapidly as glucose decomposition.
BIBLIOGRAPHY.

Austin,
J. H., Cullen, G. E., Hastings,
A. B., McLean, F. C., Peters, J. P.,
and Van Slyke, D. D., J. Biol. Chem., 64, 121 (1922).
Slator, A., J. Chem. Sot., @, 129 (1906).
Van Slyke, D. D., and Hawkins,
J. A., J. Biol. Chem., 79, 739 (1928).
Van Slyke, D. D., and Hawkins,
J. A., J. Biol. Chews., 83, 51 (1929).
Van Slyke, D. D., and Neill, J. M., J. Biol. Chem., 61, 523 (1924).
Van Slyke, D. D., and Sendroy,
J., Jr., J. Biol. Chem., 73, 127 (1927).

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so that the glucose equivalent

of CO2 formed by fermentation,
even of indefinite length, is actually slightly greater than corresponds to the equation.
However,
multiplying
all the figures
in the last column of figures for each series by a value a few per
cent above unity would not change significantly
the interpretation of the results with regard to the lag in CO2 formation.
CO2
formation
begins more slowly than does disappearance of reducing
sugar, and CO2 formation
is still progressing at an appreciable
rate during the last half hour in the first two series, after glucose
removal from the solution has become complete.