TEMPERATURE COEFFICIENTS OF ENZYMIC ACTMTY

AND THE BEAT DESTRUCTION OF PANCREATIC

AND M&T AMYLASES.*
BY DONALD H. COOK.
(FTo~ the Department of Chemislru, Columbia University, New York.)

(Received for publication, June 4, 1925.)

There are in the literature many references to the action of heat

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on enzymic processes. In general these are not specific, and where
they are they deal, as a rule, with o?e isolated point such as the
temperature of optimum activity or that of destruction. Not
much reliance can be placed on the data given in some of the
earlier work (1,2), as at that time very little was known as to the
conditions for the optimum activity of enzymes. The effect of
heat on enzymic activity through a wide range of temperature
has been little studied. As enzymes are, in general, unstable
substances, the effect of heat upon any reaction catalyzed by them
is made up of a twofold effect. Euler (3) points out that a rise in
temperature first accelerates the chemical velocity and second,
inactivates the enzyme. Thus in plotting curves of the activity
of an enzyme with increase in temperature the curve always tends
to rise to a maximum. Sgrensen (4) says: “Therefore, the curve
of the temperature should be Consideredas if it were two entirely
different curves, namely, a curve of the real temperature effect
which increases in proportion to the temperature elevation, not
only below but probably above the optimum temperature, and
on the other side a curve of the destruction of the enzyme.” The
maximum of this temperature curve varies according to the enzyme
employed, the acidity, concentration of the substrate, presence
of salts, type of solvent, etc. Much of the confusion arising from
apparently conflicting data in the literature is due to the lack of
fulfilling the conditions of bptimum activity of the enzyme,
* Published as Contribution No. 484 from the Department of Chemistry,
Columbia University, New York.
135
136 Pancreatic and Malt Amylases

especially in regard to hydrogen ion concentration and activating

salts. Much of the work of various investigators cannot be com-
pared quantitatively because of failure to insure the optimum
hydrogen ion concentration. At any other hydrogen ion concen-
tration the enzyme is less active and also, according to Liiers and
Lorinser (5), less stable. Sherman, Thomas, and Baldwin (6)
determined the hydrogen ion concentrations at room tempera-
ture which enable pancreatic and malt amylases and taka-diastase
to exert their optimum activities when acting upon soluble starch
at 40°C. for 30 minutes under definitely specified conditions.

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It has been st.ated as a general rule that for every 10°C. rise in
temperature, the velocity of a chemical reaction increases two- to
threefold. Harcourt and Esson (7) found for the liberation of
iodine from HI by HzOz that between 0” and 50” the velocity
was about doubled for ever.y 10” rise. Hudson and Paine (8),
working with invertase,, found the coefficient of destruction by
acids and alkalies was between 2 and 3 for every 10’ for the
interval 045”. Auld (9) working with amygdalin emulsion
showed a temperature coefficient of 2 for every 10” between 15-
35°C. Vernon (lo), using starch and amylase, found that the
velocity of hydrolysis was twice as great at 30’ as at 20”. Kendall
and Sherman (11) give 2 as the approximate temperature coeffi-
cient of pancreatic amylase for an interval of 10” between 20”
and 40”.
In the light of this previous work it appeared that it might be of
interest to study the effect of temperature not only on the speed
of hydrolysis but also on the rate of inactivation of two typical
starch-splitting enzymes. Pancreatic and malt amylases were
chosen for investigation.

Materials Employed.
The starch used in this work was Merck’s “soluble starch
according to Lintner.” It was washed nine times with distilled
water, six times with triply distilled water, and air-dried.
Ordinary distilled water was twice redistilled, first over alkaline
permanganate solution, then over very dilute solution of ortho-
phosphoric acid; the distillate was condensed in a block tin con-
denser. This product was always used for making up starch and
stock solutions and for the final rinsing of all glassware.
 Donald H. Cook 137
The primary and secondary phosphates used as buffers were
purified according to the unpublished methods of Beans and
Kiehl. The sodium chloride used was twice recrystallized from
triply distilled water and dried at 120°C.
The two enzyme preparations were commercial products:
Parke, Davis and Company pancreatin, laboratory sample No. 9,
and a barley malt obtained from the Froedtert Malting Co. The
malt extract was prepared by grinding 100 gm. of malt and extract-
ing with 250 cc. of triply distilled water at ice box temperature
for 2 hours. The extract was then filtered and kept in the re-

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frigerator in a steamed Nonsol bottle. The total solids and
reducing substances present in the extract were determined for
each new preparation. This stock solution of malt extract was
tested from day to day and did not change in activity during the
time of the experiments with it. The pancreatin was prepared
fresh for each day’s experiment and was made up by weighing out
50 mg. of the dry substance and making up to a definite volume
with a cold solution of phosphate of the required concentration.
The activities of these preparations were determined according
to the method of Sherman, Kendall, and Clark (12). The pan-
creatin had an average activit’y of 300 on their scale and the malt
extract an activit,y of 48.
EXPERIMENTAL.

1 per cent starch solutions were used as the substrates in these

experiments. They were adjusted to pH 7 for the work with the
pancreatin and contained 0.05 molar sodium chloride and 0.0005
molar disodium phosphate. In the experiments with malt
amylase the starch solutions contained 0.03 molar dihydrogen
phosphate and were adjusted to pH 4.5.
The measurements of pH were made either clectrometrically
or calorimetrically at room temperature (about 22°C.). Although
the above conditions, which were chosen because they had been
shown by Sherman and coworkers to give the best results with
these enzymes in tests of activity carried out at 40” for experi-
mental periods of 30 minutes, may not represent the optimum
conditions for testing the activities of these enzymes at other
temperatures and for different periods of time, they give a basis
for quantitative comparisons of the influence of temperature when
starting with the same standard initial conditions.
 Pancreatic and Malt Amylases
The direct tests of activity at the various temperatures were carried
out as follows: The starch solutions prepared as described above were
thoroughly mixed and poured into 100 cc. measuring cylinders which were
then immersed in the thermostat at the temperature at which the digestion
was to be carried out. 50 mg. of the enzyme preparation, in the case of
pancreatin, were then weighed out and made into a smooth paste with a
drop of cold water containing the same concentration of sodium chloride
and disodium phosphate as the starch solution. This was then washed
into a 100 cc. volumetric flask with the cold water-salt solution, made up
to the mark, and kept at 10” or colder until used. In the case of malt ex-
tract, 2.5 cc. of the extract were made up to 100 cc. with cold triply distilled
water without salts. Varying amounts of these enzyme solutions, de-

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pending on the activity, were pipetted with an accurately calibrated 1 cc.
pipette into dry 200 cc. Erlenmeyer flasks. The starch mixtures in the
cylinders were then poured into the flasks at 15 second intervals and the
flasks immersed in the bath for + hour. At the expiration of this period
50 cc. of Fehling’s solution were added to each flask at 15 second intervals
and in the same order in which the starch was poured on the enzyme. The
flask,s were then immersed in a boiling water bath and held at the boiling
point for 15 minutes. The cuprous oxide formed was rapidly filtered
through Gooch crucibles, washed with hot water, alcohol, and ether, dried
at UWC., and weighed. Maltose was calculated from Defren’s table.

The temperatures chosen were 20”, 30”, 40”, 50”, GO”, and 70°C.
As a rule five determinations were made simultaneously, four at
a lower temperature and one at a temperature 10” higher. Thus,
each new series of experiments overlapped the previous work and
was a constant check on the technique and the activity of the
enzyme. Blank experiments were made not only with the enzyme
preparations, but also with the starch and reagents in each series
of experiments.
As the presence of starch appears to exert a protective action
upon these enzymes it was also of interest to study the influence
of changes in temperature upon the enzymic activities when the
solutions were held at the various temperatures in the absence of
the substrate before being allowed to act upon it.
For these inactivation experiments the above procedure was modified
as follows: 300 cc. of a 2 per cent starch solution containing the molar con-
centrations of sodium chloride and sodium phosphate given above were pre-
pared and placed in 50 cc. portions in the thermostat. 300 cc. of a water-
salt solution containing the same concentrations of these salts were also
prepared. As both the water-salt solution and the starch solution
contained the same concentration of activating salts they would
give the standard 1 per cent substrate upon being combined. The water-
 Donald H. Cook
salt solution was divided into 50 cc. portions which were immersed in the
thermostats in test-tubes and after reaching the desired temperatures
were poured on the enzyme solutions at 15 second intervals. The enzyme
solutions were then placed in the thermostats and allowed to remain for
30 minutes. At the end of this time the starch solutions previously brought
to the same temperatures were added to the enzyme-water mixtures at 15
second intervals and in the same order as the water-salt solutions had been
added. The enzyme was allowed to act upon the starch for 30 minutes.
Fehling’s solution was then added and the rest of the procedure as out-
lined above was carried out. The method of overlapping experiments
from day to day was followed here in order to guard against any deteriora-
tion of the enzyme or other possible source of error. The 30 minute period

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of heating without substrate was, at the higher temperatures, shortened to
15, 10, and 5 minutes in order to follow the rapid rate of destruction of the
enzyme which was found to occur,

RESULTS.

As this investigation covered a wide range of temperatures it

was necessary to increase the quantity of enzyme used, at 20”
and 30”, in order to obtain enough cuprous oxide to weigh, while
at the higher temperatures the activity of the enzymes was so
increased that much smaller amounts were sufficient. In order
to have a basis for the comparison of results and the plotting of
curves it was decided to calculate the amounts of maltose formed
by a fixed quantity of enzyme. The amount of enzyme chosen,
which appeared to yield the most satisfactory values, was taken
as 0.1 mg. of the dry pancreatin or of the dry matter of the malt
extract. The data shown in Tables I and II were obtained in
this way and give the average amount of maltose per 0.1 mg. of
pancreatin or of the solids of the malt extract for each temperature.
The values given under the headings “Without previous heating”
in Tables I and II show the influence of temperature upon the
activities of pancreatic and malt amylases in the hydrolysis of
starch when acting upon the standard 1 per cent starch mixtures
described above for 30 minutes at temperatures ranging from 20” to
70°C. The other data given in Tables I and II show the influence
upon the enzymic activities of heating portionsof the enzyme solu-
tions in the absence of substrate in 50 cc. of the water-salt solutions
described above for 30 minutes at each of the temperatures before
allowing them to act upon the substrates at those temperatures.
As in the latter experiments the enzyme is rapidly inactivated at,
140 Pancreatic and Malt Amylases

the higher temperatures it was necessary to decrease the time of

preliminary heating of the enzyme in the absence of substrate.
TABLE I.
Experiments with Pancreatin.
(Average of all results calculated to 0.1 mg. of enzyme.
-
Temperature............................ 200 70”
. -
Without previous heating.
Cu,O, mg. 22.9 48.7 87.1 123.7 95.8 5.7
Maltose,mg.................. 18.3 39.1 70.5 100.2 77.5 4.5
After 30 min. heating.

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CuzO, mg.................... 23.3 48 9 68.8 0.0
Maltose,mg.................. 18.7 39.4 55.7 0.0
After 15 min. heating.
Cu,O, mg.................... 72 8 0.0
Maltose, my.................. 58.8 0.0
After 5 min. heating.
Cu,O, mg.................... 2.0
Maltose, mg.. .. 1.6

TABLE II.
Experiments with Malt Amylase.
(Average of all results calculated to 0.1 mg. of enzyme.)
-
Temperature.. . 200 409 500 60’ 70”
- -
Without previous heating.
CuzO, mg.. 4.5 84 13.6 17.9 13.7 0.7
Maltose, mg.. 3.6 6.7 10.9 14.4 11 .o 0.6
After 30 min. heating.
CurO, mg.. .I 4.4 7.5 10.6 13.5 0.5 00
Maltose, mg.. 3.5 6.0 8.5 10 8 0.4 0.0
After 15 min. heating.
CuzO, mg.. . . 14.3 2.0
Maltose, my.. 11.5 1.6
After 10 min. heating.
CunO, mg.. :. 3.6
Maltose, mg.. 3.0
After 5 min. heating.
CuaO, mg.. 6.8
Maltose, mg.. 5.5
-
This time was progressively decreased from 30 minutes to 15,
10, and 5 minutes. With these progressively smaller times
 Donald H. Cook 141
the rates of destruction at the lower temperatures became
unmeasurable.
From the data in these tables the rate of destruction of pan-
creatic and malt amylases under the conditions of these experi-

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0 5 lo 15 20 25
Time li, Minutes
FIG. 1. The upper curve shows the rate of destruction (inactivation)
of pancreatic amylase when heated in water-salt solution at 50” previous
to acting on starch. The lower curve shows the rate of inactivation under
the same conditions at 40”.
142 Pancreatic and Malt Amylases

ments as shown in Figs. 1 and 2 were calculated as follows: The

amount of sugar produced in the hydrolysis of the starch by the
enzyme solutions which had not had previous heating at a given
temperature was called 100 per cent. That produced after the

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5 /O /5 20 25 30
Time in Minutes
FIG. 2. The upper curve shows the rate of destruction (inactivation)
of malt amylase when heated in water-salt solution at 50” previous to acting
on starch. The lower curve shows the rate of inactivation under the same
conditions at 40’.
 Donald H. Cook 143

different times of preliminary heating was calculated to per cent

and subtracted from 100. The curves obtained from these values
therefore represent the relative velocity of inactivation of these
enzymes under these experimental conditions. At 50” the pan-
creatic amylase is about 97 per cent inactivated in 5 minutes
while the malt amylase has lost only 25 per cent of its original
activity after 4 hour. At 60”, which is much above the point
of complete inactivation of pancreatic amylase, the malt amylase
retains a slight activity even after 30 minutes.

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DISCUSSION.

It is of interest to find that increasing temperature influences

the hydrolytic activities of both these enzymes in a similar manner.
The values showing the activity of each obtained in the direct
tests of hydrolysis at each temperature rise to a maximum at 50”
with both enzymes. Evidently the increase of chemical velocity
is greater up to 50°, than the inactivation of the enzymes. Be-
yond 50°, however, the rate of inactivation under the conditions
of these experiments increases to such an extent that it becomes
t,he predominating factor and thus the values drop very rapidly.
The effect of increasing temperature upon the activities of the
enzyme solutions when they have been held without substrate
at the various temperatures previous to t,heir action upon starch
at those temperatures is also of interest.
With pancreatic amylase the activity is entirely lost upon 15
minutes heating at 50”, and at 40” the values obtained upon 15
and 30 minutes heating are much below the values obtained in the
direct tests of hydrolysis of starch at that temperature. This
would indicate that the rate of inactivation of this enzyme in-
creases rapidly with time and temperature. The results with
malt amylase while similar to those with pancreatic amylase are
less marked and indicate that malt amylase is much more stable
than pancreatic amylase. This is confirmed by unpublished data
from this laboratory and work carried on by Ernstrijm (13).
At temperatures sufficiently low where the inactivation of the
enzyme plays a small part, the ratio of the sugar formed by equal
amounts of enzyme at the lower temperature compared to that
formed at the next higher temperature is a fair measure of the
velocity of the action at these two temperatures. Thus for pan-
144 Pancreatic and Malt Amylases

creatin in the interval 2G30” the ratio is 2.1 and between 30” and
40” it is 1.8, while for malt at the same points it is 1.9 and 1.6.
From these result,s it would appear that the temperature coefficient
of the hydrolysis of starch by these two enzymes is about that of
inorganic reactions. These results are also in accord with the
data of other investigators as cited above. The velocity of the
inactivation of the enzyme is, however, much more rapid
t,han this; pancreatin, for instance, under the conditions of these
experiments is destroyed thirty times as rapidly at 50” as at 40’
if we compare times of equal destruction.
Malt amylase shows a greater stability toward heat, as the

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maximum value obtained in the inactivation experiments (Tables
I and II) lies at 50” instead of at 40” as is the case with pancrcatin.
It also seems that the presence of starch has less protective action
wit,h malt amylase than with pancreatic amylase. That this is
due to the lower activity of the malt amylase and the probable
presence of impurities that may act as protecting agents is hardly
a valid assumption in the light of preliminary experiments carried
out wit,h an old preparation of pancreatin of about one-third the
power of sample No. 9, which would presumably contain decom-
position products which would act as protective agents. This gave
identical points of maxima on bot,h the velocity and destruction
curves. It would seem that malt amylase is inherently more
stable than pancreatic amylasc.
There are two possible explanations.
1. The inactivation of the enzyme may be an hydrolysis. The
concentrat,ion of the enzyme is at all times very low. Even with
the commercial preparations worked with there is never more than
2 mg. of t,he enzyme preparation in t,he 50 cc. of water used in the
experiments upon heat destruction. It was first thought that
possibly the dilution was a factor of importance and a series of
tests were made in which the enzyme concentration was decreased
to one-half, and one-fourt,h, but no change in the rate of inactiva-
tion could be noticed. This was interpreted to mean that the
dilution was already so high t,hat hydrolysis was at a maximum.
Due to the activity of the sample and the rapid rate of starch
cleavage it was impossible to increase the concentration of the
enzyme much beyond 6he amount used. As was pointed out the
dcst,ruction of pancreatic amylase at 50” in these experiments
 Donald H. Cook 145

was much more rapid than the ordinary hydrolytic reactions which
have been studied previously-Arrhenius (14).
2. If the amylases are protein in nature it is possible that their
inactivation might be due to heat coagulation, which, with most
proteins, occurs between 50” and 70”. Sherman and Schlesinger
(15), working with purified preparations of pancreatic and malt
amylases, found a faint opalescence in their solutions at 50”, with
increasing coagulation up to 70”, at which point, after filt.ering,
no further precipitation would occur, even at high temperatures.
It is quite possible that the previous formation of this coagulum

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would, on the later addition of starch, prevent any action of the
enzyme. On the other hand, since enzyme action is probably a
surface reaction, in the presence of the st,arch the enzyme at coagu-
lation temperatures would be precipitated on the starch molecule,
and though its activity would probably be less, still the action
would continue beyond the point where it could react with the
substrate after a preliminary heating that brought about coagula-
tion. This theory is in accord with the evidence shown.
From the data at hand neither theory is susceptible to proof.
The work of Sherman, Walker, Caldwell, and Naylor (16, 17, 18)
on the protective action of certain amino acids on the amylases
supports the theory that hydrolysis plays an important role in
the heat destruction of the enzymes, but this is entirely con-
sistent with the view that there may also be destruction by heat
coagulation.
SUMMARY.

The rates of starch hydrolysis by pancreatic and malt amylases

used in the forms of good grades of commercial pancreatin and
malt have been determined under certain specified conditions for
the temperature range of 20” to 70°C. At temperatures below
the point where destruction of the enzyme plays an important role
the rate of hydrolysis is about doubled for every 10” rise
in temperature.
The temperature and rate of destruction of these enzymes in
water-salt solutions have been determined and malt amylase is
found to be much more stable than pancreatic amylase, the latter
being completely destroyed in 15 minutes heating at 50”, while
malt amylase still shows a trace of activity after 30 minutes at
146 Pancreatic and Malt Amylases

60°C.; pancreatic amylase is apparently inactivated approximately

thirty times as fast at 50’ as is malt amylase.
The results obtained for the rate of destruction of these enzymes
show a wide divergence from those giving the rates of destruction
of vitamins B (19) and C (20), and make it appear doubtful
that any advantage is to be gained by classing vitamins as en-
zymes as has sometimes been suggested.
The results obtained support the view that the heat destruction
of the enzyme may be a process of the nature of the coagulation
of a protein, probably accompanied by partial hydrolysis also.

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BIBLIOGRAPHY.

1. Klempin, P., Biochem. Z., 1908, x, 210.

2. Biedermann, W., Fermentjorschung, 1914-16, i, 385,474; 1919, ii, 458.
3. Euler, H., General chemistry of the enzymes, translated from the re-
vised and enlarged German edition by Pope, T. H., New York and
London, 1st edition, 1912, 235.
4. Sorensen, S. P. L., Compl. rend. trav. lab. Carlsberg, 1909-10, viii, 5.
5. Liiers, H., and Lorinser, P., Biochem. Z., 1922, cxxxiii, 487.
6. Sherman, II. C., Thomas, A. W., and Baldwin, M. E., J. Am. Chem.
Sot., 1919, xli, 231.
7. Harcourt, A. V., and Esson, W., Phil. T T . , 1867, clvii, 117.
8. Hudson, C. S., and Paine, H. S., J. Am. Chem. Sot., 1910, xxxii, 985.
9. Auld, S. J. M., J. Chem. Sot., 1908, xciii, 1275.
10. Vernon, H. M., J. Physiol., 1901-02, xxvii, 190.
11. Kendall, E. C., and Sherman, H. C., J. Am. Chem. Sot., 1910, xxxii,
1087.
12. Sherman, H. C., Kendall, E. C., and Clark, E. D., J. Am. Chem. Sot.,
1910, xxxii, 1073.
13. ErnstrGm, E., Z. pl~ysiol. Chem., 1922, cxix, 191.
14. Arrhenius, S., Quantitative laws in biological chemistry, London, 1915,
Chapters 2 and 3.
15. Sherman, H. C., and Schlesinger, M. D., J. Am. Chem. $‘oc., 1912: xxxiv,
1104; 1915, xxxvii, 1305.
16. Sherman, H. C., and Walker, F., J. Am. Chem. Sot., 1919, xli, 1866;
1921, xliii, 2461.
17. Sherman, H. C., and Caldwell, M. L., J. Am. Chem. Sot., 1922, xliv,
2923,2926.
18. Sherman, H. C., and Naylor, N. M., J. Am. Chem. Sot., 1922, sliv, 2957.
19. Sherman, H. C., and Grose, M. R., J. Am. Cl&em. Sot., 1923, xlv, 2728.
20. La Mer, V. K., Campbell, H. L., and Sherman, H. C., J. Am. Chem.
Sot., 1922, xliv, 172.
 TEMPERATURE COEFFICIENTS OF
ENZYMIC ACTIVITY AND THE HEAT
DESTRUCTION OF PANCREATIC AND
MALT AMYLASES
Donald H. Cook
J. Biol. Chem. 1925, 65:135-146.

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