CLIN.CHEM. 34/10, 2016-2018 (1988)
Some ThermodynamicParameters and Temperature-ConversionFactorsfor Determining
a-Amylase Concentrationin Serum
J. C. M. Hafkenscheldand M. Hesseis
We measured the relationship between measured activity
concentration and temperature for a-amylase (EC 3.2.1.1),
using 10 different substrates. At 25-37 #{176}C
the Arrhenius plot
was linear. The activation energy ranged from 33.3
kJ mol1 with maltoheptaose
as substrate to 54.4
kJ mor1 with the Blue-Starch method. Activation energy
was lowest for substrates having seven glucose moieties, the
ones most suitable for determining a-amylase--i.e., the
presence of more or fewer glycosyl units increased the
activation energy. Substrates with blocked groups at the
nonreducing end of the oligosacchande chain were not
considered here, because the relative reaction rates obtained with these substrates were less than those obtained
with nonblocked substrates. We also determined tempera-
ture-conversionfactorsfor a-amylase reactionwith the various substrates. The results are discussed in relation to
thermodynamicparameters of some other enzymes, e.g.,
creatine kinase and alkaline phosphatase.
Additional Keyphrases:
variation,source of
plots
activationenergy
enzyme activity
oligosaccharidesubstrates
Arrhenius
In the last two decades, interest
in the pathology of the
pancreas has increased enormously. Besides such technologically based techniques
as computer-assisted
tomography
and endoscopic retrograde
cholangiopancreatography,
determinations of the catalytic activity concentrations
of certain enzymes such as ca-aniylase (1,4-a-n-glucan
glucanohydrolase; EC 3.2.1.1), trypsin (EC 3.4.21.4), and lipase (EC
3.1.1.3) are also of value in diagnosing pancreatic disorders.
Numerous methods have been described for determining
a-amylase concentrations
in serum, urine, or duodenal fluid
(1-5). Polysaccharides
and oligosaccharides,
alone or coupled with 4-mtrophenol,
can be used as substrates
for aamyhase and can conveniently be purchased as kits from
manufacturers.
Nearly all activity concentrations obtained
with the different substrates
can be expressed
in UIL.
Here we describe the influence of temperature
on the
measured
activity concentration of a-amylase in serum from
normal
individuals, as determined
with 10 different
substrates.
MaterIals and Methods
Blood was taken by vempuncture
from normal individuals. After clotting, the blood was centrifuged
and the sera
were stored at -20 #{176}C
and promptly assayed.
Some sera
were pooled to obtain enough material for a-amylase
assay.
We used a Model 25 spectrophotometer
(Beckman
Instruments, Irvine, CA) for all experiments.
The variable tern-
perature-control
accessory
allowed us to warm the sample
and reference cell compartments so that catalytic
activities
could be measured at 25,27,30,32,35,37,40,42,
and 45#{176}C.
The changes in pH of the reaction mixture caused by
increasing
temperatures
were so small (0.05)
that no
corrections
were made.
The substrates 07, PG7, and EPS-PG7 were purchased
from Boehringer
Mannheim, Mannheim,
F.R.G.; 2-Cl-PG5
from Human, Taunusstein-Neuhof,
F.R.G.; 2-Cl-PG7 from
Merck, Darmstadt,
F.R.G.; PG5-PG6 from Behringwerke
AG, Marburg, F.RG.; G4 from Instruchemie,
Hilversum,
The Netherlands;
blocked starch and Phadebas Blue Starch
from Phadebas Diagnostic
AB, Uppsala,
Sweden; and BePG7 from bioM#{233}rieux,Charbonni#{232}res-les-Bains, France.1
The measurements
were carried out exactly according to the
manufacturers
instructions
except for the volume fraction
of sample. At each temperature
we used the same volume
fraction of sample, a compromise between those recommended for use at 25 #{176}C
and 37#{176}C,
to avoid matrix effects. The
observed change in absorbance per minute was multiplied
by a factor so that the results could be expressed in U/L. The
molar lineic absorbances (molar absorptivities)
incorporated
in this factor are temperature
independent
(NADH, 2chloro-4-nitrophenol)
except when the product
measured
was 4-nitrophenol,
in which case we used a different molar
lineic absorbance
at the different temperatures.
After calculating the catalytic
activity
concentrations
of
the serum samples at the indicated
temperatures,
we plotted them as hi catalytic
activity
vs l/T, where T is the
absolute temperature. The slopes of the Arrhenius plots and
the thermodynamic
activation
parameters
were calculated
as described by Rej and Vanderlinde
(6). Each plot is the
result of investigations
with several (usually nine) sera on
different days.
Results and DIscussion
In preliminary
experiments
we tested all substrates
between 25#{176}C
and 45#{176}C
and observed a linear Arrhenius plot
between 25#{176}C
and 37#{176}C.
At higher temperatures
there was
a deviation from linearity,
possibly caused by inactivation
or denaturation
of the enzyme. Therefore, we measured
the
temperature relationship from 25#{176}C
to 37#{176}C
at six different
temperatures.
Figure 1 shows characteristic
Arrhenius
plots for aamylase, obtained using three different substrates: G4, EPSPG7, and G. There is no significant
deviation from linearity, as was also true for the plots obtained on using the other
substrates. The different slopes indicate a range of temperature coefficients of activity.
‘Nonstandard
abbreviations:
G4, mahtotetraose; G7, mahtohep-
taose; 2-Cl-PG5, 2-chloro-4-nitrophenyhmaltopentaoside;
PG5-PG6,
4.nitrophenylmaltopentaoside/hexaoside;
PG7, 4-mtrophenylmalto-
Clinical Chemical Laboratory, St. Radboud Hospital, University
of Nijmegen, P.O. Box 9101, 6500 FIB Nijmegen, The Netherlands.
Received April 15, 1988; accepted June 8, 1988.
2016 CLINICAL CHEMISTRY,Vol. 34, No. 10, 1988
heptaoside; 2-Cl-PG7, 2-chloro-4-nitrophenylmaltoheptaoside;
EPSP07, 4,6-ethylidene-protected substrate-4-nitrophenylmahtoheptaoside; and Be-PG7, benzylidene-4-nitrophenylmaltoheptaoside.
activation
Ln activity (U/LI
5.0o
energy
kJ.moI
60
50
40
30
3.00
3.20
3.2/.
128
132
20
336
3
xl03tK
Fig. 1. Arrhenius plot for a-amyiasewiththreedifferentsubstrates
O-O 04: y = -6105x + 23; r: -0.9994
Ei-t
EPS-PG7: y = -4979x + 20; r -0.9980
#{149}-#{149}
G: y = 4012x + 18; r: -0.9916
Each plot is the mean of nine detesTninatiOnS
Table 1 lists the slopes of the Arrhenius plots and some
thermodynamic
activation parameters
of a-amylase. There
are large differences in Arrhenius slope, from -4012 K1 for
(37 as substrate to -6556 K’ for Blue Starch. Because the
slopes of the plots are different, the activation energy for
formation of the activated
complex is also different. We
calculated
activation energies of a-amylase ranging
from
33.3 to 54.4 kJ moF’, depending on the substrate used.
Our values agree with published values (7) for a-amylase
from human saliva or pancreas, 42.9 and 25.4 kJ mol’,
.
respectively.
Figure 2 shows the relationship
between the activation
energy and the number of glucosyl units in the substrate.
Table 1. Arrhenlus Slope and Some ThermodynamIc
Parameters of a-Amylase as Determined wIth 10
Different Substrates
Anhenlus
Substrate
G4
2-Cl-PG5
A.
167
(2.7)d
-4479 ± 543
-6105
b
kJmoI
slope, K’
±
kJmol1
50.6
±
1.4
48.1
±
1.4
37.2
±
4.5
34.7
±
4.5
40.9
±
2.9
38.4
±
2.9
41.3
±
1.7
38.8
±
1.7
(12.1)
PG5-PG6
EPS-PG7
-4930 ± 350
(7.1)
-4979 ± 205
4
5
6
7
8
9
number of glucosyl units
S
in
substrates
Fig. 2. Relationshipbetween activation energy of a-amlase andthe
numberof glycosyl units in the substrate
4 = G4;5
2-CI-PG5; 5.5 PG5-PG6;7 = G7;PG7and2-Cl-PG7; 9 = blocked
starch;and S = BlueStarch
=
=
The activation energy is lowest when a substrate with seven
glucosyl units is used ((37, PG7. or 2-Cl-P(37).
The structure of a-amylase has been described extensively (8). a-Amylase has extended binding sites for substrates
with four to nine glucose units. This enables the enzyme to
stress the substrate
and thus lower the activation energy for
hydrolysis. Our results make it clear that a substrate with
seven glucose units has the lowest activation
energy.
A
comparison
study by Wahlefeld (9) suggests that oligosaccharides with a chain length exceeding six glucose units are
best suited for the design of a-amylase
assays in human
specimens. For practical reasons PG7 is the most suitable
substrate, and it belongs to the group with the lowest
activation energy.
The relative reaction
rates for a-amylase
increase with
increasing
number of glucosyl units (10-12).
Therefore,
EPS-PG7 and Be-PG7 have not been included in Figure 2,
because the relative reaction rate obtained with both these
substrates is equivalent to that of a substrate with a chain
length of five or six glucosyl moieties (12). Both substrates
have no free glucose unit at the nonreducing end of the
chain. If the nonreducing end is modified, the action of the
indicator enzyme, a-glucosidase,
is blocked (9).
Table 2 shows the temperature
conversion factors of aainylase for the different substrates
used. Our results agree
(4.1)
Be-PG7
-5806
295
48.2 ± 2.4
45.7
±
2.4
07
(5.1)
-4012 ± 287
33.3 ± 2.4
30.8
±
2.4
38.3±3.2
35.8±3.2
±
(7.2)
388
PG7
-4617
2-Cl-PG7
(8.4)
-4490 ± 344
(7.7)
±
37.2
±
2.9
34.7
±
2.9
45.5 ± 1.7
43.0 ± 1.7
-5486 ± 207
(3.8)
54.4 ± 3.7
51.9 ± 3.7
-6556 ± 449
BlueStarch
(6.9)
avalues are expressed as mean ± 1 SD for nine determinations. bEA:
Blocked
starch
apparent energy of activation. C
of variation is given In parentheses.
apparent
enthalpy
changes.
dl5dent
Table 2. Temperature-ConversIon
Factors of a-Amylase
as Determined with Different Substrates
Substrate
30/25 ‘C
37/25 ‘C
37/30 ‘C
G4
1.42 ± 0.04
2.22 ± 0.05
1.57 ± 0.05
1.34 ± 0.11
1.73 ± 0.15
1.30 ± 0.06
2-Cl-PG5
1.36 ± 0.07
1.89 ± 0.07
1.39 ± 0.05
PG5-PG6
1.37 ± 0.02
1.90 ± 0.07
1.39 ± 0.06
EPS-PG7
1.40 ± 0.05
1.50 ± 0.07
2.12 ± 0.07
Be-PG7
1.33 ± 0.05
1.71 ± 0.06
1.29 ± 0.06
G7
1.35 ± 0.06
1.80 ± 0.10
1.33 ± 0.04
PG7
1.34 ± 0.04
1.78 ± 0.06
1.32 ± 0.04
2-Cl-PG7
Blocked starch
1.43 ± 0.06
2.04 ± 0.06
1.43 ± 0.05
1.43 ± 0.07
2.32 ± 0.13
1.63 ± 0.06
Blue Starch
a Valuesare expressed as mean ± 1 SD.
CLINICAL CHEMISTRY, Vol. 34, No. 10, 1988
2017
with those obtained by others (13). Because the slopes of the
different Arrhenius plots differ, it is obvious that the temperature conversion factors are not equal for the different
substrates-i.e.,
the temperature
conversion factors for aamylase obtained with one of these substrates
cannot be
used for another substrate.
It is well known that Km values for substrates
are
temperature
dependent. As shown in Figure 1 the Arrhenius relationships
are linear at 25-37 #{176}C,
indicating that the
changes in Km did not substantially
affect the measured
velocities. Thus zero-order
reaction rate is very important at
various
temperatures.
Furthermore,
activation energies
were determined
and calculated
for the total a-amylase
reaction,
including auxiliary and indicator enzymes. This
can cause differences in values for thermodynamic
parameters. Nevertheless,
the activation energy of a-amylase and
the number of glucose moieties are related.
Buhl et al. (14) observed a linear Arrhenius
relationship
for the isoenzymes of lactate dehydrogenase
(EC 1.1.1.27)
determined
in different buffer systems but otherwise with
the same reaction conditions. They found a difference in
activation
energy of lactate dehydrogenase
isoenzymes
1
and 5. Different activation energies have also been shown
for isoenzymes
of creatine kinase (EC 2.7.3.2) measured in
the same reaction mixture (15, 16). In contrast,
Rej and
Vanderlinde
(6) observed the same activation
energy for the
mitochondrial
and cytoplasmic
isoenzymes
of aspartate aminotransferase (EC 2.6.1.1). Copeland et al. (17) found different activation energies for the isoenzymes
of alkaline phosphatase (EC 3.1.3.1) by one method of determination
but no
difference by another.
We measured
the activation energy of two a-aznylase
isoenzymes,
pancreatic
and salivary, together in serum
under different reaction conditions, and large differences
were observed.
Results of activation energy and hence
temperature-conversion
factors must be handled
carefully
because different reaction methods
and the presence
of
isoenzymes can be responsible for different values for these.
We thank Prof. Dr. J. M. F. Trijbels and Dr. R. A. Wevers for
valuable discussions.
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2018 CLINICALCHEMISTRY, Vol. 34, No. 10, 1988
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