Differentiation of Trypsin-LikeEnzymes in Human Plasma
Herbert D. Gullick
The arginine amidase and arginine esterase activity of human plasma is compared with that of
trypsin,
thrombin,
plasmin,
and kallikrein
by
using the homologous
synthetic amino acid sub-
strates, benzoyl arginine amide (BAA) and benzoyl
arginine ethyl ester (BAEE). Hydrolyses of BAA
and BAEE in plasma are distinguished by several
features: pH optimum, heat stability, storage
stability, effect of dilution, surface contact activation, streptokinase activation, inhibition by proteolytic enzyme inhibitor, and range of normal variation. The differential sensitivity of trypsin, throm.
bin, plasmin, and kallikrein toward these substrates and the similarity in reaction characteristics of plasma-BAA and trypsin-BAA provide
evidence that the arginine amidase activity of
plasma represents trypsin, while the arginine
esterase activity of plasma is nonspecific.
Additional Keyphrases
coagulation
#{149}
pancre-
atitis
#{149} plasma
kinins
#{149} kallikrein
#{149} streptokinase
#{149} benzoyl
arginine amide and benzoyl arginine
ethyl ester as substrates
#{149} amylase
#{149} esterase
lipase
#{149} plasmin
#{149} complement
#{149} throm bin
inhibitor in plasma
#{149} normal
values
The specificity
of measurements
of proteolytic
enzymes in blood purported
to be trypsin has been
debated for many years. The question of identity of
the trypsin-like
enzymes
in plasma
or serum is
important
for several
reasons.
First,
in vitro
studies and injections
of trypsin in animals have
demonstrated
that trypsin activates blood coagulation (1-6), fibrinolysis
(7-9), and the formation
of
plasma kinins (10-13), which produce vasodilata-
From the Department
of Surgery, State University
of New
York Upstate Medical Center; and the Veterans Administration
Hospital, Syracuse, N. Y. 13210.
Received April 6, 1972; accepted Aug. 17, 1972.
tion, hypotension,
smooth muscle contraction,
and
increased
capillary
permeability.
Second, on the
basis of the above, many of the manifestations
of
clinical
and experimental
pancreatitis,
such as
hypotension
or shock and abnormalities
of hemostasis, are postulated
as being due to the release of
trypsin
or trypsin-like
enzymes
into body fluids
(14-18). Third, of the three major groups of exocrine pancreatic
enzymes-amylolytic,
lipolytic,
and proteolytic-only
two, amylase and lipase, are
widely used in clinical diagnosis;
since abnormal
elevations
of blood enzymes in pancreatic
disease
do not always occur in parallel, a reliable test for
trypsin,
as the most prominent
of the proteolytic
group
of enzymes,
should
enhance
diagnostic
capability.
The lack of specificity
of natural
protein
substrates
for measuring
blood enzymes
led to the
introduction
of synthetic
substrates
with characteristic
chemical groups susceptible
to hydrolysis
by certain enzymes and insensitive
to the action of
other enzymes.
Trypsin
hydrolyzes
argiine
and
lysine bonds. Several synthetic
amino acid substrates
with the requisite
features
for trypsin
hydrolysis
have been prepared.
Enzymes
often
exhibit multiple
substrate
specificity
and certain
substrates
cross-react
with
different
enzymes.
Consequently,
in biologic fluids that contain
a
mixture
of proteases
and pre-enzymes,
enzyme
differentiation
may depend on the physicochemical
characteristics
of various enzyme-substrate
combinations.
Also, spontaneous
blood enzyme activity
must be distinguished
from in vitro activation
of
enzyme precursors.
The term “trypsn-like”
refers to enzymes that
hydrolyze
the same peptide
linkages
as trypsin.
These enzymes are in the general class of peptidyl
peptide
hydrolases
(EC 3.4.4).1
The substrates
1 Enzyme
Nomenclature,
Recommendations
of the International Union of Biochemistry,
Elsevier, New York, N. Y., 1964.
CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972 1385
that have been most commonly
used in clinical
studies of trypsin-like
activity
in human plasma
and serum are the methyl ester of toluene sulfonyl
argiine
(TAME),2
the ethyl
ester
of benzoyl
argiine
(BAEE),
and the amide of benzoyl arginine
(BAA).
The esterase
assays are usually preferred
because
of their sensitivity
and convenience.
In
addition
to trypsin
(EC 3.4.4.4),
proteolotyic
enzymes
in blood
known
to exhibit
arginine
esterase
activity
include thrombin
(EC 3.4.4.13)
(19-21),
plasmin (EC 3.4.4.14) (20, 22, 23), kallikrein (EC 3.4.4.21)
(24-27),
C’!,, component
of
complement
(28), and clotting
factors
Xa (29)
(thrombokinase)
and XI (PTA) (30). Information
is
not available
regarding
the arginine
amidase
activity of all the above enzymes, but it is generally
agreed that amide substrates
are less sensitive than
esters for all enzymes thus far studied-including
trypsin,
thrombin,
and plasmin (19, 22, 31, 32).
The studies in this paper compare
the spontaneous
occurrence,
activation
behavior,
reaction
characteristics,
and inhibition
of BAEE esterase and
the BAA amidase of normal human plasma, and are
directed toward enzyme identification
by reference
to the esterase
and amidase
activity
of trypsin,
thrombin,
plasmin,
and kallikrein.
Evidence
is
presented
that the amidase
activity
of plasma
represents
trypsin, while plasma esterase activity
is apparently
nonspecific.
Materials and Methods
Plasma Specimens
Venous
blood samples
(lithium
oxalate
anticoagulant)
were obtained from hospital and laboratory personnel
and the plasma was separated
by
centrifugation.
Except where noted in special experiments
with polyethylene
vessels, blood samples
were collected and assayed in glass tubes. Enzyme
assays were performed
on the same day the blood
was drawn except in experiments
designed to study
the effect of storage.
Reagents
Enzymes.
These were trypsin
(“Tryptar”;
Armour Pharmaceutical
Co., Chicago,
Ill. 60690),
thrombin
(“topical
bovine
thrombin”;
Parke,
Davis and Co., Detroit,
Mich. 48232), kallikrein
(10 KU/mg; Nutritional
Biochemicals,
Cleveland,
Ohio 44128),
and
streptokinase
(“Varidase”;
Lederle
Laboratories,
Pearl River, N. Y. 10965).
2
Nonstandard
abbreviations
used:
B-tA,
a-benzoyl-L-arginine
amide
methyl
hydrochloride
monohydrate;
BAEE,
a-benzoyl-r,-arginine
ester;
TAME,
toluene
sulfonyl
arginine
methyl
ester;
BANA,
benzoyl-arginine--naphthylamide;
LME,
lysine methyl
ester;
sK, streptokinase;
nu, kallikrein
units;
atu,
kallikrein
inhibitor
units.
1386 CLINICAL CHEMISTRY. Vol. 18, No. 11, 1972
All enzyme solutions
were prepared
with distilled
water and used the same day.
Try psin-kallikrein
inhibitor.
Aprotinin
(“Trasylol”; FBA Pharmaceuticals,
New York, N. Y.)
Substrates.
a-Benzoyl-L-arginine
amide hydrochloride
monohydrate
(BAA)
and
a-benzoyl-Larginine
ethyl ester (BAEE)
were obtained
from
Mann
Research
Laboratories
(now Schwartz/
Mann, Orangeburg,
N. Y. 10962).
Enzyme Assays
Argiine
amidase activity
was determined
by a
modification
(33, 34) of the procedure
described
by
Nardi (35, 36) from a method proposed by Schwert
et al. (31). The ammonia
formed by hydrolysis
of
BAA
is liberated
by contact
with alkali (K2C03),
absorbed
by boric acid, and titrated
with hydrochloric
acid by a modification
of the Conway
microdiffusion
and titration
technique
(37). The
buffer in this assay is sodium and potassium
phosphate,
p11 7.8. Results
are expressed
as nanomoles of ammonia released per milliliter of sample
per hour3 incubation
time (nmol/ml
per hour)
With this method,
there was proportionality
with
enzyme
concentration
at all concentrations
of
trypsin used in this study. Precision of the assay at
high and low levels of activity was as follows: eight
replicate measurements
at 21#{176}C
of a single normal
plasma sample was 19 ± 5.95 nmol/ml
per hour
(relative standard
deviation,
31%). The standard
deviation
is less than 8 nmol/ml
per hour, which
represents
a 0. l-l HC1 titer, the minimal titration
value for the method.
At higher values with 30
,ug/ml
crystalline
trypsin, eight replicate
measurements at 21#{176}C
gave 487 ± 12.4 nmol/ml
per hour
(relative standard
deviation,
2.5%).
Arginine
esterase
was determined
by a colonmetric method
described
by Brown (38), which
measures
residual
substrate
after hydrolysis
by
disappearance
of a colored complex (iron argiine
hydroxamate)
formed by the reaction of BAEE with
alkaline hydroxylamine
and acidic ferric chloride.
The buffer used in this assay is sodium diethylbarbiturate
HC1 (barbital),
pH 7.8. Results
are
expressed
as micromoles
of substrate
hydrolyzed
per milliliter
of sample per hour incubation
time
(umol/ml
per hour).
The
capability
of this
method
is limited by amount
of substrate
to 40
zmo1, proportionality
with enzyme concentration
‘The enzyme
unit for BAA activity
is expressed
per hour rather
than per minute
for the following
reasons:
(a) to avoid decimal
places in the low values found in normal
plasma,
(b) the standard
assay is 1 h incubation
time. The reaction
of plasma
with BAA
is slow; kinetic
assays must be done in periods
of 30 mm to 1 h
rather than in minutes.
Thus, expressing
results as per hour rather
than per minute
seems appropriate
for this assay.
Reaction
of
plasma
with BAEE is more rapid and could possibly
be expressed
in per minute,
BAA
and
BAEE
but we retained
for more direct
the expression
comparisons
per hour for both
of substrate
utilization.
being maintained
to about
30 imol.
Routine
incubation
time with this procedure
was 1 h, but
when high results were anticipated,
the incubation
time was shortened
accordingly.
Experiments and Results
pH Optimum
One molar HCl or 1 molar NaOH was added to
the buffer and substrate
solutions
individually
to
reach about the indicated
pH. Addition
of plasma
to the buffer-substrate
mixture somewhat
altered
this initial pH, which was then adjusted
with one
or two drops of acid or alkali to the precise pH
desired for the incubation
mixture. There was autohydrolysis
of BAEE above pH 8.5 if the substrate
solutions were left standing.
Consequently,
the pH
of the BAEE
substrate
and buffer solutions
was
adjusted
immediately
before
use. Some autohydrolysis
still occurred but this was compensated
in the enzyme assays by blank samples, which were
processed
simultaneously.
There
was no autohydrolysis
of BAA.
The pH curves in Figure 1 are the mean values of
plasma BAA and BAEE activity obtained from three
separate
runs, each run consisting
of the pooled
plasma of three normal subjects. Each plasma pool
had a highly uniform response
to changes in pH
with BAA,
but a rather
variable
response
with
BAEE.
Plasma BAA activity
has a sharp pH optimum at 7.8, identical to that of crystalline
trypsin
(Figure
2), with a narrow
range of detectable
activity
between
6.5 and 8.5. The plasma
BAEE
reaction has a broad range of activity,
from pH 5.5
to 10.0, with an ill-defined optimum.
Fig. 2. The effect of pH on arginine amidase activity of
crystalline trypsin
Incubation temperature of enzyme assay, 21#{176}C
Fig. 3. Effect of heat on arginine amidase and esterase
activity of plasma
The same plasma samples were assayed at 25#{176}C
with
BAEE before and after heating at 60#{176}C
for 15 mm
Heat Stability
Figure
3 shows the heat stability
dividual plasma samples simultaneously
BAA
and BAEE activity before and after
of five intested for
heating the
PLASMA
BAA
BAEE
pmol/ml/h
Is
-
nmol/ml/h
75
--
4
10
/1#{176}t0
1
5
o
pH
BAEEj
50
!\BAA
-
#{149} -
I
2
.
3
4 5 6 7
pH ot 30C
8
9
25
0
10
Fig. 1. Effect of pH on arginine amidase and esterase
activity of normal plasma
BAA
and
plasma
at 60#{176}C
for 15 mm. After heating,
the
sample tubes were immediately
placed in a cold
bath for 20 s and then assayed
at 25#{176}C.
BABE
activity
was totally eliminated
by heating,
while
BAA
activity
was preserved,
actually increasing
in
four of the five samples. Table 1 shows the effect of
heating at this same temperature
on the esterase
activity
of plasmin
generated
by streptokinase
activation
of plasma; 87% of the plasmin activity
was destroyed.
Storage Stability
Aliquots
of individual
plasmas
were placed in
glass tubes, capped, and stored for four days at
-15#{176},4#{176},
21#{176},
and 37#{176}C.
To avoid possible effects
of repeated freezing and thawing, samples for each
day’s test were stored in separate
tubes, so that
only one tube was removed from storage each day.
Six to 10 individual
plasma samples stored at each
CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972 1387
Table 1. Effect of Heat on Esterase Activity of Five
Streptokinase Activated Plasmas
BAEE activity
Stroptokinase
Before
heating
Control
PLASMA
BAA
ACTIVITY
s,.,..
120
.10
activated
plasma
After heating
(60#{176}C,
15 mm)
6.4
8.0
4.0
48.0
12.0
21.6
48.0
62.4
48.0
12.0
9.6
50.1 ± 7.1
13.9 ± 4.6
5.2
Mean ± SD
6.2 ± 1.6
20
10
0
0
.10
20
.H
Ep
‘
.i.I0
14.4
I0
.1
.05
44.0
ACTIVITY
.05
at 25#{176}C.
umoi/mI/h
7.2
BAEE
PLASMA
Th.p.,.,.
06
.,.11II
.05
a
20
10
0
1’
17
20
10
0
S’e#{176}8. Tim.
of the four temperatures
were assayed
daily for
BAA
and BAEE
hydrolysis
activity.
The results
are shown
in Figure
4. Plasma
BAA
activity,
after declining the 1st day, increased
on the 2nd
day to approximately
twice the control
value,
decreased again on the 3rd day, and disappeared
by
the 4th day. This spontaneous
reactivation
on the
2nd day was of the same magnitude
at each storage
temperature.
The enzyme activity on the 2nd day
was statistically
significantly
higher than every
other day at all storage
temperatures
(P <.05,
<.01 or <.001 each day)-except
on the 2nd day
at 37#{176}C,
which was not significant,
owing to one
plasma sample that peaked on the 3rd day rather
than the 2nd day. Plasma
BAEE
hydrolysis
activity was not affected by storage at these temperatures except for a decline on the 1st day when
stored at 37#{176}C.
The same phenomenon
of increased
activity
on
the 2nd day was exhibited by crystalline
trypsin at
four concentrations
(15, 30, 50, and 100 tg/ml)
when stored at 4#{176}C
and assayed with BAA. In one
experiment
trypsin
solutions
were made in distilled water.
In another,
trypsin
was added to
human plasma, the plasma first being diluted 50f old with buffer. The difference in enzyme activity
depicted on the two scales in Figure 5 indicates the
amount of trypsin inhibited
by the plasma. Despite
the fact that plasma at this dilution has no spontaneous
BAA
hydrolysis
activity
and contains
sufficient
inhibitor
to neutralize
20-40
g
of
trypsin per milliliter, free enzyme activity remained
after addition of even small quantities
of trypsin to
plasma. This illustrates
protein binding of trypsin
with protection
of enzyme
activity
despite
the
presence of excess inhibitor
(39). It is also apparent
that
the endogenous
plasma
inhibitor
did not
prevent the increase in activity on the second day
when trypsin was added.
0os
at four different
temperatures
on plasma arginine amidase and esterase activity
Incubation
temperature
of enzyme assay, 21#{176}C.
The mean and
standard deviation of values for each day are indicated. n
=
number of plasma samples
TRYPSIN
TI1YPSIN
BAA
ACTIVITY
TRYPSIN / PLASMO
I pig.m dilSed I’501
/ 620
SIo,oge
T.mp,,Olu,e
4C
1.10
1,60
.90
.40
.70
.20
1.00
6
.12
.80
.60
.08
.06
.40
30 pg/mI
.04
.20
30
I5 pg/mi
I
2
3
Se,og.
4
Tim.
0
I
2
.02
p8/mI
.g/mi
3
4
S Dy
Fig. 5. Effect of storage at 4#{176}C
on amidase activity of four
concentrations of crystalline trypsin
Left, trypsin in distilled water; right, the same concentrations
of trypsin added to dilute plasma. Incubation temperature
of
enzyme assay, 21#{176}C
PIizsmg
Dilulion
Fig. 6. Effect of dilution on amidase and esterase activity of plasma
Plasma Dilution
Figure 6 depicts
plasma with saline
hydrolytic
activity.
Fig. 4. Effect of storage
i,
the effect of prior dilution
of
(0.9 g/liter,)
on BAA and BAEE
The results are given as per-
1388 CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972
Plasma was diluted with normal saline. Standard volumes of
the dilute plasma were then used as called for in the enzyme
assays. Incubation for 1 h in glass tubes at 25#{176}C.
Values represent the mean of three experiments with pools of three plasma
samples each
cent of maximum
activity and represent
the mean
of three experiments,
with pools of three plasma
samples in each experiment.
With both BAA and
BAEE,
undiluted
plasma
had only 45% of the
activity
attained
after dilution.
However,
the
maximum
activity
with BAA occurred
at twofold
dilution and with BAEE at 20-fold dilution. Plasma
activity with the two substrates
contrasted
sharply
at 20-fold dilution,
because
at this point BABE
activity
was at its maximum
and BAA activity
had totally disappeared.
Surface
Contact
Activation
The
effect of surface
contact
on BAA
and
BAEE
activity
of plasma
and of crystalline
trypsin was determined
by incubation
of the test
substance
for several
hours in either
glass or
plastic
tubes. Plasma
to be incubated
in plastic
tubes
was handled
throughout
without
glass
contact-blood
was drawn in plastic syringes, and
plastic tubes were used for centrifugation
and incubation.
Trypsin solutions were handled similarly.
The data on the left side of Figure 7 represent mean
values of three separate
experiments,
such experiment composed
of a plasma pool from three subjects. The data on the right side of Figure 7 represent mean values from three separate
experiments
with crystalline
trypsin.
Completely
opposite
response of plasma on contact with glass and plastic
tubes was exhibited
with BAA and BABE substrates.
A linear reaction
with time was obtained
with
plasma BAA activity in glass tubes, and with plasma
BAEE
activity in plastic tubes. At the usual 1-h incubation
time used in clinical assays, the BAEE hydrolysis
activity
of glass-contacted
plasma
(7.1
/2mol/ml per hour) was 2.5 times that of plasma
that had not been in contact with glass (2.9 mol/mi per hour). The response of crystalline
trypsin,
as measured
with both BAA and BAEE, was similar
to that of plasma BAA, showing linear activity
in
glass tubes, but increasing
activation
with time in
plastic tubes.
Comparison
of BAA and BAEE Activity
of Purified
Proteolytic Enzymes and of Human Plasma
A comparison
of the amidase
and esterase
activity
of purified
trypsin,
thrombin,
and kallikrein is shown in Figure
8. Concentrations
of
these three enzymes showing equivalent
activities
with BAEE
substrate
(solid line, Figure 8) were
assayed
for BAA activity.
All enzymes
showed
greater
activity
toward
BAEE
than
toward
BAA.
Trypsin
is the only enzyme showing linear
activity
with BAA at all concentrations
having
BAEE
activity
(broken line, Figure 8). Thrombin
and kallikrein
are relatively
insensitive
to BAA
and no activity was obtained
at less than 50 NIH
PLASMA
C*YSTALLINC
TRYPSIN
30
P10.1*,
20
RAE!
0,,’
20
10
2!
IC
HOURS
INCUBATION
Fig. 7. Effect of incubation in glass or plastic tubes on
amid ase and esterase activity of plasma and of crystal.
line trypsin
Incubation temperature of enzyme assay, 21#{176}C.
Plasma reaction
with BAEE was limited to 2.5 h because of disappearance
of
substrate beyond hydrolysis of 30 mol
6
6
6
A
a
a
#{149}#{149}#{149}
A.
0
0
20
30
40
0
0
20
30
40
50
60
70
80
90
00
0
I
2
3
4
0
6
7
8
9
0
Th,Rn,bIo. NIH UiI./mI
K0111k,**,,Unlt,/mI
Fig. 8. Comparison of arginine amidase and esterase
activity of purified trypsin, thrombin,
and kallikrein
The abscissa scales show concentration
of the enzymes which
have equivalent
BAEE activity
(solid line). The linear reaction
of
trypsin with BAA isshown by the broken line. The ordinate
scales are constructed on a BAEE:BAA
ratio of 100:1. Incubation
temperature of enzyme assays, 25#{176}C
units per milliliter
of thrombin
or 5 KU per
milliliter
(500 tg/m1)
of kallikrein.
Above these
concentrations
activity with BAA was linear and the
BAEE : BAA
ratio
for the three
enzymes
was:
trypsin, 80; thrombin,
1000; and kallikrein,
1250.
A fourth enzyme,
plasmin,
was also measured
with BAA and BAEE as substrate.
The source of
plasmin was whole plasma activated
with streptokinase (U, U), 1000 units/mI.
Figure 9 shows the
mean results obtained
with four plasma samples
simultaneously
assayed for BAA and BAEE activity.
Plasma BABE activity
increased
fivefold after 5K
activation
(11.0 to 51 .imol/ml per hour), while BAA
activity did not change (24 to 30 nmol/ml per hour).
A separate experiment
with five other plasma samples showed an eightfold
increase
in mean BABE
hydrolysis
with SK activation
(6.2 to 50.1 jimol/ml
CLINiCAL CHEMISTRY,
Vol. 18, No. 11, 1972 1389
were about
equivalent
to the published
upper
limit
of normal
plasma
BAA
activity
of 80
nmol/ml
per hour at 21#{176}C
(33), and the upper
limit of normal serum BAEE activity of 30 mol/mi
per hour at 37#{176}C
(38, 40) (upper limit of plasma
BAEE
activity
at 21#{176}C
is about 16 ,mol/ml
per
hour). Concentrations
of Trasylol that completely
inhibited
this activity
of crystalline
trypsin
were
added to human plasma, which was then assayed
for residual
activity.
Inhibitor
and enzyme
or
plasma were incubated
together for 30 mm before
assaying.
A similar
experiment
was conducted
Effect of Trypsin-Kallikrein
Inhibitor on BAA and
with SK-activated
plasma to determine
the activity
BAEE Activity of Purified Enzymes and of Plasma
of plasmin inhibited
by Trasylol.
The bottom half
The effect of the trypsin-kallikrein
inhibitor,
of Figure 10 shows that Trasylol
does not inhibit
“Trasylol,”
on the amidase and esterase activity
either BAA or BAEE activity of thrombin,
but is an
of trypsin, thrombin,
kallikrein, plasmin, and whole
effective inhibitor of trypsin and kallikrein
as meahuman plasma is shown in Figures 9 and 10. Consured with either substrate.
The sensitivity
of
centrations
of trypsin,
thrombin,
and kallikrein
these two enzymes
to inhibition
parallels
their
were chosen from the data shown in Figure 8 that
substrate
sensitivity.
Thus, BABE is more sensitive
to kallikrein
than to trypsin, and the BABE hydrolytic activity of kallikrein is more sensitive to inhibition than is trypsin.
With BAA substrate,
the reverse is true: BAA is more sensitive to trypsin than
to kallikrein,
and trypsin-BAA
activity
is more
readily
inhibited
than
kallikrein-BAA
activity.
6
Figure 9 shows that plasmin (sK-activated
plasma)
is only partly inhibited
by Trasylol, losing 30% of
its activity.
In terms of BAEE units inhibited
(15
imol/ml
per hour), this represents
only one-half
the esterase activity of trypsin and kallikrein
that
were inhibited
(30 mol/ml
per hour) by the same
TRASYLOL,
KI.U /mI
amount of Trasylol.
The top half of Figure 10, representing
the mean
Fig. 9. Effect of trypsin-kallikrein
inhibitor, Aprotinin,
results from three individual
plasmas, shows that
on arginine amidase and esterase activity of streptokinase-activated plasma
plasma-BAA
activity
is totally
inhibited
by 75
KIu/ml
of Trasylol
while plasma-BABE
activity
Incubation
temperatureofenzyme assay in glasstubes,25#{176}C.
C = control plasma before additionof streptokinase;o = enzyloses only 17% of its activity with 100 KIU/ml.
per hour) (Table 1). BAA is essentially
insensitive
to plasmin, with a BAEE : BAA activity ratio of over
6000.
At enzyme concentrations
measurable
with BAA,
the relative amidase activity
of the four enzymes
is: trypsin,
1.000; thrombin,
0.115;
kallikrein,
0.057;
and plasmin,
0.013. The amidase
activity
of trypsin
is thus 10 times that of thrombin,
20
times that of kallikrein,
and 80 times that of plasmm.
C
matic activity
streptokinase,
of piasmmn generated from whole plasma
1000og/mI, before addition of inhibitor
by
Variation
of Normal Plasma BAA and BAEE Activity
The individual
variation
in normal plasma BAA
activity
is twice that of BABE
activity.
Eleven
plasma samples simultaneously
assayed with both
substrates
at 25#{176}C
in glass tubes showed a relative
standard
deviation
of 81.4%
with BAA (mean,
31.2; SD, ±25.4 nmol/ml
per hour) and 42.8%
with BAEE (mean, 8.4; SD, ±3,5 umol/ml per hour).
Sixteen other plasmas
independently
assayed
at
various times at 21#{176}C
showed a relative standard
deviation
of 75% with BAA (mean, 22.5; SD, ± 16.8
nmol/ml
per hour) and 29% with BABE
(mean,
9.3; SD, ±2.7 mol/ml
per hour).
T,20I0I.
K
IU / ml
Fig. 10. Effect of trypsin-kallikrein
inhibitor, Aprotinin,
on the spontaneous
activity of plasma arginine amidase
The arginine
amidase
(BAA
as substrate)
and
esterase
(BAAE
as substrate)
activity
of human
plasma are distinguished
by several physical, chemof enzyme assay in glass tubes, 25#{176}Cical, and clinical features,
including
pH optimum,
and esterase and on the amidase and esterase activity
of purified trypsin, kallikrein, and thrombin
Incubation
temperature
Discussion
1390 CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972
heat stability,
effect of dilution,
surface contact
activation,
streptokinase
activation,
Trasylol
inhibition, and range of variation
of activity in normal plasma.
The pH optimum
of 7.8 for arginine
amidase
with BAA coincides with that for purified trypsin.
In contrast,
the pH optimum
of serum argiine
amidase
with
the substrate,
N-carbobenzoxydiglycyl-arginine-2-naphthylamide,
is 8.6-8.8 (41).
The broad pH range of plasma activity with BAEE
is similar to that noted by Brown (38), and may indicate a mixture
of enzymes
with different
pH
optima. Proteolytic
enzymes with esterase activity
show a variety
of pH optima with different ester
substrates:
kallikrein,
pH 8.5 (BAEE)
(24);
plasmm, pH 6.5 (LME) (22) to 9 (TAME) (22); thrombin, pH 7 (LME) (20) to pH 8 (TAME) (20); trypsin,
pH 7.5 (LME) (22) to pH 8 (TAME)
(31). Ronwin
noted that the pH optimum with TAME for trypsin,
thrombin,
and plasmin
were all at 8.0 (21, 23),
even with different
buffers.
The heat stability
of the plasma BAA hydrolysis
reaction
that was noted earlier (33, 42) contrasts
sharply with the elimination
by heat of spontaneous and streptokinase-activated
BABE
activity
of
plasma.
A number
of esterolytic
enzymes
are
known
to be heat labile,
including
kallikrein,
plasmin,
thrombin
and the C1 component
of complement
(24, 33). The a1-globulin
trypsin
inhibitor of plasma is also heat labile (43); the increase
in plasma BAA activity after heating may be the result of destruction
of this inhibitor.
The unusual property
exhibited
by plasma BAA
activity-spontaneous
reactivation
on the second
day of storage-is
shared by crystalline
trypsin
and by plasma with added trypsin,
but does not
occur with the plasma BAEE reaction.
The virtual
disappearance
of plasma BAA activity with Storage
on all but the second day may account
for the
inability
of some laboratories
to duplicate
published reports of elevated BAA hydrolysis in patients
with pancreatitis.
In all of our clinical studies,
plasma
BAA
activity
has been determined
on
freshly drawn plasma.
Dilution
of plasma
enhances
both BAA
and
BABE
hydrolysis
to more than twice its original
value.
Assuming
that this is due to enzymeinhibitor
dissociation,
the distinctive
patterns
of
change in activity with the two substrates
indicate
quite different enzyme-inhibitor
systems, the BAAactive enzyme having a more readily dissociated
inhibitor
than the enzyme(s)
measured
by BAEE.
This increase in activity with plasma dilution is an
important
factor in clinical assays in which BAA
or BABE is used as substrate.
The BAA method used
in these studies shows linear activity with concentrations of trypsin far in excess of that measurable
with the BAEE method. By using standard
incubation times, equivalent
high activities
of plasma
enzyme can be quantitated
with BAA with use of
undiluted
plasma; the BAEE method requires that
the plasma be diluted, with attendant
falsely high
values for free enzyme activity.
Surface contact
activation
provides
one of the
most distinguishing
features of the plasma enzymes
measured
by BAA and by BABE,
because diametrically opposite effects are produced on incubation
in
glass and plastic tubes with the two substrates.
BAA
hydrolysis
is linear with time for plasma
maintained
in glass, but not with plasma in plastic,
while kinetic activation
occurs with the BAA reaction in plastic and with the BABE
reaction
in
glass tubes.
These
characteristics
are also exhibited
by crystalline
trypsin
when its activity
is measured
by either BAA or BAEE.
It is well
known that blood coagulation
(44-46), fibrinolysis
(47-49),
and the formation
of plasma kallikrein
(13, 44-46,
50) are initiated
by glass contact
through
the common
mechanism
of Hageman
factor activation.
The increase in argiine
esterase
activity
of glass-contacted
plasma has been previously
noted (30, 50, 51), but this is the first
demonstration
of the opposite
reaction
exhibited
by plasma
amidase
and by crystalline
trypsin.
These experiments
may be taken as evidence that
the BAA hydrolysis
activity
of plasma
does not
represent
activated
Hageman
factor,
kallikrein,
plasmin, or any other enzyme that is activated
by
glass contact.
The studies
presented
confirm
the esterase
activity
of streptokinase-generated
plasmin
from
whole plasma
and demonstrate
that this is not
measurable
with BAA as substrate.
The lack of
activity of commercially
prepared
plasmin toward
BAA
has also been demonstrated
(42).
Trasylol,
a polyvalent
inhibitor
of proteolytic
enzymes, is a potent inhibitor
of trypsin and kallikrein. It does not inhibit thrombin
and inhibits
only slightly
the BAEE
hydrolytic
activity
of
plasmin
generated
by SK activation
of whole
plasma. The failure of Trasylol to inhibit plasmin
activity
as measured
by TAME has also been reported (52). Trasylol does not inhibit the BAEE or
TAME
(52, 53) activity
of normal plasma, but completely inhibits its amidase activity.
These inhibition studies effectively
rule out thrombin
or plasmm as the enzymes
being measured
with the
plasma-BAA
reaction
and also rule out trypsin or
kallikrein
as being the major enzymes measured
as
BABE
hydrolysis
activity of normal plasma.
All four enzymes
studied-trypsin,
thrombin,
plasmin, kallikrein-hydrolyze
BABE
in direct proportion to enzyme concentration,
but only trypsin
hydrolyzes
BAA
at all concentrations
showing
esterase
activity.
This confirms previous
reports
that trypsin,
thrombin,
and plasmin
are more
active toward esters than toward the corresponding amides, and establishes
that the same is true for
CLINICAL CHEMISTRY,
Vol.18,No. 11,1972 1391
kallikrein.
Kallikrein
was reported
to have no
amidase
activity
as measured
with BAA (26) and
BANA
(27). In the present studies kallikrein
hydrolyzed BAA, but only in concentrations
above 5
KU/mi
(500 ig/ml).
This is a low order of activity
relative
to trypsin,
both in reference
to weight
(2.8 JAg of trypsin per milliliter produces
the same
BAA
activity
as does 500 zg of kallikrein
per
milliliter)
and in reference
to their esterase
activity.
Relative to BAA hydrolysis
activity,
the order of
reactivity
toward BABE is plasmin>
kallikrein
>
thrombin>
trypsin; relative to BAEE activity,
the
order of reactivity
toward BAA is trypsin > thrombin > kallikrein
> plasmin.
The difference
in
sensitivity
of the four enzymes toward BAA is of
such magnitude
that, on a relative
scale, if all
enzymes
were simultaneously
present
in plasma
at equivalent
concentrations
as measured by BABE,
only trypsin
would
be measurable
in normal
plasma with BAA substrate;
thrombin,
kallikrein,
and plasmin would not be detected.
The highest
plasma-BAA
activity
we have found clinically
in
patients
with pancreatitis
is 400-450 nmol/ml
per
hour (plasma
in glass tubes).
This is equivalent
to 25-30 g of trypsin
per milliliter,
400 NIH
units of thrombin
per milliliter or 50 KU (5000 JAg)
of kallikrein
per milliliter.
Because
thrombin
is
present in serum but not in plasma, it is obviously
unlikely that any of the BAA activity
of plasma is
due to this enzyme. Furthermore,
since one NIH
unit of thrombin
will clot 1 ml of blood in 15 s,
it again seems impossible
that the large quantities
of thrombin
as represented
by its BAA equivalents
would be present in plasma.
The plasma BABE: BAA activity
ratio is intermediate
to that of trypsin
on the one hand and
that of thrombin,
kallikrein,
and plasmin on the
other. If trypsin is used as reference,
the plasma
esterase
represents
enzymatic
activity
of three
times (in plastic tubes) to eight times (in glass
tubes)
that of plasma
amidase,
and it does not
appear to be measuring
solely any one of the other
three enzymes.
At present,
the identity
of the
enzyme(s)
measured
as arginime esterase in normal
plasma is undetermined.
Divergent
results have been obtained
with the
measurement
of argiine
esterase activity of body
fluids in pancreatitis.
Increased
blood esterase
activity
in clinical pancreatitis
has been reported
with BABE (40) and TAME (50, 52-55) substrates.
The abnormal
TAME
activity
was inhibited
by
Trasylol,
but the Trasylol did not decrease plasma
TAME
activity
below the normal baseline
values
(52, 53). Experimental
pancreatitis
in the dog has
been shown to produce
early and significant
elevations of BABE (18) or TAME (17) in the interstitial
pancreatic
fluid (17) and in peritoneal
fluid (17, 18),
but not in the systemic circulation
(18). The condi1392 CLINICAL CHEMISTRY.
Vol. 18, No. 11, 1972
tions of the enzyme assay may influence these results: some studies were done with plasma that had
not contacted
glass (18, 50) and others presumably
were routinely
done with use of glass vessels. With
the former, no elevations
of BAEE activity
were
found within 4 h of onset of experimental
pancreatitis
in the dog (18), but elevations
of TAME
activity
were noted in patients
with clinical pancreatitis (50). In the study by Coleman et a!. (50),
the authors adjudged
from their multiple enzyme
characterizatiod
studies that this activity was due
to an unidentified
enzyme other than trypsin
or
kallikrein.
The above reports, together with studies in this paper, attest that the normal spontaneous plasma
arginine
esterase
activity
does not
represent
trypsin
or kallikrein,
but that the abnormal
increases
of esterase
activity
in pancreatitis
do represent
a trypsin-kallikrein-like
enzyme, though it has not been positively
identified (50).
BAA
hydrolysis
activity
of plasma
may be
increased
spontaneously
(33, 35, 36, 56) or after
secretin and pancreozymin
stimulation
(33, 57) in
patients
with pancreatitis,
along with the other
pancreatic
enzymes, amylase and lipase. It is also
increased,
with amylase and lipase, after provocative duct obstruction
and secretory
stimulation
of
the pancreas
with morphine
and neostigmine
in
normal subjects.4 Szczeklik (41) reported
a lack of
spontaneous
amidase
activity
in normal
plasma,
although
it was present in normal serum, and no
elevations
in patients
with pancreatitis
(no details
given) if N-carbobenzoxydiglycyl-argiine-2-naphthylamide
was used as the substrate.
These findings, plus other experimental
data, led him to conclude that blood proteolytic
activity
measured
with amide substrates
is the result of thrombin
and plasmin
and not of trypsin.
The present
study clearly reveals that, this generalization
regarding
amide substrates
is unwarranted.
There
are apparently
differences
in enzyme
sensitivity
and kinetics
with arginine
amide
substrates
as
there are with various
arginine
and lysine ester
substrates
(20, 30, 50). The evidence
presented
here indicates
that plasma
arginine
amidase
as
measured
with BAA is probably
trypsin,
and certainly
is not thrombin,
plasmin,
or kallikrein.
The
specificity
and
differential
sensitivity
of
this assay make it preferable
to that of argiine
esterase in studies of blood trypsin activity.
Early attempts
to measure
trypsin
activity
in
blood evoked skepticism
that it could be measured
because
of the prominent
effect of proteolytic
enzyme inhibitors.
It is now established
that when
crystalline
trypsin
is added to plasma or serum,
Gullick,
H. D., Relation
of the magnitude
elevation
to severity
of exocrine
pancreatic
for publication.
of blood enzyme
disease.
Submitted
even in the presence of excess inhibitor,
enzyme
activity
toward
low-molecular-weight
substrates
is retained
because of selective binding of trypsin
in an active form to a2-macroglobulin
(39, 58-60).
While the present studies are concerned
with the
behavioral
characteristics
of endogenous
trypsinlike activity of whole plasma, it is assumed that the
ability to measure this enzyme with BAA in normal
subjects
and to quantify
abnormal
values
in
patients
with pancreatitis4
is due to a similar protective effect of the trypsin-binding
macroglobulin
on endogenous
trypsin
(61). Further
studies
are
needed to clarify this point.
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J. W., Moffat,
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