Clinical Science and Molecular Medicine (1976) 51, 87-92.
The disappearance of enzyme-inhibitor complexes
from the circulation of man
K. OHLSSON
AND
C.-B. L A U R E L L
Department of Surgery and Clinical Chemistry, University of Limnd, Malmd General Hospital, Malmd, Sweden
(Receiced 5 March 1976)
Summary
Introduction
1. Complexes of human trypsin and human granulocyte elastase with a,-anti-trypsin and az-macroglobulin were isolated and injected intravenously into
human volunteers.
2. The elimination of az-macroglobulin complexes
with trypsin and elastase followed single-exponential
functions with half-lives of 9 and 12 min respectively.
The complexes showed no tendency to dissociate.
3. Complexes of a,-anti-trypsin with trypsin persisted in the circulation much longer, with a half-life
of 3.5 h; complexes of a,-anti-trypsin with elastase
had an intermediate half-life of 1 h.
4. Dissociation was observed of a,-anti-trypsin
complexes with transfer of trypsin and elastase to
az-macroglobulin.
5 . Dialysable radioactivity appeared in the urine
soon after the injection of az-macroglobulin complexes, which suggested a breakdown of complexes
by cells in the reticuloendothelial system. Radioactivity over the liver achieved maximum values
within 3 W min after the injection of a,-macroglobulin complexes but not unlil50-70 min after the
injection of a,-anti-trypsin complexes.
6. These results support the concept of a key position for a,-macroglobulin in the protective mechanisms against endogenous proteases.
Previous studies of interactions between the pancreatic proteases and the protease inhibitors of
plasma have shown high affinities of al-anti-trypsin
and az-macroglobulin for proteases in virro (Laurell
& Jeppsson, 1975). These proteins were responsible
in the dog for the inhibition and clearance of protease from blood and extracellular fluid (Ohlsson,
1971a, b, c). The half-life of A2M-protease(') complexes in the circulation was only 5-10 min and
followed first-order kinetics. Complexes of AAT with
protease, however, persisted longer in the circulation,
with half-lives that varied between 30 and 45 min.
Transfer was observed of protease from AAT-protease complexes to AzM, but transfer did not occur
from A2M. The AzM-protease complexes were
rapidly absorbed and digested by cells of the reticuloendothelial system. The intravenous injection of
active enzyme in the dog resulted in shock and death
whenever the quantity of enzyme exceeded the binding capacity of the plasma A,M (Ohlsson, Ganrot &
Laurell, 1971). This occurred with trypsin injections
even though the circulating AAT was often less than
half saturated with enzyme at the time of full AzM
saturation. These facts suggested a key role for AzM
in protection against protease in the dog.
Observations on the interactions of proteases with
protease inhibitors in vivo in man are few and contradictory. Complexes of AIM with plasmin were
rapidly removed from the circulation but evidence
was presented by NilChn & Ganrot (1967) that the
complexes retained some activity in the circulation,
with proteolysis that was restricted to polypeptides.
Data presented by Blatrix, Amouch, Drouet &
Steinbuch (1973) suggested that AzM- trypsin com-
Key words: a-globulins, leucocytes, macroglobulins,
neutrophils, trypsin, trypsin inhibitors.
( I ) Abbreviations: A2M. az-macroglobulin; AAT, al-antitrypsin.
Correspondence: Dr Kjell Ohlsson,Department of Clinical
Chemistry, Malmo General Hospital, S-214 01 Malmo,
Sweden.
87
88
K. Ohlsson and C.-B. Laurell
plexes were rapidly eliminated from the blood. But
the functional activity of the proteins in that study
seems questionable since the inhibitors would have
been supersaturated with enzyme as reported.
Saturation of inhibitors with enzyme risks proteolysis of the inhibitors, with drastic conformational
changes invoked by cleavage.
The purpose of this investigation was to measure
persistence in the circulation of complexes between
major inhibitors of plasma and natural proteases.
Insofar as possible, the preparation of the complexes
was designed to ensure native functional states of the
proteins. Experience from dog studies permitted us
to draw conclusions about the metabolism of
enzyme-inhibitor complexes in man from a limited
number of experiments since the catabolic rates found
were closely similar in the two species. Trypsin in the
circulation is so distinctly abnormal that data on
trypsin complexes may apply only to few disease
states. But the interactions of A2M and AAT with
the elastase derived from the granulocyte must be of
fundamental importance. Enzymes released from
granulocytes and the metabolism of their enzymeinhibitor complexes could conceivably influence
morbidity and mortality in many diseases.
Gel filtration of plasma and fractions on Sephadex
G-200 was performed at 4" C in columns (2.5 cm x 55
cm) equilibrated with Tris-HC1 buffer (40 mmol/l ;
pH 7.4) containing NaCl(O.1 mol/l). Samples (3 ml)
were fractionated at an elution rate of 15 ml/h.
Fractions (4 ml) were collected and protein content
was estimated by measuring extinction at 280 nm.
Radioactivity of blood specimens was analysed in a
Gamma spectrometer with a well scintillation detector.
All preparations were sterilized by Millipore filtration.
Experimental procedures
The elimination of the AAT and A2M complexes
was first studied in the authors and then in two additional healthy volunteer subjects who were fully informed about the risks involved (for the number of
experiments see Table 1). We used fresh plasma for
TABLE
1. Half-life of radioactive substance in the
human circulation after intraoenous injection of
3 1I-labelled protease-inhibitor complexes
Complex injected
Material and methods
Preparations
Human granulocyte elastase (Ohlsson & Olsson,
1974) and human cationic trypsin (Ohlsson & Skude,
1976) were purified as previously described. The
purified enzymes were electrophoretically homogeneous on agarose and polyacrylamide disc electrophoresis (pH 4.3). AAT was isolated as described by
Jeppsson & Laurell (1974). Rabbit antisera against
human AAT and A2M were available in the laboratory. AAT and A2M in plasma were estimated with
electroimmunoassay (Laurell, 1972).
The elastase and the trypsin were labelled with l J I I
and AAT with lz5Iwith the lactoperoxidase method
(Thorell & Johansson, 1971). Low-molecular-weight
radioactive material was separated by gel filtration
on Sephadex G-25 in columns (0.9 cm x 10 cm) equilibrated with acetate buffer (50 mmol/l; pH 4.01, or
Tris-HC1 buffer (50 mmol/l; pH 7.4) for 1251-labelled
AAT, in NaCl solution ( 0 5 mol/l). The labelled enzymes retained more than 90% of their proteolytic
activity. The specific radioactivity of the labelled proteins was 0.5 fiCi/mg for trypsin, 0.6 fiCi/mg for
elastase and 0.5 pCi/mg for AAT.
1311-labelledtrypsin-A2M
1311-labelIedelastase-A2M
1311-labelledtrypsin-AAT
'311-labelledelastase-AAT
to.5 (min)
9,9.5, 10
11.5, 12, 12.5
197, 205, 220
59, 71, 85, 96
the preparation of complexes to be injected. In addition the elimination of AAT was studied in one of the
authors: the predominant type of AAT, i.e. Pi-MM,
was used in this study.
Preparation of ' 'I-labelled trypsin and "I-labelled
granulocyte elastase complexes with AAT and A M
Human serum (2 rnl) was rapidly mixed with 1 mg
of labelled human cationic trypsin (or human granulocyte elastase) in 1.0 ml of Tris-HC1 buffer (40
mmol/l; pH 7-4) containing NaCl (0.1 mol/l) and
CaCL (50 mmol/l) and incubated at room temperature for 10 min. Proteins of the resultant mixture
were then separated by gel filtration on Sephadex
G-200. Radioactivity was measured and those fractions containing complexes of trypsin (or elastase)
with AAT and A2M were separately pooled.
Clearance of enzyme-inhibitor complexes
The labelled AzM-protease complexes were available for intravenous infusion within 3 4 h and the
corresponding AAT complexes within 15 h after the
blood had been drawn from the test subject. The
investigators were of the AAT type Pi-MM, and the
enzymes added to plasma resulted in about half
saturation of the trypsin (elastase) binding capacity
of the plasma. All proteins, including AAT, were
injected in an amount corresponding to about 25
pCi of radioactivity.
Results
Intravenous administration of A I M complexesformed
in vitro with
'I-labelled trypsin (or 311-labelled
elastase)
This was not followed by any changes in blood
pressure, pulse or breathing frequency and the test
subjects did not notice any discomfort. The disappearance of radioactivity followed a singleexponential function until about 75% of the complexes
had been eliminated (Fig. 1 and Fig. 2). In general,
metabolic behaviour was similar for both enzyme
complexes. The half-lives were 9-10 min for the
'311-labelled trypsin complexes and 12 min for the
corresponding A2M-' 'I-labelled elastase complexes
100
5
-
I-.
0
89
(Table 1). An initial decrease in l3'1 was followed in
about 30 min by a period during which radioactivity
increased slightly (Fig. 1 and Fig. 2). At this time,
dialysable radioactivity appeared in the urine. Plasma
specimens obtained 5 min after the injection of AzM
complexes with trypsin or elastase were fractionated
by gel filtration with Sephadex G-200 and analysed
for radioactivity. More than 99% (99.0-99-6%) of
radioactivity in these specimens was bound to A2M.
Similar analyses of plasma obtained 2 h after the
injection of complexes failed to reveal radioactivity
in the AIM fractions. All the radioactivity present
was dialysable. Radioactivity measured by an external counter over the liver reached a maximum
about 30 min after the injection of the 1311-labelled
trypsin complexes (Fig. 1) and about 40 min after
the injection of the corresponding A2M-' 'I-labelled
elastase complexes (Fin. 2).
Intravenous administration of AAT complexesjormed
in vitro with 'I-labelled trypsin- or 1311-labelled
elastase
The injections of AAT-' 311-labelledtrypsin complexes were accompanied by slight dizziness, which
lasted about 30-60 s in all subjects studied. Simultaneously the blood pressure fell from 130/80 mmHg
H
b
0
Time ( h 1
FIG. 1. Elimination of radioactivity from the plasma of one individual after intravenous injection of AzM-'"Ilabelled trypsin complexes (.-a),
AAT-'311-labelled trypsin complexes (0-0) and 'z51-labelled AAT
(H
Radioactivity
).
measured by external counter over the liver is also shown for A2M-1311-labelledtrypsin
complexes (0 - - - 0 ) and AAT-1311-labelled trypsin complexes (0- - - 0).
90
K . Ohlsson and C.-B. Larrrell
T ~ m e( h I
FIG.2. Elimination of radioactivity from the plasma of one individual after intravenous injection of A z M - ' ~ ' I labelled elastase complexes (0-O),
and AAT-1311-labelled elastase complexes (0-0) and '251-labelled
Radioactivity measured by external counter over the liver is also shown for A2M-13'I-labelled
AAT (+H).
elastase complexes ( 0 - - - 0 ) and AAT-1311-labelledelastase complexes (0- - - 0).
to 95/60 mmHg in one of the subjects while the pulse
rate increased about 30%. These effects were transient. The injections of AAT complexes with 1 3 ' 1 labelled elastase were not associated with any reactions, symptomatic or circulatory. About half of
the radioactivity measured in the blood 5 min after
the injection of AAT-'311-labelled trypsin complexes still remained in the plasma 3 3 h later (Fig. 1).
The half-life of AAT-' 'I-labelled elastase complexes was significantly shorter, 1.Ck1.5 h (Fig. 2).
Plasma specimens obtained 5 rnin after the injection
of these complexes were fractionated within 30 min
by filtration through Sephadex (3-200. These studies
revealed that the major part was still bound to AAT.
About 5% ( 4 5 6 . 3 % ) of the total radioactivity was,
however, recovered with A,M after injection of
AAT-l3 'I-labelled trypsin complexes and about 8
(6.2-9.5 %) after the injection of AAT-' 'I-labelled
elastase complexes. Specific antisera against the inhibitors caused precipitation of more than 90%
(905-94.2%) of the radioactivity in each of these
fractions. Both trypsin and elastase were thus transferred from AAT to A2M.
Radioactivity monitored continuously over the
liver reached a maximum 60-70 min after injection
of AAT-I 311-labelled trypsin complexes, and 50-60
min after AAT-' 31Llabelledelastase complexes (Fig.
I and Fig. 2).
Intraoenous administration of
sI-labelled A A T
l2
This was not followed by any circulatory symptoms or discomfort. The elimination curve obtained
is shown in Fig. 1 and Fig. 2 for reference.
Discussion
Preliminary experiments in this laboratory had revealed that trypsin could alter the electrophoretic
properties of isolated, pure AAT and of AAT-trypsin
complexes even before the trypsin concentration exceeded the binding capacity of the AAT. But this
effect was small with albumin concentrations similar
to those found in plasma. We produced protease
complexes for the present experiments by the addition of protease to fresh serum in quantities calculated not to exceed half saturation of AAT. Serum
was preferable to purified inhibitors for the formation of complexes in order to avoid cleavage of the
AAT and AAT-protease complexes and to minimize
the risks of undesirable reactions on injection.
Our results agree with our earlier findings in the
dog (Ohlsson, 1971a, b; Ohlsson ef al., 1971), and
show that A,M-protease complexes are cleared from
the circulation in man much faster than AAT-protease complexes. In the dog, we found that the reticuloendothelial cells absorb the complexes (Ohlsson,
1971~).This high rate of elimination suggests that
Clearance of enzyme-inhibitor complexes
blood is cleared of A2M complexes quantitatively on
single passage through those organs with efficient
reticuloendothelial-sieve mechanisms such as the
liver, bone marrow and spleen. The liver scan data
are also consistent with this assumption.
This high rate of clearance deserves special consideration because it indicates that the interaction
between the large inhibitor (rnol. wt. 725 000) and
these small enzymes (mol. wt. 24000 and 34000)
results in a conformational change of the inhibitor
molecule that is immediately recognized by thereticuloendothelial cells. It has been proposed that the
proteases occupy a surface gap (or pocket) on the
A2M molecule which traps the enzyme molecule as
soon as it digests a sensitive peptide in the pocket of
the inhibitor (Barret & Starkey, 1973). It is interesting that such an apparently small alteration can
result in immediate molecular suicide. Whether this
triggers a chain reaction analogous to the sequence
which eliminates antigen-antibody complexes from
the circulation is not known. Receptors in the reticuloendothelial cells may possibly identify deformed
inhibitor molecules for removal but such receptors
have not been identified. The slightly higher rate of
elimination for the smaller enzyme, trypsin (rnol. wt.
24OOO), over that of the larger elastase (mol. wt.
34 OOO) suggests that the enzyme size may influence
the degree of closure of the A2M pocket. If the
smaller enzyme provoked the more pronounced surface change we should expect the result observed.
This concept seems worthy of testing with enzymes
of more widely differing sizes.
The elimination of AAT-protease complexes from
the circulation was considerably slower than the
elimination of A2M-protease complexes. Even so,
there was a great difference between the elimination
of AAT complexes (to.s= 60-90 min with elastase)
and the curve obtained for free AAT, which is in
agreement with the normal metabolic turnover
(to., = 4 days) (Laurel1 & Jeppsson, 1975). The implications are similar for AAT to those already discussed for A2M. The clearance of AAT-protease
complexes is not adequately expressed as a simple
exponential decay. The explanation may be simply
that AAT-enzyme complexes are only gradually
eliminated from the circulation, or that the complexes dissociate. The data fit either possibility (Fig.
1 and Fig. 2). Evidence for dissociation was obtained
when the injection of AAT-protease complexes
resulted in the appearance of A2M-protease complexes in the blood 5 min after injection. We cannot
91
exclude the possibility that enzyme-transfer occurred
at the time of, or just after, the blood samples were
obtained. But the transfer of enzyme from AAT to
A2M is consistent with our other observations that
AAT-enzyme complexes readily yield enzyme to the
bovine lung trypsin inhibitor aprotinin (Trasylol), an
inhibitor which binds enzymes with higher energy
than AAT. We think there is a slow dissociation of
AAT-protease complexes, and there may be some
differences in rate with different proteases. The association of AAT with elastase, for example, seems of
significantly higher energy than the association of
AAT with trypsin.
It seems logical to propose that every protease
molecule released from AAT will be bound either by
another AAT molecule, by A2M, or encounter substrate. In the case of A2M, the result is immediate
clearance from plasma. Thus elimination will be
influenced by the rate of dissociation of complex and
by the ratio of free AAT to AIM molecules in the
plasma. Implicit in this concept is the idea that AAT
serves as a carrier protein for proteases, particularly
in the extracellular space where the concentration of
A2M is low. The diffusion of AAT is much greater
than A2M because it has a much smaller hydrodynamic volume. The hypothesis that AAT is mainly
a carrier protein has support from the results of our
preliminary studies on dogs. We injected complexes
which had double labels, '2sII-labelled AAT and
'I-labelled trypsin. The turnover rate of AAT from
these complexes as reflected by the disappearance of
lZsIwas much slower than the turnover of trypsin as
reflected by disappearance of 13'1 (Ohlsson &
Delshammar, 1975).
An additional point in need of clarification is
whether the reversible linkage of proteases to AAT
occasions chemical alterations that impair inhibitor
function. The key role of A2M for protection against
protease is emphasized by the present results in man.
Further studies of A2M may document this role in
diseases associated with the release of protease into
the tissues.
Acknowledgments
This investigation was supported by the Swedish
Medical Research Council (project no. B75-17X3910-03B and project no. B75-13X-581-11A),
Torsten and Ragnar SoderbergsStiftelser, the Swedish
Tobacco Company, Bayer AG, West Germany, and
the Medical Faculty of Lund.
92
K. Ohlsson and C.-B. Laurel1
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