Evaluation of a Microassay for Human Kininogens as Cysteine Protease Inhibitors
AASEN,
TOVE S. KARLSRUD, ANSGAR 0.
Several methods kininogens article
have been described
based
presents
on both
a rapid,
by cysteine hydrolyzes
enzyme
activity
the added
enzyme
substrate,
that causes significant method,
inhibition
kininogen
to the test system. The within-run
was 1.7% when the inhibition day variation
as low as 2.3%
Applications kininogens
of the method in plasma,
Key Words: mogenic
Kininogens;
a yellow
of papain
coefficient
performed
color
are presented,
a papain
studying
that is read in
when
of variation
with
kininogens.
amount
to be 0.01
is established
accurately
based
subsequently
the smallest
or high molecular
of This
is activated
by added
approximately
weight
kininogen
is
(%) of the method
of papain was in the range 45-70% when
ascites,
papain
in this reaction
is very sensitive,
the assay performs
weight
characteristics.
assay of kininogens
will be inhibited
at 405 nm. This method
pg. As a quantitative
and quantification
The target enzyme
S-2302, generating
of kininogen
added
microplate
that is not inhibited
reader
JOHANSEN
and functional
proteases.
a microplate
0.1 kg of low molecular
T.
for the identification
and simple
cysteine
HCI and the activated
residual
HARALD
immunochemical
cheap
on their ability to inhibit The
AND
and the day to
inhibition
chromatographic
of 80%. separated
and urine.
Cysteine
protease
inhibition;
proteins
synthesized
Microplate
assay; Chro-
substrates
INTRODUCTION Kininogens
are multifunctional
by the liver and present
in
plasma in a total concentration
of 250-300 pg/mL. A domain-like structure has been postulated, and three biological functions have been located to different parts of the protein structure. A vasoactive peptide bradykinin (or Lys-bradykinin), of 9 (IO) amino acids, can be released from kininogens by the action of proteolytic enzymes such as kallikreins. The kinin moiety, with its multiple pharmacological actions, is released from the interior of the protein leaving an amino terminal heavy chain and a carboxy terminal light chain, linked by disulfide bonds (Movat, 1979). Two different kininogens are present in human plasma: High molecular weight kininogen (H-kininogen, M, - 120 000 D) and low molecular weight kininogen (LFromthe Department of Pharmacology, Institute of Pharmacy, University of Oslo (TSK, HTJ) and Department of Surgery B and Institute for Surgical Research, The National Hospital (AOA), Oslo, Norway. Address reprint request to: Tove Sigstad Karlsrud, Department of Pharmacology, Institute of Pharmacy, University of Oslo, Box 1068 Blindern, 0316 Oslo 3, Norway. Received October 1990; revised and accepted March 1991. 113 Journal of Pharmacological
Methods
0 1591 Elsevier Science Publishing
26, 113.124 (1991) Co., Inc., 655 Avenue of the Americas, New York, NY 10010
0160.5402/91/$3.50
114
T. S. Karlsrud et al.
kininogen, M, -65 000 D). H-kininogen plays an important role as a nonenzymatic cofactor in the activation of coagulation factor XII (Colman et al., 1975; Saito et al., 1975; Wuepper et al., 1975); the entire coagulant activity located to the light chain of the molecule (Thompson et al., 1978). The light chain of L-kininogen is very short and with no known function. The heavy chains of H-kininogen and L-kininogen are identical and contains two domains with the ability to inhibit cysteine proteases such as cathepsins, calpain and papain (Ohkubo et al., 1984; Muller-Ester1 et al., 1985). Kininogens are major extracellular cysteine protease inhibitors (CPls) in the body. The CPI function of kininogens holds a pharmacological potential in its ability to regulate destructive actions of cysteine proteases postulated to be of pathophysiological importance in various diseases such as cancer (Goldfarb and Liotta, 1986), muscular dystrophy (Colman et al., 1989), and joint diseases (Lenarcic et al., 1988). In analogy with the therapeutic use of antithrombin III (and heparin), Cl inhibitor and a,-antitrypsin to control serine proteases in cascade systems in plasma, kininogens or, rather, fragments of the kininogen heavy chain, could represent valuable new pharmacological tools toward excessive cysteine protease activity. This article describes a microplate method employing a chromogenic substrate that assays the CPI function of kininogens. The method allows the study of kininogens (or fragments of kininogens) in biological fluids such as plasma, ascites, and urine. The method is sensitive, easy to perform and makes it possible to quantify the two kininogens independently if purified standard preparations of L-kininogen and H-kininogen are available. MATERIALS AND METHODS Materials Papain type III (suspension in 0.05 M sodium acetate buffer pH 4.5 containing 0.01% thymol) and the chromogenic substrate Bz-Arg-pNA (BAPNA) were purchased from Sigma Chemical Co. (St. Louis, MO). The substrates Bz-lle-Glu-Gly-Arg-pNA HCI (S-2222) and H-D-Pro-Phe-Arg-pNA 2HCI (S-2302) were from Kabi Vitrum (Oslo, Norway), while the substrate Bz-Phe-Val-Arg-pNA HCI (S-2160) was generously donated by Kabi Vitrum (Stockholm, Sweden). L-cysteine HCI was provided from Nutritional Biochemicals Co. (Cleveland, OH), and plasma deficient in both kininogens were from George King Inc. (Overland Park, Kansas City, KS). DEAE-Sepharose Fast Flow and Superose 12 prep grade were from Pharmacia AB (Stockholm, Sweden). All other reagents were obtained from commercial sources and were of analytical grade. Ninety-six well flat-bottom microplates made of polystyrene were produced by Greiner (Nurtingen, West Germany). The microplate reader (Model 3550 EIA Reader) and the data analysis software Microplate Manager were from Bio-Rad Corp., Richmond, CA. Purified Kininogens High molecular weight kininogen (H-kininogen) was purified according to Dittmann et al. (1981). Fresh frozen human plasma (600 mL) was thawed at 37”C, im-
Kininogens as Cysteine Protease Inhibitors
mediately added enzyme inhibitors and applied to a DEAE-Sephadex A-50 column. A single protein peak containing both kininogens was eluted by a sodium acetate buffer pH 6.2 containing 0.6 M NaCI. Following dialysis the fractions containing kininogens were applied to a CM-Sephadex C-50 column. The column was eluted by an increasing NaCI-gradient, giving a single protein peak containing H-kininogen. The fractions containing H-kininogen were lyophilized, dissolved in water and finally applied to a Sephadex G-50 column. Low molecular weight kininogen (L-kininogen) was purified principally as described by Johnson et al. (1987). Fresh frozen human plasma (100 mL) was thawed at 37”C, immediately added enzyme inhibitors and applied to a Cm-papain Sepharose affinity column. The adsorbed proteins were eluted in a single peak at pH 11.5. Fractions were collected in tubes containing a sodium acetate buffer pH 4.2, giving a final pH of 6 in the fractions. Fractions containing kininogens were pooled and dialyzed before applied to a DEAE-Sepharose Fast Flow column. The kininogens were separately eluted by an increasing NaCI-gradient. The levels of immunoreactive kininogens in the pooled fractions were quantified by rocket immunoassay (Laurell, 1966) using locally produced antiserum raised in rabbits against human H-kininogen. This antiserum was reactive against both types of kininogens. Samples Employed in the Assay Blood was collected into l/IO volume of 0.10 M sodium citrate dihydrate solution with 0.05 M benzamidine and 0.05 M EDTA-2Na, and centrifuged at 3000 g for 30 min at 22°C. Plasma samples from 10 males and 10 females were pooled and stored at -70°C. Ascitic fluid was obtained from an 80-year-old woman suffering from advanced cancer mammae. Ascites were collected from the peritoneal cavity into 0.13 M sodium citrate solution (9:l v/v) and centrifuged 10 min at 1900 rpm. The titrated ascites were stored in aliquots fresh-frozen at -70°C. Urine samples were collected from both a patient suffering from chronic glomerulonephritis having severe proteinuria and from healthy normals. Urine was sampled without addition of benzamidine, citrate or EDTA. Principle of the Assay The proteolytic enzyme papain is activated by the reducing agent cysteine HCI in a phosphate buffer pH 7.5. After activation, part of the enzyme activity is inhibited by addition of kininogens from different biological fluids. The enzyme activity that is not inhibited in this reaction subsequently hydrolyzes the added substrate (S-2302), generating a yellow color that is read in the microplate reader at 405 nm. Microassay Procedure The procedure
was performed
at room temperature.
1. To each microplate well was added 50 PL standard or diluted sample. 2. A sufficient volume of papain for the experiment was activated separately for IO min in a test tube, e.g., 70 PL papain (60 kg/mL in 0.05 M acetate buffer pH 4.5)
115
116
T. S. Karlsrud et al.
added to 750 ~.LL0.05 M cysteine HCI and 1680 PL 0.03 M phosphate buffer pH 7.5. 3. 50 PL activated papain was added with a multichannel pipette to each well containing standard or sample. The mixture was incubated for 5 min. 4. 50 (IL S-2302 (2 mM) was added to each well and allowed to incubate for 30 min. The increase in absorbance over this period was measured at 405 nm in a microplate reader. Estimation of Kinetic Parameters for Chromogenic
Substrates for Papain
Five FL papain (60 f.rg/mL in 0.05 M acetate buffer pH 4.5) was incubated with 60 PL 0.05 M cysteine HCI and 335 ~.LL0.03 M phosphate buffer pH 7.5 for 10 min at 30°C. Substrate was dissolved in water, 200 PL of different concentrations was added, and the rate of cleavage spectrophotometrically measured at 405 nm. The kinetic parameters were calculated from a Michaelis Menten plot in the software program “Enzfitter” from Elsevier-BIOSOFT (Cambridge, United Kingdom). Chromatographic Separation of H-Kininogen Plasma and Ascites
and L-Kininogen
From
0.5 mL plasma or ascites was applied to a DEAE-Sepharose Fast Flow column (1.0 x 5.0 cm) equilibrated with 0.1 M tris buffer pH 8.0, containing 5 mM benzamidine, 5 mM EDTA-2Na and 0.01% Triton X-100. Proteins that adsorbed to the column were eluted by a salt gradient in the same buffer. The separation was performed with a 30 mL linear gradient from 0 to 0.25 M NaCI, and a second gradient (5 mL) from 0.25 to 0.5 M NaCl. The gradients were created by a FPLC-system (Pharmacia AB, Uppsala, Sweden). Flow rate was set to 1 mL/min. Fractions of 1 mL were collected. Gel Filtration of Pathologic and Normal Urine The urine samples were dialyzed against a 0.1 M tris buffer pH 8.0 containing 5 mM benzamidine, 5 mM EDTA-2Na and 0.01% Triton X-100. Gel filtration experiments were performed at a flow rate of 0.1 mL/min on a Superose 12 prep grade column (HR IO/301 connected to a Pharmacia FPLC system. The column was equilibrated with the column buffer (0.1 M tris pH 8.0 containing 0.6 M NaCI, 0.02% sodium azide and 0.01% Triton X-100), and 500 ~.LL dialyzed urine was applied to the column from a sample loop. Eluent was photometrically monitored at 280 nm and fractions of 400 FL were collected. The molecular weight standards used for calibration were dissolved in column buffer and applied to the column in the same manner as the urine samples. RESULTS Evaluation of Chromogenic
Peptide Substrates for Papain
Kinetic parameters were determined for the chromogenic peptide substrates S-2160, S-2222, and S-2302 with results as shown in Table 1. The substrate S-2222 was hardly cleaved by papain at all, the catalytic rate of the reaction was too low to make a reliable determination of K,. S-2160 was cleaved by papain, and could
Kininogens as Cysteine Protease Inhibitors TABLE 1 Kinetic parameters for chromogenic against papain
K, (mM)
k,, (s-l) S-2160 s-2222 S-2302
substrates
0.11 ND 1.12
0.65 -0 1.29
Different dilutions of the substrates (S-2160: 0.00259-0.333 mM, S-2222: 0.00530-0.674 mM, S-2302: 0.0266-I ,704 mM) were added to activated papain as described in materials and methods. The kinetic parameters were estimated from a Michaelis-Menten plot by using the software program “Enzfitter.” ND not determined.
probably
have been used in the present
assay. However,
assays performed
with this
substrate were not reproducible, and with large concentrations we could observe what appeared to be substrate inhibition of the enzyme. S-2302 was rapidly cleaved by papain, and the reproducibility was far better compared to S-2160. cleavage of S-2302 made it possible to run this assay over an acceptable time.
The
chromogenic
associated S-2302,
in Figure IA.
was recorded,
amounting
As shown produced
HCI,
of cysteine
Beyond
From this experiment Time Course
of Papain
of cysteine
S-2302. Addition
to about
times
to secure
papain
the addition
Curves,
Different
dilutions
to
of purified
a minimal
effect against the
a rapid activation developing activation
as
in activity
time in the assay.
H-kininogen
apparently
complete
to papain after 1 min.
employed.
A 5 min incubation
time
was
of papain.
and Sensitivity
H-kininogen
and L-kininogen
were used to establish
of papain. Linear correlation coeffiversus papain activity were regularly
0.99 when based on 4-6 different dilutions of purified kininogen. 28 show representative curves of the inhibition of papain caused and H-kininogen, respectively. of the method
of papain,
increase
inhibition of papain could be detected. The by H-kininogen seemed stable even when
inhibition
Reproducibility,
HCI
of 0.1 pg purified
standard curves for their activity as inhibitors cients of micrograms L-kininogen/H-kininogen
The precision
was
and compared
by Kininogens
of the enzyme,
of up to 30 min were a maximum
Standard
showed
as a practical
of Papain
a rapid 50% inhibition
incubation
This substrate
10% of the activity at the end of the assay time.
Beyond this time no significant further residual papain .activity not inhibited chosen
by Cysteine
7.5 min only a slowly
of the Inhibition IB,
as well.
the solubility,
HCI caused
10 min was chosen
in Figure
was studied
concerning
by papain.
of the Activation
Prior to the addition substrate
BAPNA
problems
it was slowly cleaved
Time Course
shown
substrate
with considerable
The rapid period of
within-run
was evaluated
Figures 2A and by L-kininogen
on fractions
from
DEAE-
117
118
T. S. Karlsrud et al.
c
1A
4
so-
g F
2
BO-
z p’
70.
2 g
60-
: w” P
10
0
20
30
40
50
60
70
60
SO
50 -
100
0
5
10
MINUTES
15
20
25
30
MINUTES
FIGURE 1. (A) 5 PL papain (15 PgImL in 0.05 M acetate buffer pH 4.5) was activated by addition of 15 (11 cysteine HCI (0.05 M) and phosphate buffer pH 7.5. The activity of papain against the substrate S-2302 was assayed at different time intervals up to 90 min. (B) Purified H-kininogen (0.1 Pg) was added to 0.075 Pg activated papain and incubated for 1, 2.5,5,7.5, 14,20, and 30 min. Substrate (S-2302) was added, and the residual papain activity was assayed as described.
Sepharose Fast Flow chromatography containing L-kininogen. The coefficient of variation (CV, %) was 1.7% when the inhibition of papain was in the range 45 to 70%,
and 6% when
estimated
chromatography formed
papain
was only 25%
after five repeated with
containing
papain
inhibited.
assays on a fraction H-kininogen.
inhibition
of 80%,
The day to day variation from
DEAE-Sepharose
The CV was as low as 2.3% and 5.4%
when
the papain
was
Fast Flow when
per-
inhibition
was
50%. Clearly, accurately
as a quantitative method this CPI-assay of kininogens is performed more when the papain inhibition is 50% or higher. This represents approxi-
2
0.6
ti f
0.4
c 2
0.2
s
I 0.0
I
0.5
1.0
1.5
L-KININOGEN
2.0
lpg/mll
2.5
3.0
0.0
I 0.0
I
0.5
1.0
1.5
H-KININOGEN
2.0
2.5
3.0
3.5
(pg/ml)
FIGURE 2. Representative standard curves showing the inhibition of papain caused by different dilutions of purified L-kininogen (A) (O-2.83 Pg/mL)) and H-kininogen (B) (O-3.21 Pg/mL).
Kininogens as Cysteine Protease Inhibitors mately 0.1 ~g of L-kininogen
or H-kininogen,
corresponding
to a concentration
of
2 Fg/mL, added to the test system described. However, a qualitative judgement of whether CPI activity from kininogens is present in a sample requires much smaller amounts. The sensitivity of the method, defined as the smallest amount of kininogen that caused a significant Assay of Kininogens
inhibition
of papain, is established
to be 0.01 pg (0.2 kg/mL).
in Plasma and Ascites
Normal plasma, plasma deficient in both kininogens, and ascites were applied to a DEAE-Sepharose Fast Flow column as described in methods. The fractions from this chromatography assay presented in Figure
were tested by rocket immunoelectrophoresis
in this article.
3. Fractions
concentrations
diluted
from the separation
sufficiently
(from
covered by the standard curves. When
in the fractions, used during
were
The results
corrections
and by the CPI-
of plasma are shown
3 to IO times) calculating
to reach the
pg/mL kininogens
were made for effects on papain activity by the buffers
the chromatography.
This
was done by performing
a “blank”
chro-
matographic run without sample, and making an identical and parallel CPI assay of the blank fractions. The plasma deficient in both kininogens showed no significant CPI activity
and, predictably,
developed
no rockets
in agarose gels containing
an-
tibodies toward kininogens. As seen from the rocket immunoelectrophoresis the peaks of kininogens present in normal plasma were observed in two ranges; in fractions 10 to 18, and in fractions 21 to 28. The corresponding ranges from the separation of ascites were fractions 10 to 17, and fractions 24 to 28, respectively (data not shown).
The
quantitative
amounts
of kininogens
in ascites were,
as ex-
pected, lower than in normal plasma. When the measured CPI activity was plotted into the same diagram, it completely fell together with the mentioned rocket areas.
0
5
10 FRACTION
15
20
25
30
NUMBER
FIGURE 3. Immunological levels of kininogens and CPI activity in fractions after separation of 0.5 ml normal plasma and plasma deficient in kininogens on a DEAE-Sepharose Fast Flow column. Curve with solid squares (W) shows CPI activity in normal plasma and open squares (Cl) CPI activity in kininogen deficient plasma. Vertical bars represent rocket heights after immunoelectrophoresis of fractions from normal plasma. No rockets were detected in fractions from kininogen-deficient plasma.
119
120
T. S. Karlsrud et al. 1.35 1
I
I
0
5
10
15 FRACTION
20
25
30
35
NUMBER
FIGURE 4. CPI activity in fractions collected after gel filtration of 0.5 ml urine on a Superose 12 prep grade column. Curve with solid squares (H) shows activity in normal urine and open squares (0) activity in urine from a patient suffering from chronic glomerulonephritis. Vertical arrows indicate relative elution volume of standard proteins. The molecular weight in kilodaltons is given above each arrow.
This, together with the lack of CPI activity in kininogen deficient plasma, strongly indicates that the CPI activity measured in these fractions is solely due to kininogens. Assay of Kininogens
in Urine
Samples
Figure 4 shows the results from assay of CPI activity in fractions of 0.5 mL urine found
in fraction
samples.
In a normal
12, corresponding
urine
sample
one major
to an M, of 106,000.
after gel filtration
peak of activity
Both rocket
was
immunoelec-
trophoresis and immunoblotting experiments showed that this peak corresponded to L-kininogen (data not shown). H-kininogen was not present in detectable amounts
in normal
urine.
Urine
sample
from a patient
with chronic
glomerulone-
phritis contained less kininogen than normal urine and a nearly complete disappearance of CPI activity with molecular weight above 100,000. Instead a major activity peak appeared in fraction 23 corresponding to an M, of 11,000. Fractions 15 to 18 comprising an M, range from 60,000 to 30,000 also had significant CPI activity. Immunoblotting
experiments
appeared to be extensive lower Mr. On immunoblots from
13,000
to 17,000
of patient
urine
prior to chromatography
showed
what
proteolytic breakdown of kininogens to fragments with 5 bands in the M, range of 39,000 to 47,000 and 4 bands
appeared.
The kininogen
fragments
mentioned
were
found
in fractions 15 (mainly 39,000-47,000 fragments) to 18 (mainly 13,000-17,000 fragments). Fraction 23 (M, - 11,000) contained no kininogen and it seems probable that this CPI activity represents intracellular cystatins excreted in the urine during disease
(Abrahamson
et al., 1986).
DISCUSSION This article describes a microplate method as cysteine protease inhibitors. Evaluation
developed for the study of kininogens of the method and applications on
Kininogens as Cysteine Protease Inhibitors plasma,
ascites,
and urine
has proven
the method
very sensitive,
able to identify
as little as 0.2 kg/mL kininogens. Performed in a microplate the reproducibility very good and likely to be superior to alternative methods employed in studies
is of
kininogens. Specific
identification
other biological
and quantification
of polypeptides
fluids are usually based on either
of specific functions
of the proteins.
in plasma,
immunological
Several immunoassays
urine,
methods
and
or assays
of kininogens
have been
published (Kleniewski and Donaldson, 1977; Bouma et al., 1980; Proud et al., 1980; Kerbiriou-Nabias et al., 1984; Adam et al., 1985; Hoem et al., 1989). However, the presence L-kininogen
of common
antigen
complicates
determinants
in the heavy chain of H-kininogen
the development
of specific
assay systems.
and
Light chain-
specific antibodies have been produced (Miller-Ester1 et al., 1988), but for L-kininogen this is difficult because the light chain only comprises 38 amino acids (Lottspeich et al., 1984). When is not associated
kininogens
with a reduced
release
kinins after proteolytic
immunoreactivity.
digestion,
Thus, it is not possible
this
to assess
the biological potential of this polypeptide based on immunoassays. On the contrary, an increased level of H-kininogen has been observed after incubation with plasma kallikrein Methods described.
when assayed in a rocket immunoassay
that quantify It is possible
kininogens
system (Hoem
by their ability to release
to make a selective
et al., 1989).
kinins have also been
release of kinins from H-kininogen
and
L-kininogen using different proteolytic enzymes (Uchida and Katori, 1979). However, the following assay of released kinins is difficult. Release of kinins has been described
in several diseases and will obviously
result in an underestimation
content of kininogens in these samples. A coagulation test based on the ability of the light chain in H-kininogen as cofactor in activation of coagulation factor XII is in widespread H-kininogen. This test requires plasma deficient of H-kininogen,
of the
to function
use to quantify which has pro-
longed coagulation when measuring a kaolin-induced partial thromboplastin time. Because of the need for H-kininogen-deficient plasma, the coagulation test is expensive to perform, and it generally suffers from poor reproducibility. The cofactor function of H-kininogen is also the basis of an indirect method described by Scott et al. (1987) that is based on the ability of purified FXI. FXla generated Glu-Pro-Arg-pNA (S-2366). The most recently
discovered
is then function
of H-kininogen assayed
with
of kininogens,
to stimulate
the activation
the chromogenic
substrate
their ability to inhibit
cys-
teine proteases, has so far been of little use in quantitative assay systems. In designing such an assay system the natural choice of target enzyme is papain, which is cheap, easily available and well characterized. Kininogens are potent inhibitors of papain (Ohkubo et al., 1984), the molecular interaction between bovine kininogens and its derivatives with papain was described by Sueyoshi et al. (1988). Calculations concerning stoichiometries for the inhibition using data from the present standard curves indicate H-kininogen and L-kininogen
the molar ratio of the complexes formed between to papain to be 1.06:1 and 1.95:1, respectively. How-
ever, titration of papain was not performed, and these calculations are based on a 40% active papain sample. The calculated stoichiometry is in agreement with results presented by Sueyoshi and coworkers concerning bovine H-kininogen, but not
121
122
T. S. Karlsrud et al. bovine
L-kininogen.
function
In our system human
twice as efficiently
the interaction
between
as L-kininogen.
the cysteine
H-kininogen It should
protease
as a CPI, on a molar be mentioned
calpain
basis,
that studies
and human
dicate that H-kininogen on a molar basis is twice as effective as L-kininogen inhibiting papain (Ishiguro et al., 1987; Bradford et al., 1990). Papain NMec
activity
(Vogel
has been
assayed with fluorogenic
et al., 1988) or simple
(Sasaki et al., 1981). As spectrophotometers most
laboratories,
a method
substrates
chromogenic
substrates
or microplate
based on absorbance
of
kininogens
in-
when
such as Z-Phe-Argsuch as Bz-Arg-pNA
readers
are available
at 405 nm of released
in
parani-
troaniline would be preferable. In our search for a better substrate than Bz-ArgpNA we found that HD-Pro-Phe-Arg-pNA (S-2302) performed satisfactorily. S-2302 is a good substrate for plasma kallikrein, and the presence of this enzyme could interfere
with
and extensive
the method. dilution
Addition
of the samples
of the serine eliminated
protease
inhibitor
the possibility
benzamidine
of plasma kallikrein
interfering with the assay. Based on estimated K, values, it can be calculated that the present assay was not performed in the presence of excess substrate. However, this did not seriously affect the method as a linear increase in absorbance at 405 nm was recorded over the 30 min period (data not shown), and excellent standard curves were obtained with both purified H-kininogen and L-kininogen. Using the CPI function of kininogens as the basis for identification and quantification
is appropriate
teolytic
breakdown
ever, result what extent
as the function (Vogel
has been
shown
et al., 1988). The digestion
to be very resistant
to pro-
of the heavy chain may, how-
in release of CPI domains and possibly an increased CPI activity. To this may occur in vivo is not known but the possibility must be con-
sidered when using the method in a quantitative manner. Experiments presented in this article with plasma, ascites, and urine are performed as measurements of CPI activity in fractions from an initial chromatographic fractionation significant samples
of the biological CPI activity,
both
intact L-kininogen
no intact H-kininogen. in the patient suffering
fluids.
indicating
Plasma deficient
that the method and fragments
in kininogens
contained
is specific for kininogens. of kininogens
were
no
In urine
identified,
but
In addition, a low molecular weight CPI activity was present from chronic glomerulonephritis having severe proteinuria.
This activity could possibly be of intracellular origin. Abrahamson et al. (1986) described the presence of six CPls in urine from a patient with mixed glomerulartubular proteinuria. The dominant CPI was identified as cystatin C, while normal urine contained mainly L-kininogen. Results presented in this article are in complete accordance with the observations of Abrahamson and coworkers, and prove our method suitable for assay of low molecular weight cystatins as well. Accordingly, to make a judgement of relative amounts, some kind of separation of kininogens and cystatins is necessary The microplate method
when present for kininogens
together in a sample. described here represent
easy and accessible method of studying kininogens in biological human and other species. As such we hope it will stimulate further on kininogens as part of the defence mechanism use of CPI domains as pharmacological tools.
an inexpensive, samples research
from both
in the body, and on the possible
Kininogens as Cysteine Protease Inhibitors The this
authors
wish
to thank
Mr. A. Babinski
for
his
expert
technical
assistance
throughout
investigation.
REFERENCES Abrahamson
M, Barrett
(1986) Isolation itors
from
AJ, Salvesen
of six cysteine
human
urine.
G, Grubb
proteinase
A
inhib-
I Biol Chem 261:11282-
chimont
P (1985) Human
high molecular immunoassay values. Bouma
Kerbiriou
DM,
Vlooswijk
C/in Med Bradford human
kininogens.
AH, Colman
inhibition
RW,
of
Bagdasarian
Williams with
abnormalities pathways. RW,
hibitor with
Human levels
by
RC, Scott CF, JV, Kaplan AP defiwith
of the Hageman Factor-dependent
Bradford
HN, Warner
of thiol proteases, muscular
AH (1989) High
the extracellular
in-
in hamsters
Thromb
B, Steger
convenient lecular
large-scale
weight
Hoppe-Seyler’s Goldfarb
A, Wimmer
RH,
preparation
kininogen
from
Z Physiol
Chem
Liotta
johansen
weight
Advances
oIogy.
Kinins New
337-343.
human
mo-
plasma.
HT,
Johannesen
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in
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S, Briseid
human Medicine
K Abe,
K
and Bi-
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and S pp.
kininogen inhi-
Proc Sot fxp Biol Med 156:113-
117. teins
by electrophoresis
Lanarcic B, Gabrijelcic M, Turk
D, Rozman
proteinase
light
inhibitors joint
F, Kellermann W
(CPls)
B and
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(1984)
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V (1988) Human
matory and metabolic Hope
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in agarose gel contain-
Anal Biochem
Human
Amino-acid
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Eurl
molecular
sequence
with
Biochem
A, Rauth G,
low
other
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In:
Mtiller-Ester1 Brzin
York: W,
J, Kotnik
speich
F (1985)
identical Evidence
Fritz
Ed.,
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Kallidin, EC Erdiis.
and
M, Turk Human
pp. l-89.
W,
Ritonja
V, Kellermann plasma
immunological,
Kalli-
Berlin-Hei-
Springer-Verlag, H, Machleidt
A,
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se-
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Movat HZ (1979) the plasma kallikrein-kinin
delberg-New
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H Moriga
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Laurel1 C-B (1966) Quantitative
krein
of high and low mo-
and plate-
VH (1977) Quantification
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A]
M-J (1984)
in plasmas
and its interrelationship
12:294-309.
N-O,
In:
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(1989) Rocket immunoassay lecular
H (1981) A
LA (1986) Proteolytic
in cancer invasion Haemost
R, Fritz
Barrett kininogens.
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high
(HMW-KGN)
mass
Dittmann
MA,
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human
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Br / Haemato/56:273-286.
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Mechanism
DM, Garcia FO, Larrieu
lecular weight lets.
molecular
Res 48:187-193.
sorok
56:1650-1662.
weight kininogen,
C,
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Salvesen
Rapid
bition
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II
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JA, Pierce
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and
calpain
A, Talamo
diminished
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RW (1990) Ki-
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high
I, Mur-
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Thromb
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Biochemistry Johnson
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96:693-709.
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31:423-426.
JH (1980) Immunological kallikrein,
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C/in Chem
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Adam A, Albert A, Calay G, Closset
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I, Kurachi
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K, Takasawa
M (1984) Isolation
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low molecular
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K, Shiokawa
of a human
and its identity
kininogen.
H,
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for with
Biochemistry
JV, Pisano
of human
ogen in normal
JJ (1980)
high molecular
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Radioimmu-
H,
Ratnoff
weight
plasmas.
nolysis, bility Sasaki
agent, Fitzgerald
generation plasma
(PF/dil).
kinin-
/ Lab C/in
human
forms
for high
JP un-
factor, participating
reactions of kinins, enhancing
of clotting,
fibri-
and the property vascular
K, Minakata of thiol
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Scott CF, Pixley
R, Abraham
of a hitherto
permea-
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M, Taniguchi
molecular
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trait. Deficiency
in surface-mediated of diluted
T, Hara A, Shimada
of bovine
RA, Colman
molecular
weight
K (1981)
proteinase
Thromb
Multiin
89:169-177.
the
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in human
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of
kininogen.
high
weight
high molecular activity
kinin-free
weight
of kininogens.
high
molecular
(1988)
molecular
Res 15:127-133.
I, Ester1 A, Machleidt
W,
Proteinase-sensitive
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ki-
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Thromb
Vogel R, Assfalg-Machleidt W
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/ Exp Med 147:488-499.
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Bio/ Chem 263 : 12661-I 2668. Wuepper
RW (1987) A new assay
T, Kimura
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Procoagulant
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RE, Mandle
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(1975) Fitzgerald recognized
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T, Kato H, lwanaga S (1988) Molecular
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Sueyoshi
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23:5691-5697. Proud
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/
AASEN,
TOVE S. KARLSRUD, ANSGAR 0.
Several methods kininogens article
have been described
based
presents
on both
a rapid,
by cysteine hydrolyzes
enzyme
activity
the added
enzyme
substrate,
that causes significant method,
inhibition
kininogen
to the test system. The within-run
was 1.7% when the inhibition day variation
as low as 2.3%
Applications kininogens
of the method in plasma,
Key Words: mogenic
Kininogens;
a yellow
of papain
coefficient
performed
color
are presented,
a papain
studying
that is read in
when
of variation
with
kininogens.
amount
to be 0.01
is established
accurately
based
subsequently
the smallest
or high molecular
of This
is activated
by added
approximately
weight
kininogen
is
(%) of the method
of papain was in the range 45-70% when
ascites,
papain
in this reaction
is very sensitive,
the assay performs
weight
characteristics.
assay of kininogens
will be inhibited
at 405 nm. This method
pg. As a quantitative
and quantification
The target enzyme
S-2302, generating
of kininogen
added
microplate
that is not inhibited
reader
JOHANSEN
and functional
proteases.
a microplate
0.1 kg of low molecular
T.
for the identification
and simple
cysteine
HCI and the activated
residual
HARALD
immunochemical
cheap
on their ability to inhibit The
AND
and the day to
inhibition
chromatographic
of 80%. separated
and urine.
Cysteine
protease
inhibition;
proteins
synthesized
Microplate
assay; Chro-
substrates
INTRODUCTION Kininogens
are multifunctional
by the liver and present
in
plasma in a total concentration
of 250-300 pg/mL. A domain-like structure has been postulated, and three biological functions have been located to different parts of the protein structure. A vasoactive peptide bradykinin (or Lys-bradykinin), of 9 (IO) amino acids, can be released from kininogens by the action of proteolytic enzymes such as kallikreins. The kinin moiety, with its multiple pharmacological actions, is released from the interior of the protein leaving an amino terminal heavy chain and a carboxy terminal light chain, linked by disulfide bonds (Movat, 1979). Two different kininogens are present in human plasma: High molecular weight kininogen (H-kininogen, M, - 120 000 D) and low molecular weight kininogen (LFromthe Department of Pharmacology, Institute of Pharmacy, University of Oslo (TSK, HTJ) and Department of Surgery B and Institute for Surgical Research, The National Hospital (AOA), Oslo, Norway. Address reprint request to: Tove Sigstad Karlsrud, Department of Pharmacology, Institute of Pharmacy, University of Oslo, Box 1068 Blindern, 0316 Oslo 3, Norway. Received October 1990; revised and accepted March 1991. 113 Journal of Pharmacological
Methods
0 1591 Elsevier Science Publishing
26, 113.124 (1991) Co., Inc., 655 Avenue of the Americas, New York, NY 10010
0160.5402/91/$3.50
114
T. S. Karlsrud et al.
kininogen, M, -65 000 D). H-kininogen plays an important role as a nonenzymatic cofactor in the activation of coagulation factor XII (Colman et al., 1975; Saito et al., 1975; Wuepper et al., 1975); the entire coagulant activity located to the light chain of the molecule (Thompson et al., 1978). The light chain of L-kininogen is very short and with no known function. The heavy chains of H-kininogen and L-kininogen are identical and contains two domains with the ability to inhibit cysteine proteases such as cathepsins, calpain and papain (Ohkubo et al., 1984; Muller-Ester1 et al., 1985). Kininogens are major extracellular cysteine protease inhibitors (CPls) in the body. The CPI function of kininogens holds a pharmacological potential in its ability to regulate destructive actions of cysteine proteases postulated to be of pathophysiological importance in various diseases such as cancer (Goldfarb and Liotta, 1986), muscular dystrophy (Colman et al., 1989), and joint diseases (Lenarcic et al., 1988). In analogy with the therapeutic use of antithrombin III (and heparin), Cl inhibitor and a,-antitrypsin to control serine proteases in cascade systems in plasma, kininogens or, rather, fragments of the kininogen heavy chain, could represent valuable new pharmacological tools toward excessive cysteine protease activity. This article describes a microplate method employing a chromogenic substrate that assays the CPI function of kininogens. The method allows the study of kininogens (or fragments of kininogens) in biological fluids such as plasma, ascites, and urine. The method is sensitive, easy to perform and makes it possible to quantify the two kininogens independently if purified standard preparations of L-kininogen and H-kininogen are available. MATERIALS AND METHODS Materials Papain type III (suspension in 0.05 M sodium acetate buffer pH 4.5 containing 0.01% thymol) and the chromogenic substrate Bz-Arg-pNA (BAPNA) were purchased from Sigma Chemical Co. (St. Louis, MO). The substrates Bz-lle-Glu-Gly-Arg-pNA HCI (S-2222) and H-D-Pro-Phe-Arg-pNA 2HCI (S-2302) were from Kabi Vitrum (Oslo, Norway), while the substrate Bz-Phe-Val-Arg-pNA HCI (S-2160) was generously donated by Kabi Vitrum (Stockholm, Sweden). L-cysteine HCI was provided from Nutritional Biochemicals Co. (Cleveland, OH), and plasma deficient in both kininogens were from George King Inc. (Overland Park, Kansas City, KS). DEAE-Sepharose Fast Flow and Superose 12 prep grade were from Pharmacia AB (Stockholm, Sweden). All other reagents were obtained from commercial sources and were of analytical grade. Ninety-six well flat-bottom microplates made of polystyrene were produced by Greiner (Nurtingen, West Germany). The microplate reader (Model 3550 EIA Reader) and the data analysis software Microplate Manager were from Bio-Rad Corp., Richmond, CA. Purified Kininogens High molecular weight kininogen (H-kininogen) was purified according to Dittmann et al. (1981). Fresh frozen human plasma (600 mL) was thawed at 37”C, im-
Kininogens as Cysteine Protease Inhibitors
mediately added enzyme inhibitors and applied to a DEAE-Sephadex A-50 column. A single protein peak containing both kininogens was eluted by a sodium acetate buffer pH 6.2 containing 0.6 M NaCI. Following dialysis the fractions containing kininogens were applied to a CM-Sephadex C-50 column. The column was eluted by an increasing NaCI-gradient, giving a single protein peak containing H-kininogen. The fractions containing H-kininogen were lyophilized, dissolved in water and finally applied to a Sephadex G-50 column. Low molecular weight kininogen (L-kininogen) was purified principally as described by Johnson et al. (1987). Fresh frozen human plasma (100 mL) was thawed at 37”C, immediately added enzyme inhibitors and applied to a Cm-papain Sepharose affinity column. The adsorbed proteins were eluted in a single peak at pH 11.5. Fractions were collected in tubes containing a sodium acetate buffer pH 4.2, giving a final pH of 6 in the fractions. Fractions containing kininogens were pooled and dialyzed before applied to a DEAE-Sepharose Fast Flow column. The kininogens were separately eluted by an increasing NaCI-gradient. The levels of immunoreactive kininogens in the pooled fractions were quantified by rocket immunoassay (Laurell, 1966) using locally produced antiserum raised in rabbits against human H-kininogen. This antiserum was reactive against both types of kininogens. Samples Employed in the Assay Blood was collected into l/IO volume of 0.10 M sodium citrate dihydrate solution with 0.05 M benzamidine and 0.05 M EDTA-2Na, and centrifuged at 3000 g for 30 min at 22°C. Plasma samples from 10 males and 10 females were pooled and stored at -70°C. Ascitic fluid was obtained from an 80-year-old woman suffering from advanced cancer mammae. Ascites were collected from the peritoneal cavity into 0.13 M sodium citrate solution (9:l v/v) and centrifuged 10 min at 1900 rpm. The titrated ascites were stored in aliquots fresh-frozen at -70°C. Urine samples were collected from both a patient suffering from chronic glomerulonephritis having severe proteinuria and from healthy normals. Urine was sampled without addition of benzamidine, citrate or EDTA. Principle of the Assay The proteolytic enzyme papain is activated by the reducing agent cysteine HCI in a phosphate buffer pH 7.5. After activation, part of the enzyme activity is inhibited by addition of kininogens from different biological fluids. The enzyme activity that is not inhibited in this reaction subsequently hydrolyzes the added substrate (S-2302), generating a yellow color that is read in the microplate reader at 405 nm. Microassay Procedure The procedure
was performed
at room temperature.
1. To each microplate well was added 50 PL standard or diluted sample. 2. A sufficient volume of papain for the experiment was activated separately for IO min in a test tube, e.g., 70 PL papain (60 kg/mL in 0.05 M acetate buffer pH 4.5)
115
116
T. S. Karlsrud et al.
added to 750 ~.LL0.05 M cysteine HCI and 1680 PL 0.03 M phosphate buffer pH 7.5. 3. 50 PL activated papain was added with a multichannel pipette to each well containing standard or sample. The mixture was incubated for 5 min. 4. 50 (IL S-2302 (2 mM) was added to each well and allowed to incubate for 30 min. The increase in absorbance over this period was measured at 405 nm in a microplate reader. Estimation of Kinetic Parameters for Chromogenic
Substrates for Papain
Five FL papain (60 f.rg/mL in 0.05 M acetate buffer pH 4.5) was incubated with 60 PL 0.05 M cysteine HCI and 335 ~.LL0.03 M phosphate buffer pH 7.5 for 10 min at 30°C. Substrate was dissolved in water, 200 PL of different concentrations was added, and the rate of cleavage spectrophotometrically measured at 405 nm. The kinetic parameters were calculated from a Michaelis Menten plot in the software program “Enzfitter” from Elsevier-BIOSOFT (Cambridge, United Kingdom). Chromatographic Separation of H-Kininogen Plasma and Ascites
and L-Kininogen
From
0.5 mL plasma or ascites was applied to a DEAE-Sepharose Fast Flow column (1.0 x 5.0 cm) equilibrated with 0.1 M tris buffer pH 8.0, containing 5 mM benzamidine, 5 mM EDTA-2Na and 0.01% Triton X-100. Proteins that adsorbed to the column were eluted by a salt gradient in the same buffer. The separation was performed with a 30 mL linear gradient from 0 to 0.25 M NaCI, and a second gradient (5 mL) from 0.25 to 0.5 M NaCl. The gradients were created by a FPLC-system (Pharmacia AB, Uppsala, Sweden). Flow rate was set to 1 mL/min. Fractions of 1 mL were collected. Gel Filtration of Pathologic and Normal Urine The urine samples were dialyzed against a 0.1 M tris buffer pH 8.0 containing 5 mM benzamidine, 5 mM EDTA-2Na and 0.01% Triton X-100. Gel filtration experiments were performed at a flow rate of 0.1 mL/min on a Superose 12 prep grade column (HR IO/301 connected to a Pharmacia FPLC system. The column was equilibrated with the column buffer (0.1 M tris pH 8.0 containing 0.6 M NaCI, 0.02% sodium azide and 0.01% Triton X-100), and 500 ~.LL dialyzed urine was applied to the column from a sample loop. Eluent was photometrically monitored at 280 nm and fractions of 400 FL were collected. The molecular weight standards used for calibration were dissolved in column buffer and applied to the column in the same manner as the urine samples. RESULTS Evaluation of Chromogenic
Peptide Substrates for Papain
Kinetic parameters were determined for the chromogenic peptide substrates S-2160, S-2222, and S-2302 with results as shown in Table 1. The substrate S-2222 was hardly cleaved by papain at all, the catalytic rate of the reaction was too low to make a reliable determination of K,. S-2160 was cleaved by papain, and could
Kininogens as Cysteine Protease Inhibitors TABLE 1 Kinetic parameters for chromogenic against papain
K, (mM)
k,, (s-l) S-2160 s-2222 S-2302
substrates
0.11 ND 1.12
0.65 -0 1.29
Different dilutions of the substrates (S-2160: 0.00259-0.333 mM, S-2222: 0.00530-0.674 mM, S-2302: 0.0266-I ,704 mM) were added to activated papain as described in materials and methods. The kinetic parameters were estimated from a Michaelis-Menten plot by using the software program “Enzfitter.” ND not determined.
probably
have been used in the present
assay. However,
assays performed
with this
substrate were not reproducible, and with large concentrations we could observe what appeared to be substrate inhibition of the enzyme. S-2302 was rapidly cleaved by papain, and the reproducibility was far better compared to S-2160. cleavage of S-2302 made it possible to run this assay over an acceptable time.
The
chromogenic
associated S-2302,
in Figure IA.
was recorded,
amounting
As shown produced
HCI,
of cysteine
Beyond
From this experiment Time Course
of Papain
of cysteine
S-2302. Addition
to about
times
to secure
papain
the addition
Curves,
Different
dilutions
to
of purified
a minimal
effect against the
a rapid activation developing activation
as
in activity
time in the assay.
H-kininogen
apparently
complete
to papain after 1 min.
employed.
A 5 min incubation
time
was
of papain.
and Sensitivity
H-kininogen
and L-kininogen
were used to establish
of papain. Linear correlation coeffiversus papain activity were regularly
0.99 when based on 4-6 different dilutions of purified kininogen. 28 show representative curves of the inhibition of papain caused and H-kininogen, respectively. of the method
of papain,
increase
inhibition of papain could be detected. The by H-kininogen seemed stable even when
inhibition
Reproducibility,
HCI
of 0.1 pg purified
standard curves for their activity as inhibitors cients of micrograms L-kininogen/H-kininogen
The precision
was
and compared
by Kininogens
of the enzyme,
of up to 30 min were a maximum
Standard
showed
as a practical
of Papain
a rapid 50% inhibition
incubation
This substrate
10% of the activity at the end of the assay time.
Beyond this time no significant further residual papain .activity not inhibited chosen
by Cysteine
7.5 min only a slowly
of the Inhibition IB,
as well.
the solubility,
HCI caused
10 min was chosen
in Figure
was studied
concerning
by papain.
of the Activation
Prior to the addition substrate
BAPNA
problems
it was slowly cleaved
Time Course
shown
substrate
with considerable
The rapid period of
within-run
was evaluated
Figures 2A and by L-kininogen
on fractions
from
DEAE-
117
118
T. S. Karlsrud et al.
c
1A
4
so-
g F
2
BO-
z p’
70.
2 g
60-
: w” P
10
0
20
30
40
50
60
70
60
SO
50 -
100
0
5
10
MINUTES
15
20
25
30
MINUTES
FIGURE 1. (A) 5 PL papain (15 PgImL in 0.05 M acetate buffer pH 4.5) was activated by addition of 15 (11 cysteine HCI (0.05 M) and phosphate buffer pH 7.5. The activity of papain against the substrate S-2302 was assayed at different time intervals up to 90 min. (B) Purified H-kininogen (0.1 Pg) was added to 0.075 Pg activated papain and incubated for 1, 2.5,5,7.5, 14,20, and 30 min. Substrate (S-2302) was added, and the residual papain activity was assayed as described.
Sepharose Fast Flow chromatography containing L-kininogen. The coefficient of variation (CV, %) was 1.7% when the inhibition of papain was in the range 45 to 70%,
and 6% when
estimated
chromatography formed
papain
was only 25%
after five repeated with
containing
papain
inhibited.
assays on a fraction H-kininogen.
inhibition
of 80%,
The day to day variation from
DEAE-Sepharose
The CV was as low as 2.3% and 5.4%
when
the papain
was
Fast Flow when
per-
inhibition
was
50%. Clearly, accurately
as a quantitative method this CPI-assay of kininogens is performed more when the papain inhibition is 50% or higher. This represents approxi-
2
0.6
ti f
0.4
c 2
0.2
s
I 0.0
I
0.5
1.0
1.5
L-KININOGEN
2.0
lpg/mll
2.5
3.0
0.0
I 0.0
I
0.5
1.0
1.5
H-KININOGEN
2.0
2.5
3.0
3.5
(pg/ml)
FIGURE 2. Representative standard curves showing the inhibition of papain caused by different dilutions of purified L-kininogen (A) (O-2.83 Pg/mL)) and H-kininogen (B) (O-3.21 Pg/mL).
Kininogens as Cysteine Protease Inhibitors mately 0.1 ~g of L-kininogen
or H-kininogen,
corresponding
to a concentration
of
2 Fg/mL, added to the test system described. However, a qualitative judgement of whether CPI activity from kininogens is present in a sample requires much smaller amounts. The sensitivity of the method, defined as the smallest amount of kininogen that caused a significant Assay of Kininogens
inhibition
of papain, is established
to be 0.01 pg (0.2 kg/mL).
in Plasma and Ascites
Normal plasma, plasma deficient in both kininogens, and ascites were applied to a DEAE-Sepharose Fast Flow column as described in methods. The fractions from this chromatography assay presented in Figure
were tested by rocket immunoelectrophoresis
in this article.
3. Fractions
concentrations
diluted
from the separation
sufficiently
(from
covered by the standard curves. When
in the fractions, used during
were
The results
corrections
and by the CPI-
of plasma are shown
3 to IO times) calculating
to reach the
pg/mL kininogens
were made for effects on papain activity by the buffers
the chromatography.
This
was done by performing
a “blank”
chro-
matographic run without sample, and making an identical and parallel CPI assay of the blank fractions. The plasma deficient in both kininogens showed no significant CPI activity
and, predictably,
developed
no rockets
in agarose gels containing
an-
tibodies toward kininogens. As seen from the rocket immunoelectrophoresis the peaks of kininogens present in normal plasma were observed in two ranges; in fractions 10 to 18, and in fractions 21 to 28. The corresponding ranges from the separation of ascites were fractions 10 to 17, and fractions 24 to 28, respectively (data not shown).
The
quantitative
amounts
of kininogens
in ascites were,
as ex-
pected, lower than in normal plasma. When the measured CPI activity was plotted into the same diagram, it completely fell together with the mentioned rocket areas.
0
5
10 FRACTION
15
20
25
30
NUMBER
FIGURE 3. Immunological levels of kininogens and CPI activity in fractions after separation of 0.5 ml normal plasma and plasma deficient in kininogens on a DEAE-Sepharose Fast Flow column. Curve with solid squares (W) shows CPI activity in normal plasma and open squares (Cl) CPI activity in kininogen deficient plasma. Vertical bars represent rocket heights after immunoelectrophoresis of fractions from normal plasma. No rockets were detected in fractions from kininogen-deficient plasma.
119
120
T. S. Karlsrud et al. 1.35 1
I
I
0
5
10
15 FRACTION
20
25
30
35
NUMBER
FIGURE 4. CPI activity in fractions collected after gel filtration of 0.5 ml urine on a Superose 12 prep grade column. Curve with solid squares (H) shows activity in normal urine and open squares (0) activity in urine from a patient suffering from chronic glomerulonephritis. Vertical arrows indicate relative elution volume of standard proteins. The molecular weight in kilodaltons is given above each arrow.
This, together with the lack of CPI activity in kininogen deficient plasma, strongly indicates that the CPI activity measured in these fractions is solely due to kininogens. Assay of Kininogens
in Urine
Samples
Figure 4 shows the results from assay of CPI activity in fractions of 0.5 mL urine found
in fraction
samples.
In a normal
12, corresponding
urine
sample
one major
to an M, of 106,000.
after gel filtration
peak of activity
Both rocket
was
immunoelec-
trophoresis and immunoblotting experiments showed that this peak corresponded to L-kininogen (data not shown). H-kininogen was not present in detectable amounts
in normal
urine.
Urine
sample
from a patient
with chronic
glomerulone-
phritis contained less kininogen than normal urine and a nearly complete disappearance of CPI activity with molecular weight above 100,000. Instead a major activity peak appeared in fraction 23 corresponding to an M, of 11,000. Fractions 15 to 18 comprising an M, range from 60,000 to 30,000 also had significant CPI activity. Immunoblotting
experiments
appeared to be extensive lower Mr. On immunoblots from
13,000
to 17,000
of patient
urine
prior to chromatography
showed
what
proteolytic breakdown of kininogens to fragments with 5 bands in the M, range of 39,000 to 47,000 and 4 bands
appeared.
The kininogen
fragments
mentioned
were
found
in fractions 15 (mainly 39,000-47,000 fragments) to 18 (mainly 13,000-17,000 fragments). Fraction 23 (M, - 11,000) contained no kininogen and it seems probable that this CPI activity represents intracellular cystatins excreted in the urine during disease
(Abrahamson
et al., 1986).
DISCUSSION This article describes a microplate method as cysteine protease inhibitors. Evaluation
developed for the study of kininogens of the method and applications on
Kininogens as Cysteine Protease Inhibitors plasma,
ascites,
and urine
has proven
the method
very sensitive,
able to identify
as little as 0.2 kg/mL kininogens. Performed in a microplate the reproducibility very good and likely to be superior to alternative methods employed in studies
is of
kininogens. Specific
identification
other biological
and quantification
of polypeptides
fluids are usually based on either
of specific functions
of the proteins.
in plasma,
immunological
Several immunoassays
urine,
methods
and
or assays
of kininogens
have been
published (Kleniewski and Donaldson, 1977; Bouma et al., 1980; Proud et al., 1980; Kerbiriou-Nabias et al., 1984; Adam et al., 1985; Hoem et al., 1989). However, the presence L-kininogen
of common
antigen
complicates
determinants
in the heavy chain of H-kininogen
the development
of specific
assay systems.
and
Light chain-
specific antibodies have been produced (Miller-Ester1 et al., 1988), but for L-kininogen this is difficult because the light chain only comprises 38 amino acids (Lottspeich et al., 1984). When is not associated
kininogens
with a reduced
release
kinins after proteolytic
immunoreactivity.
digestion,
Thus, it is not possible
this
to assess
the biological potential of this polypeptide based on immunoassays. On the contrary, an increased level of H-kininogen has been observed after incubation with plasma kallikrein Methods described.
when assayed in a rocket immunoassay
that quantify It is possible
kininogens
system (Hoem
by their ability to release
to make a selective
et al., 1989).
kinins have also been
release of kinins from H-kininogen
and
L-kininogen using different proteolytic enzymes (Uchida and Katori, 1979). However, the following assay of released kinins is difficult. Release of kinins has been described
in several diseases and will obviously
result in an underestimation
content of kininogens in these samples. A coagulation test based on the ability of the light chain in H-kininogen as cofactor in activation of coagulation factor XII is in widespread H-kininogen. This test requires plasma deficient of H-kininogen,
of the
to function
use to quantify which has pro-
longed coagulation when measuring a kaolin-induced partial thromboplastin time. Because of the need for H-kininogen-deficient plasma, the coagulation test is expensive to perform, and it generally suffers from poor reproducibility. The cofactor function of H-kininogen is also the basis of an indirect method described by Scott et al. (1987) that is based on the ability of purified FXI. FXla generated Glu-Pro-Arg-pNA (S-2366). The most recently
discovered
is then function
of H-kininogen assayed
with
of kininogens,
to stimulate
the activation
the chromogenic
substrate
their ability to inhibit
cys-
teine proteases, has so far been of little use in quantitative assay systems. In designing such an assay system the natural choice of target enzyme is papain, which is cheap, easily available and well characterized. Kininogens are potent inhibitors of papain (Ohkubo et al., 1984), the molecular interaction between bovine kininogens and its derivatives with papain was described by Sueyoshi et al. (1988). Calculations concerning stoichiometries for the inhibition using data from the present standard curves indicate H-kininogen and L-kininogen
the molar ratio of the complexes formed between to papain to be 1.06:1 and 1.95:1, respectively. How-
ever, titration of papain was not performed, and these calculations are based on a 40% active papain sample. The calculated stoichiometry is in agreement with results presented by Sueyoshi and coworkers concerning bovine H-kininogen, but not
121
122
T. S. Karlsrud et al. bovine
L-kininogen.
function
In our system human
twice as efficiently
the interaction
between
as L-kininogen.
the cysteine
H-kininogen It should
protease
as a CPI, on a molar be mentioned
calpain
basis,
that studies
and human
dicate that H-kininogen on a molar basis is twice as effective as L-kininogen inhibiting papain (Ishiguro et al., 1987; Bradford et al., 1990). Papain NMec
activity
(Vogel
has been
assayed with fluorogenic
et al., 1988) or simple
(Sasaki et al., 1981). As spectrophotometers most
laboratories,
a method
substrates
chromogenic
substrates
or microplate
based on absorbance
of
kininogens
in-
when
such as Z-Phe-Argsuch as Bz-Arg-pNA
readers
are available
at 405 nm of released
in
parani-
troaniline would be preferable. In our search for a better substrate than Bz-ArgpNA we found that HD-Pro-Phe-Arg-pNA (S-2302) performed satisfactorily. S-2302 is a good substrate for plasma kallikrein, and the presence of this enzyme could interfere
with
and extensive
the method. dilution
Addition
of the samples
of the serine eliminated
protease
inhibitor
the possibility
benzamidine
of plasma kallikrein
interfering with the assay. Based on estimated K, values, it can be calculated that the present assay was not performed in the presence of excess substrate. However, this did not seriously affect the method as a linear increase in absorbance at 405 nm was recorded over the 30 min period (data not shown), and excellent standard curves were obtained with both purified H-kininogen and L-kininogen. Using the CPI function of kininogens as the basis for identification and quantification
is appropriate
teolytic
breakdown
ever, result what extent
as the function (Vogel
has been
shown
et al., 1988). The digestion
to be very resistant
to pro-
of the heavy chain may, how-
in release of CPI domains and possibly an increased CPI activity. To this may occur in vivo is not known but the possibility must be con-
sidered when using the method in a quantitative manner. Experiments presented in this article with plasma, ascites, and urine are performed as measurements of CPI activity in fractions from an initial chromatographic fractionation significant samples
of the biological CPI activity,
both
intact L-kininogen
no intact H-kininogen. in the patient suffering
fluids.
indicating
Plasma deficient
that the method and fragments
in kininogens
contained
is specific for kininogens. of kininogens
were
no
In urine
identified,
but
In addition, a low molecular weight CPI activity was present from chronic glomerulonephritis having severe proteinuria.
This activity could possibly be of intracellular origin. Abrahamson et al. (1986) described the presence of six CPls in urine from a patient with mixed glomerulartubular proteinuria. The dominant CPI was identified as cystatin C, while normal urine contained mainly L-kininogen. Results presented in this article are in complete accordance with the observations of Abrahamson and coworkers, and prove our method suitable for assay of low molecular weight cystatins as well. Accordingly, to make a judgement of relative amounts, some kind of separation of kininogens and cystatins is necessary The microplate method
when present for kininogens
together in a sample. described here represent
easy and accessible method of studying kininogens in biological human and other species. As such we hope it will stimulate further on kininogens as part of the defence mechanism use of CPI domains as pharmacological tools.
an inexpensive, samples research
from both
in the body, and on the possible
Kininogens as Cysteine Protease Inhibitors The this
authors
wish
to thank
Mr. A. Babinski
for
his
expert
technical
assistance
throughout
investigation.
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