Clinical Chemistry 47: 215-222, 2001;
(Clinical Chemistry. 2001;47:215-222.)
© 2001 American Association for Clinical Chemistry, Inc.
Macromolecular Chromogenic Substrates for Measuring Proteinase Activity
Glen L. Hortin1,a,
Ilka Warshawsky1 and
Maryline Laude-Sharp2
1
Clinical Pathology Department, National Institutes of Health, Bldg. 10, Room 2C-407, 10 Center Dr., Bethesda, MD 20892-1508.
2
Department of Pediatrics, Washington University School
of Medicine, St. Louis, MO 63110.
a Author for correspondence. Fax 301-402-1885; e-mail
ghortin{at}cc.nih.gov.
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Abstract
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Background: Proteinase activities are often measured using
chromogenic substrates that are much smaller than physiological
substrates.
Methods: The hydrodynamic size of macromolecular substrates
(macrosubstrates) prepared by linking small chromogenic substrates to
polyethylene glycol was determined by gel filtration. Efficiency of
macrosubstrate cleavage by proteinases and
2-macroglobulin-proteinase complexes was monitored
spectrophotometrically.
Results: Macrosubstrates had hydrodynamic radii of 
20 Å,
similar to proteins with a molecular weight of 18 000.
Different macrosubstrates served as efficient substrates for
chymotrypsin, trypsin, and thrombin. Linking small substrates to a
polymer variably affected substrate efficiency, with the impact on
activity ranging from a 60-fold decrease to a 30-fold increase.
Proteinases complexed with 2-macroglobulin had
10-fold lower activity vs macrosubstrates than small substrates.
Conclusions: Macrosubstrates are efficient substrates that allow
decreased measurement of sterically hindered proteinase
molecules such as 2-macroglobulin-proteinase complexes.
Thus, macrosubstrates may provide more accurate functional assays of
proteinases such as coagulation factors.
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Introduction
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Small peptide substrates have contributed greatly to understanding
the catalytic function of proteases, beginning with studies in Emil
Fischer’s laboratory in the early 1900s (1) and continuing
with studies of peptide esters through the 1950s (2)(3)(4). The
development of 4-nitroanilide (4-NA)1
substrates
(5)(6)(7) provided greatly improved tools for protease
measurements that allowed continuous spectrophotometric monitoring of
reactions. The discovery that substrate cleavage by some proteinases
was affected by the sequence of several residues in the substrate
preceding the site of cleavage (7)(8)(9)(10)(11) opened the way to
developing substrates that were relatively specific for assaying
particular proteinases. Changes in the amino acid residues located two
to four positions before the sites of cleavage can change the affinity
and rates of cleavage of a substrate by particular proteases
>1000-fold. The search for optimal small peptide substrates has led to
hundreds of highly sensitive and relatively specific chromogenic and
fluorogenic substrates for proteinases
(12)(13)(14)(15)(16)(17).
A major drawback of peptide substrates for measuring the activity of
physiological proteinases such as components of the coagulation,
complement, and fibrinolytic pathways, however, is that the substrates
do not reproduce the size of the natural substrates, which are
proteins. Small synthetic substrates may be cleaved by proteinases that
cannot easily access physiological protein substrates because
of complexing of the proteinase with other components, such as
cofactors, inhibitors, binding proteins, antibodies, or surfaces. A
major pathway for proteinase inactivation is by entrapment within the
inhibitor 2-macroglobulin
(2-M), which does not block the active site or
peptidase activity of proteinases bound to it (18)(19)(20)(21)(22)(23)(24). The
peptidase activity of proteinases entrapped within
2-M interferes with measurement of the
proteinase activity with small substrates. As a result of these
factors, measurements of peptidase activity using small peptide
substrates can differ substantially from functional proteinase
activity, and this has been recognized as a major problem for assays of
proteinases in plasma specimens (23)(24).
There are several approaches to overcome this shortcoming and to
measure proteinase action on protein substrates. Some of the earliest
methods for measuring proteinase activity analyzed the cleavage of
proteins such as casein or hemoglobin (25)(26),
and pepsin activity still is occasionally expressed in units of
hemoglobin hydrolysis and plasmin activity as casein units. However,
these activity measurements are relatively nonspecific, measure only
endpoints, require physical separation steps, and are hard to define
kinetically because there are multiple potential sites of cleavage,
each with different kinetic properties. For many physiological
proteinases, it is possible to measure functional activity in clotting,
fibrinolysis, or complement activation, but these assays involve
complex multistep reactions.
In the present study, we synthesized and evaluated synthetic
macromolecular substrates that had a large molecular size but still
offered continuous monitoring and defined cleavage specificity of small
chromogenic peptide substrates.
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Materials and Methods
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materials
Bovine chymotrypsin and trypsin were purchased from Worthington
Biochemicals. Active 2-M was obtained
from Calbiochem. The substrates S-2288 (D-Ile-Pro-Arg-4-NA)
and S-2238 [D-Phe-pipecolic acid (Pip)-Arg-4-NA] were
purchased from DiaPharma Group, Inc. Sarcosyl (Sar)-Pro-Arg-4-NA,
carbobenzoxy (CBZ)-Lys-Arg-4-NA, Glu-Gly-Arg-4-NA, Gly-Arg-4-NA, and
Arg-4-NA were from Bachem Bioscience. Human thrombin was from Sigma or
from Enzyme Research Laboratory. Polyethylene glycol (PEG), PEG
bis(amine) (Mr 3400),
succinyl-Ala-Ala-Pro-Phe-4-NA, 4-amidinophenylsulfonyl
fluoride, Sephadex G-25 coarse, 4-nitrophenyl-4'-guanidinobenzoic acid,
a standard solution of 4-nitrophenol, and proteins used as molecular
weight calibrators were obtained from Sigma. Bio-Gel P-6 and P-60 were
from Bio-Rad. Methoxypolyethylene glycol (mPEG) derivatives
(mPEG-CH2-CH2-CO-N-hydroxysuccinimide
and mPEG-O-CO-hydroxybenzotriazole esters) were obtained from
Shearwater Polymers. The percentage of substitution of each mPEG with
active ester was determined by the manufacturer, using nuclear magnetic
resonance spectroscopy, to be 
95% for all products except the
hydroxybenzotriazole carbonate ester of
Mr 2000 mPEG, for which the
substitution was 89%.
preparation of peg-substrate conjugates
Coupling of mPEG derivatives was performed by dissolving substrate
to a concentration of 100 mmol/L in 2 mL of dimethylformamide
containing 100 g/L N-ethylmorpholine and adding
0.5–1.0 molar-equivalent of mPEG active ester. Reactions were
incubated for 2 h at room temperature, and then diluted with 2 mL
of water; product was isolated in the void volume of a 2.5 x 15
cm Sephadex G-25 (coarse) column in 1 g/L acetic acid. Products
were lyophilized to a dry powder, and absorbance measurements at 342 nm
indicated a typical content of 4-NA of 80% expected from dry
weight, indicating a degree substitution of products 80%, with the
remainder being free mPEG or associated water.
Other products were prepared by coupling reactions with
diisopropylcarbodiimide using ratios of reactants to yield
predominantly monofunctional products, although the PEGs used for these
reactions contained two potential coupling sites. Monofunctional
products were desired to avoid mixtures of mono- and bifunctional
products that might not have exactly the same kinetic properties as
substrates. Succinyl-Ala-Ala-Pro-Phe-4-NA (46 mg) in 0.5 mL of
dimethylformamide was activated with 0.1 mol/L diisopropylcarbodiimide
for 10 min at room temperature and mixed with 150 mg of PEG bis(amine)
in 0.5 mL of dimethylformamide. After 1 h, the reaction mixture
was chromatographed on a Bio-Gel P-6 column in 0.1 mol/L ammonium
acetate, pH 6.0, and product was collected in the void volume.
bis-Succinimide PEG was prepared by the action of succinic anhydride on
PEG bis(amine). The bis-succinimide derivative was activated with
diisopropylcarbodiimide and coupled with substrates containing free
amino groups, such as S-2288 and S-2238. Products were isolated by
chromatography on Bio-Gel P-6. Acetylation of S-2238 was performed with
acetic anhydride.
molecular size analysis
Size exclusion chromatography was performed with a 2.5 x 26
cm Bio-Gel P-60 column monitored by spectrophotometric analysis of
column fractions. The buffer was 0.1 mol/L ammonium acetate.
Calibration is described in the legend of Fig. 1
. Hydrodynamic
sizes of macrosubstrates were estimated by comparison with protein
calibrators under nondenaturing conditions, where there is a
linear relationship of log molecular weight vs
KAV, the partition coefficient of
molecules on the column. KAV =
(Ve -
Vo)/(Vi
- Vo), where
Ve,
Vo, and
Vi are the elution, void, and included
volumes of the column.
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Figure 1. Hydrodynamic sizes of macrosubstrates as analyzed by
gel-exclusion chromatography.
Elution positions of calibrators are indicated by
arrows: arrow 1, ovalbumin
(Mr 45 000); arrow 2,
trypsinogen (Mr 24 000); arrow
3, trypsin inhibitor (Mr 20 000);
arrow 4, aprotinin (Mr 6500).
Elution of macrosubstrates (solid line) and free
substrate (dashed line) in separate chromatographic runs
was monitored at 318 nm for succinyl-Ala-Ala-Pro-Phe-4-NA
(A), D-Phe-Pip-Arg-4-NA (B),
and D-Ile-Pro-Arg-4-NA (C).
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enzyme assays
Measurements of protease activity at 37 °C were performed and
analyzed as described previously (27) with a Cobas FARA
analyzer (Roche Diagnostic Systems). All assay buffers contained 10 g/L
PEG. Thrombin assays were performed in 140 mmol/L NaCl, 10 mmol/L
HEPES, pH 7.4. Chymotrypsin and trypsin assays were performed in 10
mmol/L CaCl2, 100 mmol/L Tris, pH 7.8. Reactions
had a total volume of 100 µL and were monitored at 405 or 410 nm.
Initial rates of reactions were evaluated with Lineweaver-Burk plots.
Substrate solutions were prepared in either water for thrombin and
trypsin substrates or in 500 mL/L dimethylformamide for
succinyl-Ala-Ala-Pro-Phe-4-NA. Substrate concentrations were determined
by absorbance at 342 nm, using a molar absorptivity of 8260, and
concentrations were confirmed by absorbance at 410 nm after substrate
hydrolysis, using a molar absorptivity of 8600 for the 4-NA product
(28). Active site titration of thrombin was performed using
4-nitrophenyl-4'-guanidinobenzoic acid with standardization of the
absorbance of 4-nitrophenol under reaction conditions using a standard
solution of 4-nitrophenol. Preincubation of enzymes with
2-M was at 37 °C. The
2-M was pretreated with a 200-fold molar
excess of 4-amidinophenylmethylsulfonylfluoride to inactivate any
protease trapped within the inhibitor and left at 4 °C for 72 h
before use. Without this pretreatment, inhibitor preparations had
substantial endogenous peptidase activity for trypsin and thrombin
substrates.
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Results
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synthesis and size analysis of macrosubstrates
Covalent coupling of three 4-NA substrates,
succinyl-Ala-Ala-Pro-Phe-4-NA, D-Phe-Pip-Arg-4-NA, and
D-Ile-Pro-Arg-4-NA (Mr
500), to PEG derivatives
(Mr 3400) yielded products with a much
larger hydrodynamic size than the substrate alone. Analysis of three
macrosubstrates by gel filtration on a Bio-Gel P-60 column (Fig. 1
, solid lines) showed that each eluted slightly after
chymotrypsinogen (Mr 24 000) and
soybean trypsin inhibitor (Mr
20 000). The calibration plot indicated that the hydrodynamic size of
each conjugate was comparable to a protein of
Mr 18 000 (Fig. 1D
), corresponding
to an effective radius in solution of 20 Å (29). There
was a single major peak preceded by a minor component that may
represent dimers of the PEG derivative. The peptide and PEG components
of the macrosubstrates were joined by stable amide bonds that were
stable in aqueous solution, and no free substrate was detectable. The
elution positions of the free substrates are shown in Fig. 1
as dashed
lines from parallel runs.
It was not possible to estimate the hydrodynamic sizes of the free
substrates because they adsorbed weakly on the column and eluted in a
volume slightly greater than the column volume. The adsorption of the
free substrates reflects the hydrophobic character of 4-NA substrates,
which in some cases require organic solvents such as dimethyl sulfoxide
to prepare concentrated stock solutions. The vastly different elution
volumes of macrosubstrates and their peptidyl-4-NA components
during gel filtration provided a simple means of purifying the
macrosubstrates after reactions and obtaining material that was
completely free of uncoupled substrate for kinetic analysis of
substrate activity. The analyses in Fig. 1
confirmed that there was no
unconjugated substrate in products. A favorable property of the
macrosubstrates is high solubility in water, reflecting the dominance
of the PEG component on the physical characteristics of the
macrosubstrate. The size estimates of macrosubstrates were consistent
with previous estimates of the size of free PEG
(30)(31). PEGs have an extended random coil
structure with a relatively large hydrodynamic size per molecular
weight compared with globular proteins (30). This explains
how a macrosubstrate with a PEG of only
Mr 3400 can model the size of a small
globular protein of Mr 18 000.
kinetics of macrosubstrate cleavage
The kinetic properties of small 4-NA substrates were compared with
macrosubstrates containing the same substrate sequences (Table 1
). Control reactions were performed with PEG derivatives
without coupled substrates, and these were observed to have little
effect on enzyme reactions. Addition of PEG derivatives to a
concentration of 10 g/L caused only a 2–3% decrease in rates
of substrate cleavage by trypsin or thrombin. The maximal rates of
cleavage (Vmax) of several substrates
per molecule of chymotrypsin, trypsin, and thrombin decreased modestly
when substrates were linked to PEG to form macrosubstrates. The
affinity of enzymes for substrates was affected more substantially. The
Kms of macrosubstrates, the
concentrations yielding half-maximal rates, were higher than for the
homologous free substrate: 1.7-fold higher for a chymotrypsin
substrate, 1.4- and 4.2-fold higher for two trypsin substrates, and
2.4- and 6.2-fold higher for two thrombin substrates. Even with the
drop in affinity, the Kms for all of
the macrosubstrates were quite low, in the range of 12–110
µmol/L.
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Table 1. Comparison of kinetic properties at 37 °C of peptidyl
substrates and homologous macrosubstrates with PEG
(Mr
3400).1
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Primarily as a result of the increase in
Km, the efficiencies of these
substrates decreased approximately two- to sixfold for proteinases such
as trypsin and chymotrypsin, which have a relatively accessible
catalytic site. Efficiency of cleavage of substrates by thrombin
decreased by 3- and 20-fold when a substrate was linked to PEG,
possibly reflecting the greater steric hindrance of the thrombin active
site (32) or its extended substrate specificity
(7)(12)(13)(14). Most of the substantial loss of
efficiency in the action of thrombin on the substrate
D-Phe-Pip-Arg-4-NA appeared to result from
blockage of the N-terminal amino group, which is recognized to
contribute to the efficiency of tripeptide thrombin substrates having
D-amino acids at their NH2
terminus (14). Addition of an acetyl group to this substrate
had an even greater effect than the addition of PEG. Optimization of
macrosubstrates for thrombin will require reanalysis of structures. It
is apparent that the presence or absence of a free
NH2 terminus on the third (P3) residue preceding
the cleavage site has a major effect on substrate efficiency,
and the type of group added to this amino acid residue is likely to
exert a strong effect on substrate efficiency.
improved specificity of macrosubstrates for proteinase activity
We hypothesized that a major advantage of macrosubstrates relative
to small chromogenic substrates would be the ability to distinguish
between proteinase and peptidase activity. We examined this hypothesis
using a model system that has been described in many previous reports
(18)(19)(20)(21)(22)(23)(24). 2-M binds to and entraps
proteinases without blocking their catalytic sites. Consequently,
protein substrates are sterically hindered from reaching catalytic
sites and proteinase activity is nearly completely inhibited. Peptidase
activity, however, is retained when peptide substrates with molecular
weights <8000 are used because they are still able to reach the
catalytic site. When increasing amounts of 2-M
were added to a fixed amount of chymotrypsin before activity
measurements, activity measured with a chromogenic substrate was
maximally inhibited by 60% in the presence of excess
2-M (Fig. 2
), indicating the residual peptidase activity of the
inhibitor-proteinase complex. Activity measured with the same substrate
as a component of a macrosubstrate was inhibited by >95%. Analysis of
the inhibition of trypsin and thrombin activity by
2-M using pairs of substrates and
macrosubstrates yielded similar results (Fig. 3
). Trypsin and thrombin activities measured with tripeptide
substrates were inhibited only up to 40–50% by excess inhibitor. The
activities of macrosubstrates incorporating the same peptide sequences
were inhibited >95%. These results provide clear evidence that the
macrosubstrates selectively measured proteinase activity and that
cleavage of these substrates by the peptidase activity trapped within
2-M-proteinase complexes was greatly
reduced. Reactivity of the macrosubstrates with
2-M-proteinase complexes was decreased
10-fold relative to free tripeptide substrates.
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Figure 3. Inhibition of trypsin (top) or thrombin
(bottom) activity by increasing amounts of
2-M as measured with the small substrate
D-Ile-Pro-Arg-4-NA (S-2288) and its PEG conjugate
(top) or the small substrate
D-Phe-Pip-Arg-4-NA (S-2238) and its PEG conjugate
(bottom).
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mPEG DERIVATIVES
Use of a monofunctional polymer carrier, mPEG, has several
potential advantages over the use of PEG derivatives that have two
potential sites for attachment of substrate groups: It assures that all
macrosubstrate molecules have a single substrate group and that there
are no complex cleavage kinetics resulting from variable mixtures of
mono- and bifunctional substrates, the ratio of which would change
during the course of an enzyme reaction. There are also practical
advantages in that mPEG derivatives are commercially available with a
greater range of sizes and activated linker groups for coupling to
peptides. The size and chemical linkage of polymer to peptide
components of macrosubstrates may have a large effect on substrate
efficiency, hydrodynamic size, or steric hindrance of the substrate
site.
A series of macrosubstrates containing mPEG derivatives were examined
as thrombin substrates (Table 2
). These macrosubstrates, like the ones listed in Table 1
,
exhibited simple saturation kinetics with linear Lineweaver-Burk plots
(Fig. 4
). Addition of mPEG derivatives to substrates with one or two
amino acid residues, Arg-4-NA and Gly-Arg-4-NA, produced substantial
increases in substrate efficiency. However, the efficiencies of
these substrates measured as
kcat/Km
were still more than 1000-fold less than efficient tripeptide
substrates. The mPEG component did not fully substitute for the P3
amino acid residues that serve as critical determinants of the
efficiency of substrates for thrombin. These macrosubstrates would
serve as slow or low-efficiency substrates for monitoring large
quantities of enzyme over longer periods of time. It appears necessary
to incorporate tripeptide sequences, e.g., some of the products in
Table 1
, to produce macrosubstrates with high affinity and rates of
cleavage.
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Figure 4. Lineweaver-Burk plots of cleavage of substrates and
homologous macrosubstrates by thrombin.
The y-axes show the reciprocal of the absorbance change
at 405 nm per minute, and the x-axes show the reciprocal
of the concentration of the indicated substrates.
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The size of the mPEG component did not have a strong influence on
substrate efficiency because a macrosubstrate with a
Gly-Arg-4-NA component had similar substrate efficiency whether
the molecular weight of the mPEG component was 1000 or 5000. Linkage of
Gly-Arg-4-NA to a lysine residue bearing mPEGs with a molecular weight
of 5000 on both of its amino groups produced a substrate with
substantially reduced affinity for thrombin, possibly because steric
hindrance limited access to the substrate. Addition of mPEG to
the sidechain of a lysine residue in the P2 position of
CBZ-Lys-Arg-4-NA yielded a branched product that was >30-fold more
efficient than the original substrate, which was an extremely poor
thrombin substrate. Addition of mPEG to the tripeptide substrate
Glu-Gly-Arg-4-NA, which also is a poor thrombin substrate, had little
effect on substrate efficiency, whereas addition of mPEG to
Sar-Pro-Arg-4-NA, which is a good thrombin substrate, produced a
>50-fold loss of efficiency. It is apparent that the influence of the
addition of mPEG to small peptide substrates is highly variable
depending on the individual substrates. In some cases, the substrate
efficiency is increased and in others it is dramatically decreased.
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Discussion
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This report describes macromolecular chromogenic substrates with a
single defined cleavage specificity and simple reaction kinetics
similar to those for small substrates. The macrosubstrates have a
hydrodynamic size comparable to a small protein, and it is possible to
generate substrates of various sizes with the same cleavage specificity
by varying the size of the polymeric component. This may allow analysis
of the effect of substrate size independent of other variables. It
should be possible to produce macrosubstrates for most endoproteinases
and to generate macrosubstrates with a variety of fluorogenic or
chromogenic groups in addition to the 4-NA chromophore. Polymers other
than PEG or mPEG might be adapted to serve as the polymeric components
of macrosubstrates. However, PEG and mPEG derivatives have several
highly favorable attributes, as summarized by Harris (33).
PEGs and mPEGs are inert, uncharged polymers that assume extended
random-coil structures with a high excluded volume per molecular
weight, and the polymer is highly soluble in water and in organic
solvents that are favorable for peptide coupling and derivatization.
The wide range of commercially available polymer sizes and activated
derivatives allows the preparation of a wide range of structures. A
final critical point concerning the suitability of PEGs and mPEGs as
components of macrosubstrates is that the high substrate efficiency of
some macrosubstrates indicates that the polymeric component does not
envelope or severely limit access of proteases to the substrate
component.
Use of macrosubstrates should better model the steric factors that
affect the action of many physiological proteinases, such as
complement, fibrinolytic, and coagulation factors, where the natural
substrates are proteins of substantial size. The ability to selectively
measure proteinase rather than peptidase activity may provide, in
particular, more accurate measures of the functional activity of
proteinases in serum or plasma, where 2-M can
inhibit proteinase activity without blocking the active sites of
proteinases. 2-M has been reported to
interfere with efforts using chromogenic substrates to
directly measure proteinase activity in plasma
(23)(24), and this is a general problem with
measuring proteinase activity in serum or plasma. By avoiding this
interference, macrosubstrates may substantially improve the performance
of chromogenic assays for measuring functional activities of
coagulation factors, fibrinolytic components, complement factors,
inhibitors of physiological proteinases, and cofactors of inhibitors
such as heparin. There are many other circumstances, in addition to
complexing with 2-M, where catalytically
active proteinase molecules could have reduced proteinase activity
relative to peptidase activity because they are complexed with
antibodies, surfaces, or other factors. In these circumstances,
macrosubstrates would provide better measures of functional proteinases
than small chromogenic substrates. The PEG component of macrosubstrates
also may serve as a highly effective protecting group against
exopeptidase action to assure that only endoproteinase activity is
measured.
The ability to synthesize macrosubstrates with branched structures
where the polymeric component is linked to an amino acid sidechain may
be useful in examining the steric hindrance of particular substrate
positions for specific target enzymes. This modification together with
structural variation in linker chemistries and polymer size offers a
wide range of structural variation that may serve as additional
determinants for making substrates that are more specific for the
measurement of different proteinases. Furthermore, the specificities of
many proteinases for their physiological substrates are determined by
binding interactions of substrates with exosites that are separate from
the catalytic sites of enzymes. It may be possible to develop
macrosubstrates of much higher target specificity by making
bifunctional substrates in which one end mediates binding to an exosite
and the other serves as a substrate for the catalytic site. The
polymeric component of a macrosubstrate would then serve as a spacer to
connect these two components. Several heterobifunctional derivatives of
PEG are available that would allow selective coupling of different
functional groups to opposite ends of PEG chains.
In conclusion, the development of chromogenic substrates
of substantial size may be useful in the development of
immunoinhibition assays of specific proteinases or in efforts to
discover new inhibitors of proteinases. Use of substrates of large
physical size may enhance the steric hindrance produced by the binding
of antibodies, aptamers, or other molecules to enzymes and
expand the target zone of inhibition to additional epitopes or binding
sites that are neighboring but not immediately part of the active site.
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Footnotes
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1 Nonstandard abbreviations: 4-NA, 4-nitroanilide; 2-M, 2-macroglobulin; Pip, pipecolic acid; Sar, sarcosine (N-methyl glycine); CBZ, carbobenzoxy; PEG, polyethylene glycol; and mPEG, methoxypolyethylene glycol. 
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References
|
|---|
-
Fischer E, Abderhalden E. Über das Verhalten einiger Polypeptide gegen Pankreassaft [The behavior of some polypeptides toward pancreatic juice]. Z Physiol Chem 1907;51:264-268.
-
Bergmann M. A classification of proteolytic enzymes. Adv Enzymol 1942;2:49-68.
-
Schwert GW, Neurath H, Kaufman S, Snoke JE. The specific esterase activity of trypsin. J Biol Chem 1948;172:221-239.[Free Full Text]
-
Sherry S, Troll W. The action of thrombin on synthetic substrates. J Biol Chem 1954;208:95-105.[Free Full Text]
-
Erlanger BF, Kokowsky N, Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961;95:271-278.[ISI][Medline]
[Order article via Infotrieve]
-
Tuppy H, Wiesbauer U, Wintersberger E. Aminosäure-p-nitroanilide als Substrate für Aminopeptidasen und andere proteolytische Fermente. Z Physiol Chem 1962;329:278-288.
-
Svendsen L, Blombäck B, Blombäck M, Olsson PI. Synthetic chromogenic substrates for determination of trypsin, thrombin, and thrombin-like enzymes. Thromb Res 1972;1:267-278.
-
Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 1967;27:157-162.[ISI][Medline]
[Order article via Infotrieve]
-
Morihara K, Oka T, Tsuzuki H. Comparison of -chymotrypsin and subtilisin BPN': size and specificity of the active site. Biochem Biophys Res Commun 1969;35:210-214.[Medline]
[Order article via Infotrieve]
-
Fruton JS. The specificity and mechanism of pepsin action. Adv Enzymol 1970;22:401-443.
-
Thompson RC, Blout ER. Dependence of the kinetic parameters for elastase-catalyzed amide hydrolysis on the length of peptide substrates. Biochemistry 1973;12:57-65.[Medline]
[Order article via Infotrieve]
-
Hemker HC. Handbook of synthetic substrates for the coagulation and fibrinolytic system 1983 Martinius Nijhoff Publishing The Hague. .
-
Lottenberg R, Hall JA, Blinder M, Binder EP, Jackson CM. The action of thrombin on p-nitroanilide substrates: substrate specificity and examination of hydrolysis under different reaction conditions. Biochim Biophys Acta 1983;742:539-557.[Medline]
[Order article via Infotrieve]
-
Claeson G. Synthetic peptides and peptidomimetics as substrates and inhibitors of thrombin and other proteases in the blood coagulation system. Blood Coagul Fibrinolysis 1994;5:411-436.[ISI][Medline]
[Order article via Infotrieve]
-
McRae BJ, Kurachi K, Heimark RL, Fujikawa K, Davie EW, Powers JC. Mapping of the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates. Biochemistry 1981;20:7196-7206.[Medline]
[Order article via Infotrieve]
-
Zimmerman M, Ashe B, Yurewicz EC, Patel G. Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates. Anal Biochem 1977;78:47-51.[ISI][Medline]
[Order article via Infotrieve]
-
Butenas S, DiLorenzo ME, Mann KG. Ultrasensitive fluorogenic substrates for serine proteases. Thromb Haemost 1997;78:1193-1201.[ISI][Medline]
[Order article via Infotrieve]
-
Harpel PC, Mosesson MW. Degradation of human fibrinogen by plasma 2-macroglobulin-enzyme complexes. J Clin Invest 1973;52:2175-2184.
-
Kueppers F, Abrams WR, Weinbaum G, Rosenbloom J. Resistance of tropoelastin and elastin peptides to degradation by 2-macroglobulin-protease complexes. Arch Biochem Biophys 1981;211:143-150.[Medline]
[Order article via Infotrieve]
-
Barrett AJ. 2-Macroglobulin. Methods Enzymol 1981;80:737-754.
-
Sottrup-Jensen L. -Macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem 1989;264:11539-11543.[Free Full Text]
-
Qazi U, Gettins PGW, Strickland DK, Stoop JK. Structural details of proteinase entrapment by human 2-macroglobulin emerge from three-dimensional reconstructions of Fab labeled native, half-transformed, and transformed molecules. J Biol Chem 1999;274:8137-8142.[Abstract/Free Full Text]
-
Mackie IJ, Gallimore M, Machin SJ. Contact factor proteases and the complexes formed with 2-macroglobulin can interfere in protein C assays by cleaving amidolytic substrates. Blood Coagul Fibrinolysis 1992;3:589-595.[Medline]
[Order article via Infotrieve]
-
Rijkers DTS, Wielders SJH, Beguin S, Hemker HC. Prevention of the influence of fibrin and 2-macroglobulin in the continuous measurement of the thrombin potential: implications for an endpoint determination of the optical density. Thromb Res 1998;89:161-169.[ISI][Medline]
[Order article via Infotrieve]
-
Anson ML. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol 1938;22:79-91.[Free Full Text]
-
Kunitz M. Crystalline soybean trypsin inhibitor. II. General properties. J Gen Physiol 1947;30:291-310.[Abstract/Free Full Text]
-
Hortin GL, Trimpe BL. Allosteric changes in thrombin’s activity produced by peptides corresponding to segments of natural inhibitors and substrates. J Biol Chem 1991;266:6866-6871.[Abstract/Free Full Text]
-
Lottenberg R, Jackson CM. Solution composition dependent variation in extinction coefficients for p-nitroaniline. Biochim Biophys Acta 1983;742:558-564.[Medline]
[Order article via Infotrieve]
-
Tarvers RC, Church FC. Use of high-performance size exclusion chromatography to measure protein molecular weight and hydrodynamic radius. An investigation of the properties of the TSK 3000 SW column. Int J Pept Protein Res 1985;26:539-549.[ISI][Medline]
[Order article via Infotrieve]
-
Squire PG. Hydrodynamic characterization of random coil polymers by size exclusion chromatography. Methods Enzymol 1985;117:142-153.[ISI][Medline]
[Order article via Infotrieve]
-
Bhat R, Timasheff SN. Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols. Protein Sci 1992;1:1133-1143.[Abstract]
-
Bode W, Mayr I, Baumann U, Huber R, Stone SH,
Hofsteenge J. The refined 1.9 Å crystal structure of human
-thrombin: interaction with D-Phe-Pro-Arg
chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion
segment. EMBO J 1989;8:3467–75..
-
Harris JM. Introduction to biotechnical and biomedical
applications of poly (ethylene glycol). In: Harris JM, ed.
Poly(ethylene glycol) chemistry. New York: Plenum Press, 1992:1–14..
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Abstract
Introduction
), or the homologous macrosubstrate
(
).