A general method for detecting protease activity via gelation and its application to artificial clotting

Article abstract of DOI:10.1039/c2cc31537h

A modular system for detecting protease activity via enzyme-triggered gel formation is described. Protease-specific recognition sequences are utilized to achieve enzyme specificity. Artificial blood clotting is demonstrated by activating endogenous thrombin to trigger gelation in fibrinogen-deficient blood plasma.

Full text of DOI:10.1039/c2cc31537h

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A general method for detecting protease activity via gelation and its
application to artificial clottingw
Steven C. Bremmer,a Jing Chen,b Anne J. McNeil*b and Matthew B. Soellner*a
Received 29th February 2012, Accepted 29th March 2012
DOI: 10.1039/c2cc31537h
A modular system for detecting protease activity via enzyme-
triggered gel formation is described. Protease-specific recognition
sequences are utilized to achieve enzyme specificity. Artificial
blood clotting is demonstrated by activating endogenous thrombin
to trigger gelation in fibrinogen-deficient blood plasma.
involves a protease-triggered gelation using matrix metallo-
proteinase-7 (MMP-7), a protein that is over-expressed in
some cancers.10 Goto and co-workers used a recognition
sequence to achieve selectivity and demonstrated that gelation
could be triggered under physiologically relevant concentrations.10
The limitation of this system is that the recognition sequence
remains on the gelator after the cleavage event. This requirement
complicates the application to other proteases because each
protease has its own unique recognition sequence and a novel
gelator containing the recognition sequence must be identified for
every protease of interest. Given that switching a single amino acid
residue within a peptide can disrupt gelation,11 this requirement
will significantly limit the overall utility of this method.
The discovery that small peptides can reversibly form gels has
sparked intense research into these new biomaterials.1 The gels
themselves are being investigated for applications such as tissue
engineering2 and lab-on-a-chip platforms.3 The stimuli-responsive
nature of the gelation process, on the other hand, is being used in
applications including drug delivery4 and regenerative medicine.5
Peptides are advantageous building blocks for all these applications
because of their biocompatibility and facile synthesis. Moreover,
peptide-based gels can be triggered to form (or breakdown) by
simply changing the pH, temperature, or ionic strength.
We hypothesized that an enzyme-triggered cleavage at the
N-terminus of a peptide gelator, which separates the recognition
sequence from the gelator, would represent a general and modular
strategy for detecting enzymatic activity via gelation (Scheme 1).
The recognition sequence provides specificity and optimizing the
gelator structure should enable detection of physiologically relevant
enzyme concentrations. Proteases were selected in this study based
on their important role in many biological processes, including
blood clotting, apoptosis, and pathogenesis.12
Because many diseases are correlated with overactive and/or
overexpressed enzymes, a simple visual assay based on enzyme-
triggered gelation of peptides is appealing for both detection
and diagnosis.6 Although several enzyme-triggered gelations
have been reported over the last decade,7 few systems are
suitable for detecting physiological concentrations of enzymes.
For example, Xu and co-workers developed several gelations
based on a phosphatase-triggered dephosphorylation reaction,
which induces gelation by changing the peptide solubility.8
These systems are not suitable for detection, however, because
the reaction is not selective due to the inherent promiscuity of
phosphatase enzymes (i.e., most tyrosine phosphatases will
trigger gelation) and non-physiological concentrations of
enzymes were needed. In a different example, Xu and co-workers
were able to use physiologically relevant enzyme concentrations
in a b-lactamase-triggered gelation.9 Nevertheless, the system was
not demonstrated to be generalizable to other enzymes. The only
reported enzyme-triggered gelation that is suitable for sensing
The first challenge was to identify a peptide-based gelator
with an N-terminal amine. To avoid complications arising
from protonation of the amine, p-aminobenzoic acid (PABA),
which has an approximate pKa of 5, was selected as the first
building block of the peptide-based gelator. At neutral pH, this
amine remains uncharged. In addition, peptides containing a related
anilide linkage have been successfully cleaved by proteases.13
Several different hydrophobic amino acids were then appended
to PABA and the resulting compounds were screened for gelation
in buffer (100 mM PBS, 10% DMSO, pH = 7.2) using the
heat/cool method (ESIw). Of these, PABA-F5Phe-NH2 (1) was
found to form gels at neutral pH.14 Nevertheless, the critical
gel concentration (cgc) was 54 mM, which is insufficient for
detecting physiological concentrations of enzyme. To lower
the cgc, an additional amino acid residue (Phe) was appended
and the resulting compound (2) was found to form gels in
a Departments of Medicinal Chemistry and Chemistry,
University of Michigan, 930 North University Avenue, Ann Arbor,
Michigan, 48109-1055, USA. E-mail: soellner@umich.edu
b Department of Chemistry and Macromolecular Science and
Engineering Program, University of Michigan,
930 North University Avenue, Ann Arbor, Michigan, 48109-1055,
USA. E-mail: ajmcneil@umich.edu
w Electronic supplementary information (ESI) available: experimental
data; gel rheology and morphology characterization data; control
experiments. See DOI: 10.1039/c2cc31537h
Scheme 1 Design strategy of a modular sensor for enzymatic activity
based on gelation.
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buffer (100 mM PBS, 10% DMSO, pH = 7.2) with a cgc of
1.7 mM. A similar dipeptide was then prepared, this time
with the unnatural D-amino acids (3). The rationale was that
this dipeptide would be more resistant to proteolysis while
maintaining an identical ability to gel.15 The resulting gel was
comprised of nanometer-sized fibers and exhibited characteristic
viscoelastic behavior under external stress and strain, similar to
other peptide-based gels (ESIw).1
Fig. 1 A clear gel is observed within 10 min of adding thrombin
(50 nM) to compound 4 (4.4 mM) in buffer (400 mL, 100 mM PBS,
10% DMSO, pH = 7.2).
The physiological function of thrombin is to cleave fibrinogen
into fibrin, which triggers blood clot formation.18 While rare,
deficiencies in fibrinogen can lead to fatal hemorrhages.19 We
anticipated that our gelator could be used to cause artificial
clotting in patients with low fibrinogen levels. Indeed, an
opaque gel was observed within 30 min of adding compound
4 and thrombin (59 nM) to fibrinogen-depleted human blood
plasma, indicating that gelation can occur in this complex
medium. Control samples missing either compound 4 or
thrombin failed to form a gel after 24 h (ESIw). The fibrinogen-
deficient plasma contains endogenous thrombin, in the form of
unactivated prothrombin (coagulation factor II). This endogenous
thrombin can be activated by adding either thromboplastin or
calcium.18,19 Excitingly, gelation occurred within 2 h of adding
thromboplastin and within 20 h of adding calcium to blood
plasma containing compound 4 (Fig. 2 and ESIw). Thus, we were
able to create artificial clotting in human blood plasma deficient
with fibrinogen, a key component of the blood-clotting pathway.
This gelation method was designed to be modular and easily
extended to other enzymes. To demonstrate the generality of
this approach, two additional proteases with highly distinct
recognition sequences were targeted: chymotrypsin (Ala-Ala-
Pro-Phe)20 and Glu-C (Asp-Ala-Phe-Glu).21 We reasoned
that a highly hydrophobic recognition sequence (as with
chymotrypsin) and a dianionic recognition sequence (as found
in Glu-C) would test the limits of recognition sequences
compatible with our system. Using the same gelator and
solubility factor, two new compounds were prepared (ESI).
In a series of experiments highlighted in Fig. 3, gelation was
observed to be specific to each enzyme based solely on the
recognition sequence. For example, chymotrypsin was unable
Thrombin, a blood-clotting enzyme, was initially selected
because its recognition sequence is well defined, and the
enzyme is both specific and highly active.16 To increase
solubility and prevent gelation, an oligo(ethylene glycol) unit
and three hydrophilic D-amino acids were added to the
N-terminal end of the recognition sequence. D-Amino acids
were again selected for their resistance to proteolysis.15 The
resulting compound (4) did not form a gel under any conditions
examined and was soluble in buffer (100 mM PBS, 10% DMSO,
pH = 7.2) to concentrations greater than 12 mM (Scheme 2).
To test the enzyme-triggered gelation, compound 4 (4.4 mM)
was treated with thrombin (50 nM) in buffer (400 mL, 100 mM
PBS, 10% DMSO, pH = 7.2). Within 10 min, a translucent gel
was observed (Fig. 1). Aliquots were taken at various times and
analyzed by LC-MS to determine conversion. These studies
revealed that approximately 40% of compound 4 had been
cleaved when gelation occurred, which corresponds to about
1.7 mM of released 3 (ESIw). It is important to note that
off-target cleavage products were not observed by LC-MS,
indicating that the enzyme-triggered gelation is specific to the
recognition sequence. The lowest concentration of thrombin that
we could detect using this method was 400 pM (ESIw). Of note,
physiological concentrations of thrombin in the body are low
nanomolar (0.1–3 nM), indicating that our system functions at
physiological concentrations.17 Adding a thrombin inhibitor
(1 mM PMSF) led to the expected decrease in the rate of
enzymatic cleavage and gelation was not observed even after
24 h. A second control, wherein no thrombin was added, remained
a non-viscous solution, indicating that compound 4 is stable under
these conditions. Combined, these controls demonstrate that
gelation is dependent upon specific thrombin activity.
Fig. 2 An opaque gel is observed within 2 h of adding thromboplastin
(170 mL of a 1 mg mLÀ1 solution) to compound 4 (12 mM) in fibrinogen-
deficient blood plasma (130 mL). This photo was taken after 20 h.
Because thrombin is involved in the blood-clotting cascade,18 we
examined whether an artificial clot could be formed via this method.
Fig. 3 Each vial contains B 4 mM of the indicated compound and
50 nM enzyme in buffer (400 mL, 100 mM PBS, 10% DMSO, pH = 7.2)
where THR = thrombin and CHY = chymotrypsin. Although all gels
were stable to inversion after 2 h, these photos were taken after 20 h.
Scheme 2 Thrombin-selective compound 4.
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to induce gelation of either the thrombin or Glu-C progelator.
Moreover, the enzyme concentrations (50 nM) were physiologically
relevant. These results demonstrate that the modular system
described herein is quite general to other proteases.
R. H. Zha and S. I. Stupp, Curr. Opin. Solid State Mater. Sci.,
2011, 15, 225–235.
6 For a recent review, see: A. Razgulin, N. Ma and J. Rao, Chem.
Soc. Rev., 2011, 40, 4186–4216.
7 For recent reviews of enzyme-triggered gelations, see:
(a) Y. Zhang, Y. Kuang, Y. Gao and B. Xu, Langmuir, 2011, 27,
529–537; (b) R. J. Williams, R. J. Mart and R. V. Ulijn,
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8 For recent examples, see: (a) X. Li, Y. Gao, Y. Kuang and B. Xu,
Chem. Commun., 2010, 46, 5364–5366; (b) Q. Wang, Z. Yang,
Y. Gao, W. Ge, L. Wang and B. Xu, Soft Matter, 2008, 4, 550–553.
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41, 315–326.
In summary, a modular system for detecting protease
activity using an enzyme-triggered gelation has been developed.
Important features include the ability to simply change the
recognition sequence to target other proteases and the use of
physiologically relevant enzyme concentrations. We further
demonstrated that artificial blood clots could be triggered in
human blood plasma using gel formation. One could imagine
using our thrombin-activated gelation in bandages to promote
blood clotting where severe trauma has occurred. Furthermore,
we anticipate that this system will lead to facile assays for
detection and diagnosis of disease-relevant proteases, and these
studies are currently underway.
9 Z. Yang, P.-L. Ho, G. Liang, K. H. Chow, Q. Wang, Y. Cao,
Z. Guo and B. Xu, J. Am. Chem. Soc., 2007, 129, 266–267.
10 D. Koda, T. Maruyama, N. Minakuchi, K. Nakashima and
M. Goto, Chem. Commun., 2010, 46, 979–981.
11 Seemingly minor structural changes can convert peptide-based
gelators into nongelators. For recent examples, see:
(a) M. L. Muro-Small, J. Chen and A. J. McNeil, Langmuir,
2011, 27, 13248–13253; (b) H. Wang, C. Yang, M. Tan,
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S. Revel, K. Morris, L. C. Serpell and D. J. Adams, Langmuir,
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We thank Ms. Anne Juggernauth for assistance with the
AFM measurements, the Office of Naval Research (N00014-
09-1-0848 to A.J.M.) and National Institutes of Health
(R00RR024366 to M.B.S.) for support of this work.
Notes and references
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