SERRS-based enzymatic probes for the detection of protease activity

Article abstract of DOI:10.1021/ja803655h

Measurement of protease activity, for the first time using SERRS as a detection method, is reported herein. Synthetic introduction of phenylalanine to a benzotriazole azo dye allows the SERRS response to be switched off and subsequent exposure to protease restores the SERRS response. The substrates exhibit varying reactivity for a range of proteases and allow for in situ, real-time analysis of protease reactivity. A limit of detection for one protease, Subtilisin carlsberg, was investigated and was established to be 50 ng ml^-1. Copyright

Full text of DOI:10.1021/ja803655h

Published on Web 08/14/2008
SERRS-Based Enzymatic Probes for the Detection of Protease Activity
Andrew Ingram, Louise Byers, Karen Faulds, Barry D. Moore, and Duncan Graham*
Centre for Molecular Nanometrology, WestCHEM, Department of Pure and Applied Chemistry, UniVersity of
Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, U.K.
Received May 21, 2008; E-mail: duncan.graham@strath.ac.uk
Scheme 1a
Surface enhanced resonance Raman scattering (SERRS) has
proven to be a powerful analytical tool for the detection of
enzymatic activity. Recent cases have included phosphatase,
peroxidase,1-3 lipase,4 and now for the first time determination of
protease activity, as reported herein.
From the human genome data, it is now known that more than
500 genes encode proteases.5 Proteases mediate nonspecific protein
hydrolysis, but also have a vital role in post-translational modifica-
tions of proteins, performing selective and efficient cleavage of
specific substrates.6 Synthetic probes for the detection of protease
activity are therefore essential, and most probes use fluorescence
detection. Fluorescence can provide very sensitive monitoring, but
the technique can suffer from background fluorescence when
analyzing cellular extracts.
A detection technology whereby rapid, simultaneous detection
a Conditions: (i) CMCS, NaHCO3, Bu4NHSO4, DCM/H2O, 99%; (ii) (a)
NaOH, H2O; (b) acetone, 13%; (iii) TFA, 0 °C, 99%.
of multiple enzyme activities could be measured at concentrations
found in cellular environments would be of great interest. Surface-
enhanced resonance Raman spectroscopy (SERRS) has the potential
to meet these criteria. SERRS has been used extensively for DNA
detection,7-12 but there have been few reported cases of its use as
a detection method for enzymatic activity. Phenylenediamine can
be transformed into a SERRS-active compound by reaction with
peroxidase and has been used to detect peroxidase-conjugated
antibody for use in immunoassays.1-3 Ruan et al. report that
5-bromo-4-chloro-3-indolyl phosphate (BCIP) forms a SERRS
active species upon reaction with alkaline phosphatase.13 BCIP is
traditionally used for enzyme detection by colorimtery, and it is
likely it is the indigo dye product that is SERRS active.
Previously we reported a SERRS-based technique for lipase
detection, where the surface seeking moiety of a dye, in this case
benzotriazole, was covalently alkylated, thus binding to the metal
surface was prevented and surface enhancement of the Raman
scattering was limited.4,14 By careful design of the covalent mask,
it can be recognized and selectively cleaved by specific enzymes,
thus regenerating a surface seeking species and accordingly an
increase in SERRS response clearly detectable.
This study shows how the technique has been adapted to the
synthesis of masked protease substrates. An amino acid ester, in
this case phenylalanine, was covalently attached to a SERRS active
species via chloromethyl ester 1 and 2 (scheme 1). The chloromethyl
ester was prepared by reaction of chloromethyl chlorosulfate
(CMCS) with Boc-protected phenylalanine under phase-transfer
conditions.15 Both D and L isomers of phenylalanine were attached
by this method. Furthermore a deprotection protocol for the removal
of Boc from 4, to afford amino analogue 6, was developed. It was
found that Boc deprotection would proceed smoothly in neat TFA
after 15 min treatment. The compound is stable if the amine is
quaternized as the TFA salt, but the free amine species is unstable
if stored for longer than 24 h. Compatibility with a Boc deprotection
strategy demonstrates the potential to incorporate masked amino
acids such as 4 into a longer synthetic peptide sequence, which
Figure 1. SERRS obtained from 4 and 5 upon treatment with Subtilisin
carlsberg for 30 min. (Inset) Monitoring emergence of peak at 1419 cm-1
over time for 4 and 5 upon exposure to Subtilisin carlsberg 0.1 mg ml-1
.
may be necessary if the protease in question requires a specific
multiple amino acid sequence. A colloidal silver suspension
containing either masked enantiomer 4 or 5, at a concentration of
10-7 M gave no SERRS spectra under the experimental conditions
employed. Upon treatment with Subtilisin carlsberg the SERRS
response of 4 was significantly increased (Figure 1). This was
attributed to the generation of the SERRS active compound 3 by
enzymatic cleavage, the spectra being identical to that obtained in
previous enzymatic studies where the same masked SERRS
substrate had been used,4 and also identical to SERRS spectra
recorded of 3 alone. When the experiment was repeated with D-Boc-
phenylalanine enantiomer 5, no detectable increase in SERRS was
observed, suggesting that no enzymatic hydrolysis of 5 had taken
place. This is not unexpected as the enzyme should have enan-
tiospecificity for the natural substrate and means that the D isomer
can be used as a negative control in future experiments.
When tested against a selection of proteases, a broad range of
SERRS responses was observed after 30 min incubation (Fig-
9
11846 J. AM. CHEM. SOC. 2008, 130, 11846–11847
10.1021/ja803655h CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
The detection of protease activity using SERRS and modified
substrates has been successfully achieved. The substrate was shown
to be L selective, selective for proteases over lipases, and for
subtilisin the sensitivity of the technique was at least 50 ng ml-1
.
Incorporation of different amino acid masking groups will be
straightforward, and any Boc-protected amino acid (with side-chain
protection where appropriate) can be used, most of which are
commercially available. This approach offers a highly sensitive
screening method for activity of protease variants, for example
generated through a directed evolution format. This preliminary
study offers the prospect of multiplexed protease activity measure-
ments by incorporation of varying amino acid sequences attached
to SERRS dyes with different chromophores.
Figure 2. SERRS obtained from 4 upon 30 min treatment with (a)
R-Chymotrypsin, (b) Subtilisin carlsberg, (c) Trypsin, (d) Proteinase K, (e)
Pepsin, (f) blank, in each case with the enzyme concentration at 0.01 mg
ml-1
.
Acknowledgment. The authors would like to thank the Royal
Society of Chemistry’s Analytical Chemistry Trust Fund for funding
D.G. through the award of the Analytical Grand Prix and A.I. as a
studentship.
Supporting Information Available: Full protocols for enzyme
reactions, spectroscopic experiments, additional spectroscopic experi-
ments using lipases and synthesis of masked substrates. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
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Figure 3. SERRS obtained from 4 upon exposure to Subtilisin carlsberg,
at concentration of (a) 0.5, (b) 0.25, (c) 0.05, and (d) 0.005 µg ml-1. (Inset)
Enzyme concentration against SERRS response at 1419 cm-1. Reaction
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at enzyme concentrations as low as 50 ng ml-1
.
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