Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

Article abstract of DOI:10.1016/j.ab.2013.12.032

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp = py

Full text of DOI:10.1016/j.ab.2013.12.032

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Analytical Biochemistry xxx (2014) xxx–xxx
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Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
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Selective chromogenic and fluorogenic peptide substrates for the assay
of cysteine peptidases in complex mixtures
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Tatiana A. Semashko a, Elena A. Vorotnikova b, Valeriya F. Sharikova a, Konstantin S. Vinokurov c,
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Yulia A. Smirnova d, Yakov E. Dunaevsky d, Mikhail A. Belozersky d, Brenda Oppert e, , Elena N. Elpidina d,
⇑
Irina Y. Filippova a
a Department of Chemistry, Moscow State University, Moscow 119992, Russia
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b Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow 119992, Russia
c
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ꢀ
Entomological Institute, Biology Centre AV CR, Ceské Budejovice CZ-37005, Czech Republic
d A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia
e USDA Agricultural Research Service, Center for Grain and Animal Health Research, Manhattan, KS 66502, USA
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases
of the C1 papain family, important in many biological processes. The structure of the newly synthesized
substrates is Glp-Xaa-Ala-Y (where Glp = pyroglutamyl; Xaa = Phe or Val; and Y = pNA [p-nitroanilide],
AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were
synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target com-
pounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized
substrates was evaluated by C1 cysteine peptidases from different organisms and with different func-
tions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L.
The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic,
or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates
during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle,
Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were
able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. mol-
itor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures
containing peptidases from different classes.
Received 20 September 2013
Received in revised form 20 December 2013
Accepted 23 December 2013
Available online xxxx
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Keywords:
Cysteine peptidases
Substrates of peptidases
Selective peptide substrates
Multicomponent enzyme mixtures
Enzymatic peptide synthesis
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Published by Elsevier Inc.
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Peptide hydrolases of the C1 (papain) family belong to clan
CA of cysteine peptidases containing a catalytic diad, Cys and
His. Two other active site residues are found: a Gln residue pre-
ceding the catalytic Cys and an Asn residue following the cata-
lytic His. The Gln residue is believed to help in the formation
of the ‘‘oxyanion hole,’’ and the Asn is believed to orientate
the imidazolium ring of the catalytic His [1–3]. Cysteine pepti-
dases have been identified and studied in detail in different
organisms—viruses [4,5], bacteria [6,7], protozoa [8–16], plants
[1,17–21], and mammals [3,17,22,23]. The ancestor of this fam-
ily, a plant peptidase papain from papaya, as well as closely re-
lated peptidases from the tropical fruits bromelain (pineapple)
and ficin (fig latex) have important applications in the food
industry, pharmacology, and scientific research [1,24,25]. A large
group of C1 family cysteine peptidases are lysosomal cysteine
cathepsins, described mainly in mammals and humans
[3,26,27]. Cysteine cathepsins are responsible for nonselective
lysosomal protein degradation, but they also participate in more
specific processes such as activation of zymogens, hormone mat-
uration, and antigen presentation, among others. However, cys-
teine peptidases also are indicated in the development of
different pathologies such as osteoporosis, rheumatoid arthritis,
atherosclerosis, cancer metastasis, and tumor invasion [26,28–
35]. Digestive cysteine peptidases also have been described in
some insect pests, including some families of beetles, aphids,
and thrips [36–45].
Given the fact that cysteine peptidases are widespread and
important, highly specific and effective peptide substrates are
needed. Although cysteine peptidases of the papain family have
broad substrate specificity, activity assays for these enzymes have
been limited to a set of Arg-containing substrates, starting from the
⇑
Corresponding author. Fax: +1 785 537 5584.
E-mail address: brenda.oppert@ars.usda.gov (B. Oppert).
0003-2697/$ - see front matter Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.ab.2013.12.032
Please cite this article in press as: T.A. Semashko et al., Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in
complex mixtures , Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2013.12.032

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most simple methyl p-nitrophenyl esters and amides of benzoyl-
Arg (Bz-Arg)1 [46–48]. However, these substrates are rarely used
now because of inherent problems; they lack sensitivity, have low
solubility, and sometimes spontaneously hydrolyze (in the case of
p-nitrophenyl esters).
of water, was obtained from Lekbiofarm (Russia). Dimethylform-
amide (DMF) and triethylamine (TEA) of analytical grade (Reak-
him, Russia) were further purified by the method in Ref. [62].
Trifluoroacetic acid (TFA) of analytical grade was obtained from
Fluka. Acetic acid of analytical grade was obtained from Reakhim.
Ethylenediaminetetraacetic acid (EDTA), calcium chloride, and
dithiothreitol (DTT) were obtained from Sigma–Aldrich. Sephadex
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The most popular cysteine peptidase substrates are the short
chromogenic peptides Z-Phe-Arg-pNA (where pNA = p-nitroani-
lide), Z-Arg-Arg-pNA [17,49–52], and their fluorogenic analogs
Z-Phe-Arg-AMC (where AMC = 4-amino-7-methylcoumaride), Z-
Phe-Arg-AFC (where AFC = 4-amino-7-trifluoromethyl-coumaride),
Z-Arg-Arg-AMC, and Z-Arg-Arg-AFC [50,53–56]. These compounds
are moderately soluble and highly stable in water-based solvents,
and they have kinetic characteristics desirable in peptidase assays.
With these substrates, the values of Km for the model enzyme pa-
pain vary from 0.08 to 32 mM and the values of kcat vary from 0.2
to 34 sꢀ1 [50]. However, Arg- and Lys-containing substrates are also
hydrolyzed by trypsin-like serine peptidases; therefore, these sub-
strates are not selective toward C1 cysteine peptidases. In addition,
the production of arginine-containing substrates by a complex mul-
tistage chemical synthesis is complicated [56].
Previously, we enzymatically synthesized the selective chromo-
genic peptide substrate Glp-Phe-Leu-pNA (where Glp = pyrogluta-
myl) [57]. This substrate is specific for papain-like cysteine
peptidases and was successfully used in the purification and eval-
uation of cysteine peptidases of various origins and levels of purity
[38,57]. However, the use of this substrate is limited by its low sol-
ubility in water and the need to use high concentrations of organic
solvents (20% dimethylformamide or dimethyl sulfoxide). To avoid
these problems, we found that chromogenic and fluorogenic ana-
logues of this substrate, containing an Ala residue in the P1 posi-
tion, were better substrates for cysteine peptidases [58,59], and
one of them, Glp-Phe-Ala-pNA, was successfully used to character-
ize complexes of digestive peptidases from insects [43–45,60].
In the current article, we have broadened the set of substrates
containing the Ala residue in the P1 position, modified the tech-
niques of synthesis, and demonstrated the selectivity of the sub-
strates for the characterization of C1 cysteine peptidases either in
a homogeneous state or as part of complex multicomponent mix-
tures of digestive enzymes in the stored products pests Tenebrio
molitor and Tribolium spp. (Coleoptera: Tenebrionidae).
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G-100 was obtained from Pharmacia (Sweden).
do-4-(trifluoromethyl)coumarin trifluoroacetate
L
-Alanine 7-ami-
-Ala-AFC ꢁ T-
-Ala-
L-Ala-pNA), and chromogenic
88
(L
89
FA),
L-alanine 7-amido-4-methylcoumarin trifluoroacetate (
L
90
AMC ꢁ TFA),
L-alanine p-nitroanilide (
91
substrates Bz-Arg-pNA, Z-Arg-Arg-pNA, and Z-Phe-Arg-pNA were
obtained from Bachem (Switzerland). Derivatives of other amino
acids and peptides were synthesized in our laboratory by standard
techniques [63].
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Synthesis of substrates by enzymes in solution
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98
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153
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To begin the synthesis of Glp-Phe-Ala-pNA, 1.5 mg of
trypsin was added to a solution containing 112 mg (0.53 mmol) of
-Ala-pNA and 154 mg (0.53 mmol) of pyroglutamyl-phenylalanine
a-chymo-
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L
methyl ester (Glp-Phe-OCH3) [59] in 0.53 ml of DMF and 3 ml of
0.2 M Na2CO3–NaHCO3 buffer (pH 9.9). After 5 min, a voluminous
precipitate was formed. The mixture was stirred for 1 h at 20 °C
and left for 10 h at 0 °C. The precipitate was centrifuged, washed
with 5% citric acid and water, and dried over NaOH in a vacuum.
Synthesis with subtilisin Carlsberg was performed as described
above except that 0.2 M Tris–HCl buffer (pH 8.2) was used instead
of the sodium carbonate buffer.
Glp-Phe-Ala-AFC was synthesized by the procedure similar to
that described above for Glp-Phe-Ala-pNA, using 29 mg (0.1 mmol)
Glp-PheOMe and 41 mg (0.1 mmol) CF3COOHꢂAla-AFC. All other
enzymes and buffers were the same.
163
Synthesis of substrates by immobilized enzymes
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Alternatively, substrates Glp-Phe-Ala-pNA (I), Glp-Val-Ala-pNA
(II), Glp-Phe-Ala-AMC (III), and Glp-Phe-Ala-AFC (IV) were synthe-
sized using immobilized chymotrypsin and subtilisin. Preparations
of immobilized enzymes on poly(vinyl alcohol)–cryogel carrier
were obtained according to the procedure in Ref. [64]. All sub-
strates (I–IV) were obtained in analytical quantities in an anhy-
drous medium of polar organic solvents (DMF–MeCN, 20:80,
vol%) in one cycle for 24 h according to the previously described
method [59] using Glp-X-OMe (where X = Phe or Val), and Ala-Y
(where Y = pNA, AMC, or AFC) as starting compounds. Glp-Phe-
Ala-pNA (I) and Glp-Phe-Ala-AMC (III) were also synthesized
during six and three consequent cycles of peptide production,
respectively, according to the procedure described in Ref. [64].
After each cycle, the beads of biocatalyst were rinsed twice with
the solvent mixture. Glp-Phe-Val-pNA and Glp-Phe-Ala-AFC were
also obtained in preparative quantities as described previously
[59]. The physic-chemical characteristics of substrates are pre-
sented in Table S1 of the online Supplementary material.
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Materials and methods
Enzymes
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Papain (EC 3.4.22.2), stem bromelain (EC 3.4.22.32), ficin (EC
3.4.22.3), cathepsin B (EC 3.4.22.1), cathepsin L (EC 3.4.22.15), bo-
vine trypsin (EC 3.4.21.4), and subtilisin Carlsberg (EC 3.4.21.62)
were obtained from Sigma–Aldrich (USA). Thermolysin (EC
3.4.24.27) and
a-chymotrypsin (EC 3.4.21.1) were obtained from
Fluka (Switzerland). Porcine pepsin (EC 3.4.23.1) was purified as
described previously [61].
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Chemicals
182
Analyses of synthesized compounds
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131
Acetonitrile (MeCN) for high-performance liquid chromatogra-
phy (HPLC), special purity grade containing not more than 0.01%
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Thin-layer chromatography (TLC) was performed on an East-
man Chromogram Sheet (Kodak, USA) in the following systems:
(A) n-butanol–pyridine–water–acetic acid (15:12:10:3); (B) ace-
1
Abbreviations used: Bz, benzoyl; pNA, p-nitroanilide; AMC, 4-amino-7-meth-
ylcoumaride; AFC, 4-amino-7-trifluoromethyl-coumaride; Glp, pyroglutamyl; MeCN,
acetonitrile; HPLC, high-performance liquid chromatography; DMF, dimethylform-
amide; TEA, triethylamine; TFA, trifluoroacetic acid; EDTA, ethylenediaminetetraace-
tic acid; DTT, dithiothreitol; UV, ultraviolet; TLC, thin-layer chromatography; NMR,
nuclear magnetic resonance; 3D, three-dimensional; UB, universal buffer; E-64, trans-
tone–benzene–acetic acid (100:25:4). Compounds with a free
a-
amino group were detected with ninhydrin reagent, and peptides
were visualized by spraying chlorinated plates with 0.05 M KI
and exposure to ultraviolet (UV) light (254 and 290 nm) using
UV analysis lamps (HP-UVIS, Sweden).
epoxysuccinyl-l-leucylamido(4-guanidino)butane; Abz, o-aminobenzoyl; CHTR,
chymotrypsin; SL, subtilisin Carlsberg.
a-
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dI ꢃ kfl
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HPLC (reverse phase) was carried out with an Altex model 110A
chromatograph (USA) using Nucleosil C18 column
(4.6 ꢁ 250 mm; Biokhimmak, Russia) eluted with a linear gradient
of 10 to 85% CH3CN in water for 35 min at a flow rate 1 ml/min. The
eluent contained 0.1% TFA. The elution profile was monitored at
220, 280, or 315 nm.
Amino acid analyses were performed on a Hitachi 835 auto-
matic amino acid analyzer (Japan) after acidic hydrolysis of sam-
ples with 5.7 M HCl at 105 °C in evacuated ampoules for 24 and
48 h.
Mass spectra were obtained by a Finnigan LCQ-IonTrap instru-
ment (Thermo Electron, USA) using an electrospray ionization
method.
A ¼
;
252
a
dt ꢃ cenz
where A is the activity of the preparation,
l
Msubstrate ꢁ Mꢀ1
-
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enzyme
ꢁ minꢀ1; dI/dt, units/min, is an initial rate of fluorophore release,
determined as a coefficient of a linear plot of the kinetic curve (a
function of fluorescence intensity I from time); kfl,
l
M ꢁ Uꢀ1, is
the concentration of a fluorophore at which the fluorescence inten-
sity of the solution is equal to 1 unit (defined in a special experi-
ment by plotting a calibration curve); kfl = 0.885 mM ꢁ Uꢀ1 for 7-
amino-4-methylcoumarin and 0.118
l
M ꢁ Uꢀ1 for 7-amino-4-(tri-
fluoromethyl)coumarin; and cenz is enzyme concentration in the
sample, M (for cysteine peptidases, cenz was calculated by the con-
centration of active sites of the peptidase).
Nuclear magnetic resonance (NMR) spectra were obtained in di-
methyl sulfoxide (DMSO)-d6 by a Brucker AC-300 spectrometer at
a frequency of 300 Mhz. Chemical shifts were in parts per million
(d, ppm) toward the inner standard of tetramethylsilane.
264
Determination of kinetic parameters of substrate hydrolysis
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The initial rates of hydrolysis of the chromogenic and
fluorogenic substrates were determined by similar procedures as
described above (see enzymatic assay), with substrate concentra-
tions of 0.05–0.5 mM for Glp-Phe-Ala-pNA, 0.1–1.0 mM for Glp-
208
3D modeling and molecular docking
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Homology modeling of a three-dimensional (3D) bromelain
structure was performed using chymopapain chain A (1YAL) as
the closest homolog by SwissModel server [65]. Molecular docking
was performed using SwissDock [66]. Data were visualized with
VMD [67] and PyMOL [68].
Val-Ala-pNA, and 1.25–25 lM for Glp-Phe-Ala-AMC and Glp-Phe-
Ala-AFC. Enzyme concentrations in the reaction were similar to
those in procedures described above. Calculations of the kinetic
parameters were performed by nonlinear regression using the pro-
gram OriginPro 7.5.
214
Enzyme assays using chromogenic pNA substrates
274
Active site titration of cysteine peptidases
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For assays of commercially available enzymes, 5
solution with concentrations of 0.2–0.56 M (except for bromelain
with a concentration of 1.8 M) were placed into wells of a 96-well
plate and diluted to a final volume of 195 l with 0.1 M universal
acetate–phosphate–borate buffer (pH 5.6) (UB) [69] containing
1 mM DTT. The mixture was preincubated at room temperature
(23 °C) for 20 min before the addition of substrate. To initiate the
ll of enzyme
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To identify the proportion of active cysteine peptidases in the
l
commercial preparations, 5
D280 = 0.03–0.08 OU; other commercial peptidases, D280 = 0.1 OU)
was added to 40 l of 0.1 M UB (pH 5.6) [69] containing 1 mM
DTT, and 5 l of the inhibitor trans-epoxysuccinyl-l-leucylami-
ll of enzyme solution (cathepsin L,
l
l
l
l
do(4-guanidino)butane (E-64) in 50% ethanol was combined in a
well of a 96-well plate and incubated at room temperature
(23 °C) for 20 min. The initial concentrations of E-64 were 0.17 to
reaction, 5 ll of 20 mM substrate solution was added (except for
10 mM Z-Phe-Arg-pNA in DMF), and initial absorption at 405 nm
at time zero was measured and thereafter every 5 min for 60 min
at 37 °C. The absorbance was measured by StatFax 2100 (Aware-
ness Technology, USA) and ELx808 (BioTek, USA) microplate read-
ers. Enzymatic activity was calculated by the formula
1.7
0.05–0.50
was diluted with 145
l
M for papain and ficin, 0.25 to 3.00
M for cathepsin L. To initiate the reaction, the mixture
l of 0.1 M UB containing 1 mM DTT and 5 ll
lM for cathepsin B, and
l
l
of 20 mM Glp-Phe-Ala-pNA solution in DMF at 37 °C, and initial
absorption at 405 nm at time zero was measured, and thereafter
every 5 min for 60 min. A linear plot of the dependence of enzyme
residual activity on the inhibitor concentration was approximated
by a straight line. The concentration of active sites was determined
by the intersection of this line with the abscissa axis. Calculations
were carried out by least squares regression using Microsoft Excel
2007.
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228
dD405 ꢃ kchr
A ¼
;
230
dt ꢃ cenz
where A is the activity of the preparation,
l
Msubstrate ꢁ Mꢀ1
-
enzyme
231
232
233
234
235
236
237
238
239
ꢁ minꢀ1; dD405/dt, OU/min, is an initial rate of p-nitroaniline re-
lease, determined as a coefficient of initial linear plot of kinetic
curve (a function of the change in absorbance at 405 nm over time);
kchr = 154
l
M ꢁ OUꢀ1 – concentration of p-nitroaniline at which the
294
Fractionation of T. molitor larvae midgut peptidases
absorbance of the solution is equal to 1 optical unit (defined in a
separate experiment by plotting a calibration curve); and cenz is en-
zyme concentration in the sample, M (for cysteine peptidases, cenz
was calculated by the concentration of active sites of the peptidase).
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300
301
T. molitor larvae rearing, isolation of midgut proteins extract,
and fractionation of larval digestive peptidases on a Sephadex
G-100 column were performed as described previously [43,44].
Enzymatic activity in the fractions was assayed with chromogenic
peptide substrates Glp-Phe-Ala-pNA and Z-Phe-Arg-pNA (pH 5.6
240
Enzyme assays using fluorogenic substrates
with 1 mM DTT or pH 7.9 and nonreducing) in 50-
described in the previous sections.
ll aliquots, as
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245
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The enzymatic activity with fluorogenic substrates was assayed
similar to that described for chromogenic substrates, using 1 mM
substrate stock solutions in DMF and measuring fluorescence
intensity at kex = 355 nm and kem = 460 nm for Glp-Phe-Ala-AMC
and at kex = 400 nm and kem = 505 nm for Glp-Phe-Ala-AFC. Fluo-
rescence was measured on a Cary Eclipse fluorimeter (Varian,
USA) as well as on a Fluoroskan Ascent microplate spectrofluorim-
eter (Thermo Scientific, USA). Enzymatic activity was calculated by
the formula
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Inhibitory analysis of midgut extracts from Tribolium larvae
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304
305
306
307
Rearing of Tribolium castaneum, Tribolium confusum, and Triboli-
um brevicornis larvae was as described in Ref. [45]. For inhibition
studies, aliquots of the total enzyme preparation (from the total
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250
midgut fraction) were preincubated with 0.1, 1, and 10
lM E-64
for 15 min at room temperature, and the substrates Bz-Arg-pNA,
Please cite this article in press as: T.A. Semashko et al., Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in
complex mixtures , Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2013.12.032

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373
Z-Phe-Arg-pNA, and Glp-Phe-Ala-pNA in 0.1 M UB (pH 6.8) [69]
containing 1 mM DTT were added to initiate the reaction (final
substrate concentration of 0.25 mM), and residual activity was as-
sayed as described previously.
ineffective for bromelain, making it difficult for a valid comparison
of kinetic constants for this enzyme with those of the other pepti-
dases. Of the tested enzymes, papain demonstrated the highest
efficiency of hydrolysis. In general, plant enzymes papain and ficin
were substantially more active in the hydrolysis of all substrates
than were mammalian lysosomal cathepsins, and fluorogenic sub-
strates were more efficiently hydrolyzed by all enzymes (Table 2).
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Results
Design of substrates
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For this study, we designed four chromogenic and fluorogenic
substrates—Glp-Phe-Ala-pNA (I), Glp-Val-Ala-pNA (II), Glp-Phe-
Ala-AMC (III), and Glp-Phe-Ala-AFC (IV)—specific for cysteine pep-
tidases from the papain family with an alanine residue in the P1 po-
sition. We evaluated compounds I–IV as substrates for C1 cysteine
peptidases by flexible molecular docking in comparison with the
previously described substrate Abz-Phe-Ala-pNA (where Abz = o-
aminobenzoyl) [59]. Fig. 1A–D predict the binding of substrates
with the active centers of papain, cathepsin B, and cathepsin L
3D structures and a bromelain model. Fig. 1E summarizes the bind-
ing energy modules of the enzyme–substrate complexes. The
molecular docking studies predicted that these proposed sub-
strates would be hydrolyzed effectively by C1 cysteine peptidases.
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375
Substrate selectivity: Comparison with commercial Arg-containing
substrates
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Action of model peptidases from other classes
Substrates I–IV were evaluated for their selectivity, that is, for
resistance to hydrolysis by peptidases from other classes, including
serine peptidases (trypsin, CHTR, and SL), aspartic peptidase (pep-
sin), and metallo peptidase (thermolysin). Enzymatic activity for
all enzymes was measured under conditions optimal for cysteine
peptidases: under mild acidic conditions (pH 5.6) in the presence
of a reducing agent (1 mM DTT). All substrates (I–IV) were selec-
tive for cysteine peptidases of the C1 family and were not hydro-
lyzed by peptidases of other classes (Table 3). For comparison,
we also tested the selectivity of commercial chromogenic sub-
strates that are widely used for testing the enzymatic activity of
C1 family cysteine peptidases: Z-Phe-Arg-pNA (V), Z-Arg-Arg-
pNA (VI), and Bz-Arg-pNA (VII). Z-Phe-Arg-pNA was easily hydro-
lyzed by all cysteine peptidases, with papain being the most active.
Z-Arg-Arg-pNA was cleaved by bromelain and cathepsin B, and Bz-
Arg-pNA was hydrolyzed by all cysteine peptidases but at a lower
rate. However, all commercial substrates were not selective be-
cause they were easily cleaved with trypsin.
327
Synthesis of substrates
328
329
Synthesis of the substrates was carried out by the following
scheme, using the enzymes
a-chymotrypsin (CHTR) or subtilisin
330
331
Carlsberg (SL) for peptide bond formation:
Glp-XaaOMe þ H-Ala-Y enzyme
! Glp-Xaa-Ala-Y þ MeOH
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
where Xaa = Phe or Val and Y = pNA, AMC, or AFC.
The enzymatic synthesis of substrates was performed by two
methods: (i) under the action of native CHTR and SL in DMF/aque-
ous buffer and (ii) with the use of modified enzymes in an anhy-
drous medium of polar organic solvents (DMF–MeCN, 20:80,
vol%) (Table 1). In the first method, synthesis was possible only
for Glp-Phe-Ala-pNA (I), with 82 and 66% yields, and Glp-Phe-
Ala-AFC (IV), with 50 and 18% yields, using CHTR and SL, respec-
tively. The second method, using CHTR and SL immobilized on
polyvinyl alcohol cryogel (PVAG), was more universal and resulted
in synthesis of all substrates, albeit with differing efficiencies. The
yield of Glp-Phe-Ala-pNA was high (70–80%) even after reusing the
same sample of biocatalyst six times. The efficiency of synthesis of
Glp-Phe-Ala-AMC was similar for two cycles; in 4 h, the yield was
50%, and it essentially was complete (100%) in 48 h.
All synthesized peptides were stable crystalline compounds
with high melting points (> 200 °C). All compounds were charac-
terized by mass spectrometry, NMR, and amino acid analysis as
well as chromatographic mobility values (Rf values) in several sys-
tems (TLC) and retention times (HPLC) (see Table S1 in Supplemen-
tary material). The resulting physico-chemical properties of the
synthesized compounds I–IV demonstrate their chemical homoge-
neity and spectral purity.
395
396
Evaluation of cysteine peptidases during the chromatographic
separation of a native multicomponent enzyme mixture
397
398
399
400
401
402
403
404
405
406
407
During the separation of midgut contents from T. molitor larvae,
the elution profiles of peptidase activities were monitored by the
hydrolysis of Glp-Phe-Ala-pNA and the commercial substrate Z-
Phe-Arg-pNA under conditions optimal for cysteine peptidases
(pH 5.6, 1 mM DTT) or trypsin-like peptidases (pH 7.9, no DTT)
(Fig. 2). Enzyme activity with the substrate Glp-Phe-Ala-pNA was
detected only under conditions optimal for cysteine peptidases
and only in two peaks: TmCPI and TmCPII. Activity with the sub-
strate Z-Phe-Arg-pNA was detected under both conditions and in
more fractions, which corresponded to the elution not only of cys-
teine but also of trypsin-like peptidases.
408
409
Inhibition analysis of cysteine peptidase activity in multicomponent
crude extracts
410
411
412
413
414
415
416
417
418
419
420
421
422
We used the cysteine peptidase inhibitor E-64 [70] to demon-
strate the selectivity of substrate (I) for cysteine peptidases in
crude extracts from the gut of T. molitor larvae as well as other
tenebrionid larvae of the genus Tribolium: T. castaneum, T. confu-
sum, and T. brevicornis. Cysteine peptidase activity was measured
by the hydrolysis of Glp-Phe-Ala-pNA compared with the commer-
cially available substrates Z-Phe-Arg-pNA and Bz-Arg-pNA at pH
6.8 in the presence of DTT with or without E-64 (Fig. 3). At 1 lM
E-64 concentration, which is characteristic for the inhibition of
pure cysteine peptidases, only the enzyme activity with Glp-Phe-
Ala-pNA was almost totally inhibited. Even with a 10-lM concen-
357
358
Hydrolysis of the substrates by model cysteine peptidases (from C1
family)
359
360
361
362
363
364
365
366
The hydrolysis of newly synthesized substrates I–IV, deter-
mined as kcat/KM, was evaluated by cysteine peptidases from the
C1 family, including plant enzymes papain, bromelain, ficin, and
bovine and human lysosomal cathepsins B and L, respectively (Ta-
ble 2). For all enzymes except bromelain, kinetic constants of
hydrolysis were calculated by the concentration of the active en-
zymes, which was determined by titration with the cysteine pepti-
dase inhibitor E-64 [70] (data not shown). This titrant was
tration of E-64, the residual enzyme activity with Z-Phe-Arg-pNA
and Bz-Arg-pNA was still 40 to 90%.
Please cite this article in press as: T.A. Semashko et al., Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in
complex mixtures , Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2013.12.032

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5
Fig.1. Modeling of substrates I–IV binding to papain (PDB ID 1CVZ) (A), bromelain model (UniProt ID O23799) (B), cathepsin B (PDB ID 1CSB) (C), and cathepsin L (PDB ID
1ICF) (D). Glp-Phe-Ala-pNA (I) is marked with yellow, Glp-Val-Ala-pNA (II) is marked with green, Glp-Phe-Ala-AMC (III) is marked with blue, Glp-Phe-Ala-AFC (IV) is marked
with purple, and Abz-Phe-Ala-pNA is marked with red. The binding energy modules of the enzyme–substrate complexes is shown in panel E. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
Table 1
Yields of the enzymatic synthesis of substrates by native enzymes in an aqueous–organic (DMF/aqueous buffer) mixture or by immobilized enzymes in an organic (DMF/MeCN)
mixture using chymotrypsin or subtilisin.
Substrate
Yield (%): DMF/aqueous buffer
CHTR
Yield (%): DMF/MeCNa
CHTR
SL
SL
Glp-Phe-Ala-pNA
Glp-Val-Ala-pNA
Glp-Phe-Ala-AMC
Glp-Phe-Ala-AFC
82
0
0
66
0
0
83
44
32
11
25
0
100 (46)b
40
50
18
Note. CHTR, chymotrypsin; SL, subtilisin.
a
24-h incubation, one cycle.
Yield is given for preparative variant.
b
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Substrates for papain family cysteine peptidases / T.A. Semashko et al. / Anal. Biochem. xxx (2014) xxx–xxx
Table 2
Efficiency of the hydrolysis of substrates I–IV by cysteine peptidases.
Peptidase
Papain
Substrate
kcat/KM (mMꢀ1 minꢀ1
)
Glp-Phe-Ala-pNA
Glp-Val-Ala-pNA
Glp-Phe-Ala-AMC
Glp-Phe-Ala-AFC
Glp-Phe-Ala-pNA
Glp-Val-Ala-pNA
Glp-Phe-Ala-AMC
Glp-Phe-Ala-AFC
Glp-Phe-Ala-pNA
Glp-Val-Ala-pNA
Glp-Phe-Ala-AMC
Glp-Phe-Ala-AFC
Glp-Phe-Ala-pNA
Glp-Val-Ala-pNA
Glp-Phe-Ala-AMC
Glp-Phe-Ala-AFC
Glp-Phe-Ala-pNA
Glp-Val-Ala-pNA
Glp-Phe-Ala-AMC
Glp-Phe-Ala-AFC
(I)
481 76
148 24
4376 162
5493 312
129 12
(II)
(III)
(IV)
(I)
(II)
(III)
(IV)
(I)
(II)
(III)
(IV)
(I)
(II)
(III)
(IV)
(I)
Ficin
71
6
1345 83
1259 83
Bromelain
Cathepsin B
Cathepsin L
7
8
116
90
39
19
1
1
8
4
4
1
582 78
670 33
Fig.2. Use of the synthesized substrate Glp-Phe-Ala-pNA and
a commercial
substrate, Z-Phe-Arg-pNA, for the detection of cysteine peptidases (pH 5.6, 1 mM
DTT) and trypsin-like peptidases (pH 7.9, no DTT) during the fractionation of T.
molitor larval digestive enzymes in a midgut extract on a Sephadex G-100 column.
33
25
4
2
(II)
(III)
(IV)
328 14
137
1
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
which is crucial for the specificity of C1 peptidases [71]. The intro-
duction of a Val residue in the P2 position was proposed according
to the screening of the enzymatic activity of cysteine peptidases
from the papain family, which indicated that, contrary to popular
opinion about the preference for aromatic residues, position P2
may be occupied by small aliphatic amino acid residues [72,76].
We hypothesized that short substrates containing two amino acid
residues would provide better selectivity and resistance to hydro-
lysis by peptidases from other classes despite some loss in effi-
ciency of hydrolysis by cysteine peptidases. The docking studies
indicated that all compounds bound to the active center of four dif-
ferent C1 peptidases in a conformation conducive to the further
cleavage of substrates at the amide bond proposed for the
hydrolysis.
The N-terminal group of the substrates is a pyroglutamic acid
residue (Glp). This residue is more hydrophilic than the majority
of commonly used protecting groups and was introduced to in-
crease the solubility of substrates in an aqueous mixture. A chro-
mogenic pNA or fluorogenic AMC and AFC groups were added as
C-terminal residues. These residues provided convenience and
accuracy to control enzymatic activity by detecting changes of
the spectral and fluorescent characteristics of the substrates during
hydrolysis.
423
Discussion
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
Most peptidases of the papain C1 family are monomers with a
molecular mass of 20–35 kDa, structurally composed of two do-
mains, L and R, separated by a cleft where the active site is located
[1,2,49,71]. The substrate binding site in this peptidase family con-
sists of seven subsites (S1–S4 and S01–S30 ), which are located at the
junction of the L and R domains. The S2 subsite has the highest
selectivity, with slightly less for S1 and S01; other binding subsites
have significantly lower selectivity and specificity [71]. The S2 sub-
site is the only subsite with a complete substrate binding pocket
[3].
Using substrate libraries for a series of human C1 cathepsins (L,
V, B, S, K, and F), it has been shown that the majority of cysteine
peptidases are characterized by strong affinity for substrates with
a Lys and Arg at position P1 and, to a lesser extent, for the sub-
strates with small and uncharged amino acid residues at P1 (gluta-
mine, threonine, and alanine). In position P2, preferred amino acids
are hydrophobic (Phe, Trp, Tyr, Leu, Ile, Val, and Met) [72–76]. In
general, cysteine peptidases have rather broad specificity and are
able to hydrolyze a wide range of substrates [1,3,71].
The substrates were obtained by an enzymatic approach using
CHTR or SL. These enzymes have substrate specificities that satisfy
the structure of synthesized compounds. The enzymatic peptide
bond formation guaranteed selectivity of the reaction, provided
optical purity of the target compounds, and simplified the scheme
of synthesis and isolation of products. The comparison of the two
synthesis methods, by native and immobilized enzymes, demon-
The structure of our proposed substrates Glp-Phe-Ala-pNA (I),
Glp-Val-Ala-pNA (II), Glp-Phe-Ala-AMC (III), and Glp-Phe-Ala-AFC
(IV) can be expressed by the general formula Glp-Xaa-Ala;A
(where Xaa = Phe or Val; A = pNA, AMC, or AFC; and the arrow
shows the expected position of substrate hydrolysis). These sub-
strates have the hydrophobic residue Phe or Val in position P2,
Table 3
Q2
Hydrolysis of synthesized (I–IV) and commercial (V–VII) chromogenic and fluorogenic substrates with cysteine peptidases from the C1 family and peptidases from other classes.
Peptidase
A (Msubstrate/Menzyme ⁄ min)
Glp-Phe-Ala-pNA (I) Glp-Val-Ala-pNA (II) Glp-Phe-Ala-AMC (III) Glp-Phe-Ala-AFC (IV) Z-Phe-Arg-pNA (V) Z-Arg-Arg-NA (VI) Bz-Arg-pNA (VII)
Papain
Ficin
Bromelain
Cathepsin B
Cathepsin L
Trypsin
132.1 0.0
41.6 0.0
2.2 0.0
11.8 0.0
11.1 1.4
0.1 0.0
0
39.4 0.0
18.8 0.0
2.6 0.0
7.5 0.0
4.9 1.5
0.3 0.1
0.5 0.0
0.1 0.0
0
56.7 0.0
16.3 0.0
2.2 0.0
3.0 0.0
58.8 0.0
14.8 0.0
1.9 0.0
4.8 0.0
760.8 0.0
133.9 0.0
1.8 0.0
89.5 0.0
159.1 21.1
27.3 0.6
0
2.9 0.0
2.2 0.0
8.9 0.0
17.8 0.0
13.0 4.1
50.3 1.2
0.7 0.0
0.1 0.0
0.4 0.1
0
12.1 0.0
3.5 0.0
0.3 0.0
3.0 0.0
–
1.7 0.1
0.1 0.0
0
14.7 0.1
4.7 0.5
0
0
0
0
0
0
0
0
0
0
a-Chymotrypsin
Subtilisin
Pepsin
Thermolysin
0
0
0
0.7 0.0
0
0
0
0.5 0.1
0.4 0.1
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Fig.3. Effect of E-64 on the activity of enzymes in midgut extracts from Tribolium castaneum, Tribolium confusum, Tribolium brevicornis, and Tenebrio molitor larvae with
chromogenic commercial substrates Bz-Arg-pNA and Z-Phe-Arg-pNA and synthesized substrate Glp-Phe-Ala-pNA, assayed at pH 6.8.
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
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509
510
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strated the advantage of synthesis with immobilized biocatalysts.
The second approach was more efficient and allowed the repeated
use of the catalyst. In general, the synthesis of Phe-containing
derivatives was more effective with the use of CHTR, whereas
Val-containing derivatives were more efficiently synthesized by SL.
A demonstration of the hydrolysis of the substrates by model
cysteine peptidases from the C1 family indicated that plant en-
zymes papain and ficin were substantially more active in the
hydrolysis of all substrates than were mammalian lysosomal
cathepsins. Fluorogenic substrates were more efficiently hydro-
lyzed by the model enzymes by approximately an order of magni-
tude more than chromogenic substrates.
The selectivity of our substrates was compared with commer-
cial Arg-containing substrates by the action of model peptidases
from other classes, by an evaluation of cysteine peptidases during
the chromatographic separation of a native multicomponent en-
zyme mixture, and by an inhibition analysis of cysteine peptidase
activity in crude multicomponent extracts.
least 25 genes encoding cysteine cathepsins in T. castaneum,
including those tentatively identified as cathepsins L, B, O, and K,
according to genome annotation studies [80]. Our studies indicate
that the primary structure of the major digestive cathepsin L from
T. castaneum is very close to pCAL3 AY332272 from T. molitor
[80,81].
Our data demonstrate that the substrate Glp-Phe-Ala-pNA was
superior to other commercially available cysteine peptidase sub-
strates in the evaluation of biologically active enzymes in complex
multicomponent mixtures. Overall, these studies demonstrate that
the synthesized substrates are selective for C1 cysteine peptidases
and can be useful in the analysis of complex systems containing
peptidases from different classes.
538
Acknowledgments
539
540
541
542
543
544
545
This work was supported by funds from the Russian Foundation
for Basic Research (grants 12-04-01562-a and 12-03-01057-a) and
the International Science and Technology Center (ISTC, grant
3455). The mention of trade names or commercial products in this
publication is solely for the purpose of providing specific informa-
tion and does not imply recommendation or endorsement by the
U.S. Department of Agriculture.
The synthesized substrates containing an Ala residue in the P1
position were more selective for cysteine peptidase activity than
the widely used commercial substrates that contain an Arg residue
at position P1.
The synthesized substrates have been indispensable in the
study of digestive peptidases from stored-product pests, including
larvae of the yellow mealworm T. molitor. T. molitor larvae have a
multicomponent set of digestive peptidases located in the midgut,
mostly cysteine-, trypsin-, and chymotrypsin-like peptidases [42–
45]. Cysteine digestive peptidases are engaged during the initial
stages of proteolysis of food proteins and are mainly located in
the anterior midgut with pH 5.6, and serine peptidases are primar-
ily located in the posterior midgut with pH 7.9. During the chro-
matographic fractionation of proteins from T. molitor larvae
midgut extract, only the use of our selective substrate enabled
unambiguous identification of cysteine peptidase activity in the
digestive complex of the insect.
Recently, the 3D structure of two major cathepsins L from T.
molitor was published [77]. Our data indicated that one of these
cathepsins, pCAL3 AY332272, is the major digestive enzyme in this
insect [78,79]. According to Beton and coworkers [77], its 3D struc-
ture is very close to that of human cathepsin L.
The newly synthesized substrates and a cysteine peptidase
inhibitor were used to evaluate enzymes in the most complicated
mixtures–crude midgut extracts from T. molitor larvae as well as
other tenebrionid larvae of the genus Tribolium: T. castaneum, T.
confusum, and T. brevicornis, all of which have a similar set of diges-
tive peptidases with mainly quantitative differences. There are at
546
Appendix A. Supplementary data
547
548
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ab.2013.12.032.
549
References
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[1] N.D. Rawlings, A.J. Barrett, Introduction: the clans and families of cysteine
peptidases, in: N.D. Rawlings, G.S. Salvesen (Eds.), Handbook of Proteolytic
Enzymes, vol. 2, 3rd ed., Academic Press, London, 2013, pp. 1743–1776.
[2] N.D. Rawlings, A.J. Barrett, A. Bateman, MEROPS: the peptidase database,
Nucleic Acids Res. 38 (2010) D227–D233.
[3] V. Turk, V. Stoka, O. Vasiljeva, M. Renko, T. Sun, B. Turk, D. Turk, Cysteine
cathepsins: from structure, function, and regulation to new frontiers, Biochim.
Biophys. Acta 2012 (1824) 68–88.
[4] M. Allaire, M.M. Chernaia, B.A. Malcolm, M.N. James, Picornaviral 3C cysteine
proteinases have a fold similar to chymotrypsin-like serine proteinases, Nature
369 (1994) 72–76.
[5] G.N. Rudenskaya, D.V. Pupov, Cysteine proteinases of microorganisms and
viruses, Biochemistry (Moscow) 73 (2008) 1–13.
[6] G. Dubin, B. Wladyka, J. Stec-Niemczyk, D. Chmiel, M. Zdzalik, A. Dubin, J.
Potempa, The staphostatin family of cysteine protease inhibitors in the genus
Staphylococcus as an example of parallel evolution of protease and inhibitor
specificity, Biol. Chem. 388 (2007) 227–235.
Please cite this article in press as: T.A. Semashko et al., Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in
complex mixtures , Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2013.12.032

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567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
[7] J. Potempa, R. Pike, J. Travis, The multiple forms of trypsin-like activity present
in various strains of Porphyromonas gingivalis are due to the presence of either
Arg-gingipain or Lys-gingipain, J. Infect. Immunol. 63 (1995) 1176–1182.
[8] C. Serveau, G. Lalmanach, M.A. Juliano, J. Scharfstein, L. Juliano, F. Gauthier,
Investigation of the substrate specificity of cruzipain, the major cysteine
proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates
and inhibitors, Biochem. J. 313 (1996) 951–956.
[9] C.R. Caffrey, E. Hansell, K.D. Lucas, L.S. Brinen, A. Alvarez Hernandez, J. Cheng,
S.L. Gwaltney 2nd, W.R. Roush, Y.D. Stierhof, M. Bogyo, D. Steverding, J.H.
McKerrow, Active site mapping, biochemical properties, and subcellular
localization of rhodesain, the major cysteine protease of Trypanosoma brucei
rhodesiense, Mol. Biochem. Parasitol. 118 (2001) 61–73.
[10] J. Cazzulo, V. Stoka, V. Turk, The major cysteine proteinase of Trypanosoma
cruzi: A valid target for chemotherapy of Chagas disease, Curr. Pharm. Des. 7
(2001) 1143–1156.
[11] D.C. Greenbaum, A. Baruch, M. Grainger, Z. Bozdech, K.F. Medziharadszky, J.
Engel, J. DeRisi, A.A. Holder, M. Bogyo, A role for the protease falcipain 1 in host
cell invasion by the human malaria parasite, Science 298 (2002) 2002–2006.
[12] M. Sajid, J.H. McKerrow, Cysteine proteases of parasitic organisms, Mol.
Biochem. Parasitol. 120 (2002) 1–21.
[37] W.R. Terra, C. Ferreira, Insect digestive enzymes: properties,
compartmentalization, and function, Comp. Biochem. Physiol. B 109 (1994)
1–62.
[38] C.J. Bolter, M.A. Jongsma, Colorado potato beetles (Leptinotarsa decemlineata)
adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate, J.
Insect Physiol. 41 (1995) 1071–1078.
[39] P.T. Cristofoletti, A.F. Ribeiro, C. Deraison, Y. Rahbe, W.R. Terra, Midgut
adaptation and digestive enzyme distribution in a phloem feeding insect, the
pea aphid Acyrthosiphon pisum, J. Insect. Physiol. 49 (2003) 11–24.
[40] K. Gruden, A.G. Kuipers, G. Guncar, N. Slapar, B. Strukelj, M.A. Jongsma,
Molecular basis of Colorado potato beetle adaptation to potato plant defense
at the level of digestive cysteine proteinases, Insect Biochem. Mol. Biol. 34
(2004) 365–375.
[41] A.G. Kuipers, M.A. Jongsma, Isolation and molecular characterization of
cathepsin L-like cysteine protease cDNAs from western flower thrips
(Frankliniella occidentalis), Comp. Biochem. Physiol. B 139 (2004) 65–75.
[42] P.T. Cristofoletti, A.F. Ribeiro, W.R. Terra, The cathepsin L-like proteinases from
the midgut of
Tenebrio
molitor
larvae: Sequence, properties,
immunocytochemical localization, and function, Insect Biochem. Mol. Biol.
35 (2005) 883–901.
[13] Y.A. Sabnis, P.V. Desai, P.J. Rosenthal, M.A. Avery, Probing the structure of
falcipain-3, a cysteine protease from Plasmodium falciparum: Comparative
protein modeling and docking studies, Protein Sci. 12 (2003) 501–509.
[14] V. Nogaroto, S. Tagliavini, A. Gianotti, A. Mikawa, N.T. Barros, L. Puzer, A.K.
Carmona, P. Costa, F. Henrique-Silva, Recombinant expression and
[43] K.S. Vinokurov, E.N. Elpidina, B. Oppert, S. Prabhakar, D.P. Zhuzhikov, Y.E.
Dunaevsky, M.A. Belozersky, Diversity of digestive proteinases in Tenebrio
molitor (Coleoptera: Tenebrionidae) larvae, Comp. Biochem. Physiol. B 145
(2006) 126–137.
[44] K.S. Vinokurov, E.N. Elpidina, B. Oppert, S. Prabhakar, D.P. Zhuzhikov, Y.E.
Dunaevsky, M.A. Belozersky, Fractionation of digestive proteinases from
Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein
digestion, Comp. Biochem. Physiol. B 145 (2006) 138–146.
[45] K.S. Vinokurov, E.N. Elpidina, D.P. Zhuzhikov, B. Oppert, D. Kodrik, F. Sehnal,
Digestive proteolysis organization in two closely related Tenebrionid beetles:
Red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium
confusum), Arch. Insect Biochem. Physiol. 70 (2009) 254–279.
[46] L.J. Brubacher, M.R. Zaher, A kinetic study of hydrophobic interactions at the S1
and S2 sites of papain, Can. J. Biochem. 57 (1979) 1064–1072.
[47] G. Lowe, Y. Yuthavong, Kinetic specificity in papain-catalyzed hydrolyses,
Biochem. J. 124 (1971) 107–115.
characterization of
a Xylella fastidiosa cysteine protease differentially
expressed in a nonpathogenic strain, FEMS Microbiol. Lett. 261 (2006) 187–
193.
[15] C.R. Affray, A.P. Lima, D. Steverding, Cysteine peptidases of kinetoplastid
parasites, Adv. Exp. Med. Biol. 712 (2011) 84–99.
[16] Z. Dou, V.B. Carruthers, Cathepsin proteases in Toxoplasma gondii, Adv. Exp.
Med. Biol. 712 (2011) 49–61.
[17] H.-H. Otto, T. Schirmeister, Cysteine proteases and their inhibitors, Chem. Rev.
97 (1997) 133–171.
[18] D.F. Gruis, D.A. Selinger, J.M. Curran, R. Jung, Redundant proteolytic
mechanisms process seed storage proteins in the absence of seed-type
members of the vacuolar processing enzyme family of cysteine proteases,
Plant Cell 14 (2002) 2863–2882.
[19] V.K. Dubey, M.V. Jagannadham, Procerain, a stable cysteine protease from the
latex of Calotropis procera, Phytochemistry 62 (2003) 1057–1071.
[20] K. Konno, C. Hirayama, M. Nakamura, K. Tateishi, Y. Tamura, M. Hattori, K.
Kohno, Papain protects papaya trees from herbivorous insects: role of cysteine
proteases in latex, Plant J. 37 (2004) 370–378.
[21] D.P. Dickinson, Cysteine peptidases of mammals: their biological roles and
potential effects in the oral cavity and other tissues in health and disease, Crit.
Rev. Oral Biol. Med. 13 (2002) 238–275.
[22] M. Martınez, I. Cambra, P. González-Melendi, M.E. Santamarıa, I. Dıaz, C1A
cysteine-proteases and their inhibitors in plants, Physiol. Plant. 145 (2012)
85–94.
[23] K. Brix, A. Dunkhorst, K. Mayer, S. Jordans, Cysteine cathepsins: cellular
roadmap to different functions, Biochimie 90 (2008) 194–207.
[24] Z. Grzonka, F. Kasprzykowski, W. Wiczk, Cysteine proteases, in: J. Polaina, A.P.
MacCabe (Eds.), Industrial Enzymes: Structure, Function, and Applications,
Springer, New York, 2007, pp. 181–195.
[25] K. Chobotova, A.B. Vernallis, F.A. Majid, Bromelain’s activity and potential as an
anti-cancer agent: current evidence and perspectives, Cancer Lett. 290 (2010)
148–156.
[26] V. Turk, B. Turk, D. Turk, Lysosomal cysteine proteases: facts and
opportunities, EMBO J. 20 (2001) 4629–4633.
[27] F. Lecaille, J. Kaleta, D. Brömme, Human and parasitic papain-like cysteine
proteases: their role in physiology and pathology and recent developments in
inhibitor design, Chem. Rev. 102 (2002) 4459–4488.
[28] F. Bühling, N. Waldburg, A. Reisenauer, A. Heimburg, H. Golpon, T. Welte,
Lysosomal cysteine proteases in the lung: role in protein processing and
immunoregulation, Eur. Respir. J. 23 (2004) 620–628.
[48] J.E. Mole, H.R. Horton, Kinetics of papain-catalyzed hydrolysis of N-benzoyl-l-
arginine-p-nitroanilide, Biochemistry 12 (1973) 816–822.
[49] A. Berger, I. Schechter, Mapping the active site of papain with the aid of
peptide substrates and inhibitors, Philos. Trans. R. Soc. Lond. B 257 (1970)
249–264.
[50] J. Tchoupe, T. Moreau, F. Gauthier, J. Bieth, Photometric or fluorometric assay
of cathepsin B, L, and
H
and papain using substrates with an
aminotrifluoromethylcoumarin leaving group, Biochim. Biophys. Acta 1076
(1991) 149–151.
}
[51] E. Schroder, C. Phillips, K. Harlos, C. Crawford, X-ray crystallographic structure
of a papain–leupeptin complex, FEBS Lett. 315 (1993) 38–42.
[52] A. Davy, M.B. Sorensen, I. Svendsen, V. Cameron-Mills, D.J. Simpson, Prediction
of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B
based on the kinetics of synthetic peptide hydrolysis, Plant Physiol. 122 (2000)
137–146.
[53] S. Zucker, D. Buttle, J. Nicklin, A. Barrett, The proteolytic activities of
chymopapain, papain, and papaya proteinase III, Biochem. Biophys. Res.
Commun. 828 (1985) 196–204.
[54] A. Taralp, H. Kaplan, I.I. Sytwu, I. Vlattas, R. Bohacek, A.K. Knap, T. Hirama, C.P.
Huber, S. Hasnain, Characterization of the S3 subsite specificity of cathepsin B,
J. Biol. Chem. 270 (1995) 18036–18043.
[55] M. Cezari, L. Puzer, M. Juliano, A. Carmona, A. Juliano, Cathepsin B
carboxydipeptidase specificity analysis using internally quenched
fluorescent peptides, Biochem. J. 368 (2002) 365–369.
[56] L.C. Alves, P.C. Almedia, L. Franzoni, L. Juliano, M.A. Juliano, Synthesis of Na-
protected aminoacyl 7-amino-4-methyl-coumarin amide by phosphorous
oxychloride and preparation of specific fluorogenic substrates for papain,
Pept. Res. 9 (1996) 92–96.
[57] I.Yu. Filippova, E.N. Lysogorskaya, E.S. Oksenoit, G.N. Rudenskaya, V.M.
[29] C. Jedeszko, B.F. Sloane, Cysteine cathepsins in human cancer, Biol. Chem. 385
(2004) 1017–1027.
Stepanov,
L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide—a
chromogenic substrate for thiol proteinase assay, Anal. Biochem. 143 (1984)
293–297.
[30] C. Urbich, C. Heeschen, A. Aicher, K. Sasaki, T. Bruhl, M.R. Farhadi, P. Vajkoczy,
W.K. Hofmann, C. Peters, L.A. Pennacchio, N.D. Abolmaali, E. Chavakis, T.
Reinheckel, A.M. Zeiher, S. Dimmeler, Cathepsin L is required for endothelial
progenitor cell-induced neovascularization, Nat. Med. 11 (2005) 206–213.
[31] G.J. van Acker, G. Perides, M.L. Steer, Co-localization hypothesis: a mechanism
for the intrapancreatic activation of digestive enzymes during the early phases
of acute pancreatitis, World J. Gastroenterol. 12 (2006) 1985–1990.
[32] O. Vasiljeva, T. Reinheckel, C. Peters, D. Turk, V. Turk, B. Turk, Emerging roles of
cysteine cathepsins in disease and their potential as drug targets, Curr. Pharm.
Des. 13 (2007) 387–403.
[33] Y. Rengel, C. Ospelt, S. Gay, Proteinases in the joint: clinical relevance of
proteinases in joint destruction, Arthritis Res. Ther. 9 (2007) 221.
[34] C. Palermo, J.A. Joyce, Cysteine cathepsin proteases as pharmacological targets
in cancer, Trends Pharmacol. Sci. 29 (2008) 22–28.
[35] U. Repnik, V. Stoka, V. Turk, B. Turk, Lysosomes and lysosomal cathepsins in
cell death, Biochim. Biophys. Acta 2012 (1824) 22–33.
[58] V. M. Stepanov, E. N. Lysogorskaya, I. Yu. Filippova, E. S. Oksenoit, L. A.
Lyublinskaya,
L
-Pyroglutamyl-
L
-phenylalanyl-
L-alanyne
p-nitroanilide—a
chromogenic substrate of thiol proteinases, Russian inventor’s certificate no.
1198082, Byul. Izobr. 46 (1985) (in Russian).
[59] T.A. Semashko, E.N. Lysogorskaya, E.S. Oksenoit, A.V. Bacheva, I.Y. Filippova,
Chemoenzymatic synthesis of new fluorogenous substrates for cysteine
proteases of the papain family, Rus. J. Bioorg. Chem. 34 (2008) 339–343.
[60] K.S. Vinokurov, B. Oppert, E.N. Elpidina, An overlay technique for
postelectrophoretic analysis of proteinase spectra in complex mixtures using
p-nitroanilide substrates, Anal. Biochem. 337 (2005) 164–166.
[61] T.A. Solovjeva, S.V. Belyaev, V.M. Stepanov, Purification of pepsin by ion-
exchange chromatography on Aminosilochrom, Khim. Prirod. Soed. 3 (1977)
398–403 (in Russian).
[62] A.J. Gordon, R.A. Ford, A Handbook of Practical Data, Techniques, and
References, John Wiley, New York, 1972.
[36] N.M.R. Thie, J.G. Houseman, Identification of cathepsin B, D, and H in the larval
midgut of Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera:
Chrysomelidae), Insect Biochem. 20 (1990) 313–318.
[63] R. Nyfeler, Peptide synthesis via fragment condensation, in: M.W. Pennington,
B.M. Dunn (Eds.), Peptide Synthesis Protocols, Humana Press, Totowa, NJ,
1994, pp. 303–316.
Please cite this article in press as: T.A. Semashko et al., Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in
complex mixtures , Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2013.12.032

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742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
[64] A.V. Bacheva, F.M. Plieva, E.N. Lysogorskaya, I.Y. Filippova, V.I. Lozinsky,
Peptide synthesis in organic media with subtilisin 72 immobilized on
poly(vinyl alcohol)–cryogel carrier, Bioorg. Med. Chem. Lett. 11 (2001)
1005–1008.
[65] T. Schwede, J. Kopp, N. Guex, M.C. Peitsch, SWISS–MODEL: an automated
protein homology-modeling server, Nucleic Acids Res. 31 (2003) 3381–3385.
[66] A. Grosdidier, V. Zoete, O. Michielin, SwissDock, a protein–small molecule
docking web service based on EADock DSS, Nucleic Acids Res. 39 (2011) 270–
277.
[74] L. Puzer, S.S. Cotrin, M.F. Alves, T. Egborge, M.S. Araújo, M.A. Juliano, L. Juliano,
D. Brömme, A.K. Carmona, Comparative substrate specificity analysis of
recombinant human cathepsin V and cathepsin L, Arch. Biochem. Biophys.
430 (2004) 274–283.
[75] D.N. Gosalia, C.M. Salisbury, J.A. Ellman, S.L. Diamond, High throughput
substrate specificity profiling of serine and cysteine proteases using solution-
phase fluorogenic peptide microarrays, Mol. Cell. Proteomics 4 (2005) 626–
636.
[76] Y. Choe, F. Leonetti, D.C. Greenbaum, F. Lecaille, M. Bogyo, D. Brömme, J.A.
[67] W. Humphrey, A. Dalke, K. Schulten, VMD—Visual Molecular Dynamics, J. Mol.
Graphics 14 (1996) 33–38.
[68] PyMOL, http://pymol. org.
Ellman, C.S. Craik, Substrate profiling of cysteine proteases using a
combinatorial peptide library identifies functionally unique specificities, J.
Biol. Chem. 281 (2006) 12824–12832.
[69] J.A.C. Frugoni, Tampone universale di Britton
constante, Gazz. Chem. Ital. 87 (1957) 403–407.
e
Robinson
a
forza ionica
[77] D. Beton, C.R. Guzzo, A.F. Ribeiro, C.S. Farah, W.R. Terra, The 3D structure and
function of digestive cathepsin L-like proteinases of Tenebrio molitor larval
midgut, Insect Biochem. Mol. Biol. 42 (2012) 655–664.
[78] S. Prabhakar, M.S. Chen, E.N. Elpidina, K.S. Vinokurov, C.M. Smith, B. Oppert,
Sequence analysis and molecular characterization of larval midgut cDNA
transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L,
Insect Mol. Biol. 16 (2007) 455–468.
[79] B. Oppert, A.G. Martynov, E.N. Elpidina, Bacillus thuringiensis Cry3Aa protoxin
intoxication of Tenebrio molitor induces widespread changes in the expression
of serine peptidase transcripts, Comp. Biochem. Physiol. D 7 (2012) 233–242.
[80] Tribolium Genome Sequencing Consortium, The genome of the model beetle
and pest Tribolium castaneum, Nature 452 (2008) 949–955.
[70] A.J. Barrett, A. Kembhavi, M.A. Brown, H. Kirschke, C.G. Knight, M. Tamai, K.
Hanada, L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and
its analogues as inhibitors of cysteine proteinases including cathepsins B, H,
and L, Biochem. J. 201 (1982) 189–198.
[71] D. Turk, G. Guncar, M. Podobnik, B. Turk, Revised definition of substrate
binding sites of papain-like cysteine proteases, Biol. Chem. 379 (1998) 137–
147.
[72] J.L. Harris, B.J. Backes, F. Leonetti, S. Mahrus, J.A. Ellman, C.S. Craik, Rapid and
general profiling of protease specificity by using combinatorial fluorogenic
substrate libraries, Proc. Natl. Acad. Sci. USA 97 (2000) 7754–7759.
[73] S.S. Cotrin, L. Puzer, W.A. Judice, L. Juliano, A.K. Carmona, M.A. Juliano,
Positional-scanning combinatorial libraries of fluorescence resonance energy
transfer peptides to define substrate specificity of carboxydipeptidases: assays
with human cathepsin B, Anal. Biochem. 335 (2004) 244–252.
[81] K. Morris, M.D. Lorenzen, Y. Hiromasa, J.M. Tomich, C. Oppert, E.N. Elpidina,
K.S. Vinokurov, J.L. Jurat-Fuentes, J. Fabrick, B. Oppert, Tribolium castaneum
larval gut transcriptome and proteome:
a resource for the study of the
coleopteran gut, J. Proteome Res. 8 (2009) 3889–3898.
795
Please cite this article in press as: T.A. Semashko et al., Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in
complex mixtures , Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2013.12.032