Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family

Article abstract of DOI:10.1134/S1068162008030151

A chemoenzymatic synthesis was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and Amc are o-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate (I) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate (II) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel. Hydrolysis of substrate (I) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate (II), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate (II), than for substrate (I). The lowest detectable papain concentrations were 2.4 × 10^-10 M for (I) and 1.2 × 10^-11 M for (II). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.

Full text of DOI:10.1134/S1068162008030151

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 3, pp. 339–343. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © T.A. Semashko, E.N. Lysogorskaya, E.S. Oksenoit, A.V. Bacheva, I.Yu. Filippova, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 3,
pp. 376–381.
Chemoenzymatic Synthesis of New Fluorogenous Substrates
for Cysteine Proteases of the Papain Family
T. A. Semashkoa, E. N. Lysogorskayab, E. S. Oksenoitb, A. V. Bachevab, and I. Yu. Filippovab,1
a Faculty of Bioengineering and Bioinformatics, Moscow State University, Vorob’evy gory, Moscow, 119991 Russia.
b Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119991 Russia.
Received July 02, 2007; in final form, November 15, 2007
Abstract—A chemoenzymatic synthesis was developed for new highly specific fluorogenic substrates for cys-
teine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and
Amc are o-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Sub-
strate (I) was obtained in an aqueous–organic medium using native chymotrypsin. Substrate (II) was synthe-
sized in DMF–MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl
alcohol cryogel. Hydrolysis of substrate (I) with papain, ficin, and bromelain was accompanied by a 15-fold
increase in fluorescence intensity, and that of substrate (II), by a change in the fluorescence spectrum. Unam-
biguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the
substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity
for both substrates. The activity of all proteases under study was essentially higher for substrate (II), than for
substrate (I). The lowest detectable papain concentrations were 2.4 × 10–10 M for (I) and 1.2 × 10–11 M for (II).
A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.
Key words: cysteine proteases, enzymatic peptide synthesis, fluorogenic substrates, papain
DOI: 10.1134/S1068162008030151
INTRODUCTION
family had been earlier synthesized in our laboratory
[5]. This compound was successfully used to monitor
the enzymatic activity of cysteine proteases of different
origin [5, 6]. This study is devoted to the development
of chemoenzymatic synthesis of fluorogenic analogues
of this substrate, Abz-Phe-Ala-pNA (I) and Glp-Phe-
Ala-Amc (II).
Papain and related enzymes of plant (ficin, brome-
lain) and animal origin (cathepsins B, H, and L) form
one of the most numerous families of cysteine pro-
teases (EC 3.4.22) [1, 2].2
These enzymes are of interest due to both their
important physiological role in degradation of proteins
and regulation of some metabolic processes and the
wide practical use in medicine, food industry, agricul-
ture, biotechnology, and scientific research. The studies
of proteases belonging to this group are complicated
because the synthetic substrates usually used for the
determination of their activity to more extent corre-
spond to the specificity of trypsin and related enzymes
[3–5]. This reduces both the selectivity and sensitivity
of the correct determination of the activity of cysteine
proteases.
RESULTS AND DISCUSSION
The molecule of Abz-Phe-Ala-pNA (I) contains the
N-terminal fluorogenic residue of anthranilic acid
(Abz) and the ë-terminal p-nitroanilide (pNA) group, a
fluorescence quencher. We had earlier shown the suc-
cessful use of these moieties as donor–acceptor pairs in
substrates with intramolecular quenching of fluores-
cence for aspartyl proteases [7] and metalloproteases
[8]. Fluorescent properties of Glp-Phe-Ala-Amc (II)
are determined by the presence of ë-terminal 4-amino-
7-methylcoumaride (Amc) group, one of the most well-
known and effective fluorophores [9, 10].
Chromogenic substrate Glp-Phe-Ala-pNA corre-
sponding in specificity to cysteine proteases of papain
1
Corresponding author; phone: +7 (495) 939-5529; fax: +7 (495)
932-8846; e-mail: irfilipp@genebee.msu.su
Abbreviations: Abz-OH, o-aminobenzoic acid; Amc, 4-amino-7-
methylcoumaride; ChT and iChT, native and immobilized chy-
motrypsin; DCC, N,N'-dicyclohexylcarbodiimide; Glp, pyro-
glutamyl; HOBt, N-hydroxybenzotriazole; iSL, immobilized sub-
tilisin; pNA, p-nitroanilide; PVA, polyvinyl alcohol; and TEA,
triethylamine. All amino acids are of L-series.
2
Substrates (I) and (II) were obtained by a combina-
tion of chemical and enzymatic methods (scheme). The
use of enzymes at the final step guaranteed a high opti-
cal purity of products and facilitated isolation and puri-
fication of the target peptides.
339

340
SEMASHKO et al.
(‡)
AbzOH
HOBt
Boc-AlaOH
pNA
DCC, dioxane
POCl3, pyridine, –15°C
AbzOBt
Boc-Ala-pNA
HCl · PheOMe
Et3N, DMF
3M HCl, dioxane
Abz-PheOMe
HCl · Ala-pNA
N-methylmorpholine, DMF,
aqueous buffer, ChT
Abz-Phe-Ala-pNA (I)
(b)
GlpOH
C6F5OH
HCl · PheOMe
Et3N, EtOAc
DCC, EtOAc, 0°C
PheOMe
GlpOC6F5
EtOAc
Glp-PheOMe
CF3COOH · Ala-Amc
iChT, Et3N, DMF/MeCN (20/80)
Glp-Phe-Ala-Amc (II)
Scheme 1. The synthetic scheme for (a) Abz-Phe-Ala-pNA (I) and (b) Glp-Phe-Ala-Amc (II).
The carboxyl componentAbz-Phe-OMe for the syn- in an aqueous–organic medium under the conditions of
thesis of (I) was obtained by the method of active esters
using Abz-OBt [11] (scheme 1 ‡). Enzymatic conden-
sation Abz-Phe-OMe and Ala-pNA was carried out by
the treatment with native chymotrypsin in an aqueous–
organic medium at pH 7.2 and pH 9.9 in the presence of
50% DMF. Concentrations of starting compounds of
the enzymatic reaction were 0.2 M; the concentration
of the enzyme was 80 µM ([S] : [E] = 2500 : 1; herein-
after, mol/mol). Chymotrypsin was selected as a cata-
lyst, because of the correspondence of the structure of
the peptide synthesized to the requirements of the
enzyme specificity, the presence of Phe residue in P1-
position. The shift of the equilibrium to the substrate
synthesis was ensured by the removal of the reaction
product from the reaction medium due to its precipita-
tion. The yield of the peptide was shown to be 70% at
pH 9.9 and 60% at pH 7.2.
peptide (I) synthesis failed because of very low solubil-
ity of Ala-Amc. Synthesis in 20 : 80 DMF–MeCN with
chymotrypsin and subtilisin Carlsberg preparations
immobilized on PVA cryogel used as catalysts was
more effective for obtaining of (II). Immobilized
enzymes were obtained by covalent fixation of pro-
teases on a PVA cryogel matrix activated with glutaral-
dehyde [13]. The choice of these enzymes was stipu-
lated by conformity of the structure of the peptide syn-
thesized to the requirements to their specificity. In the
case of immobilized chymotrypsin (iChT) at a 1 : 2500
enzyme–substrate ratio, the yield of the product was
already 52% after 2 h and 100% after 24 h (Fig. 1‡). We
have shown that immobilized chymotrypsin can be
used repeatedly, the efficiency of Glp-Phe-Ala-Amc
synthesis being practically the same both in the case of
4-h and at 48-h incubation (Fig. 1b).
Synthesis of Glp-Phe-Ala-Amc (II) is shown in
scheme 1b. Chemical synthesis of Glp-Phe-OMe was
carried by the method of active esters according to pro-
cedure [12]. An attempt to carry out enzymatic conden-
In the case of immobilized subtilisin Carlsberg
(iSL) at a 1 : 5400 enzyme–substrate ratio, a slower
accumulation of the product was observed. The yield of
sation between Glp-Phe-OMe and Ala-Amc fragments the peptide after 96 h was only 56% (Fig. 1‡).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 3 2008

CHEMOENZYMATIC SYNTHESIS OF NEW FLUOROGENOUS SUBSTRATES
341
(‡)
I, %
100
Product yield, %
(‡)
100
100
100
100
80
60
40
20
Abz-Phe-Ala-pNA (1)
Abz-Phe-Ala-OH + pNA (2)
iChT
iSL
80
2
56.2
52.7
60
40
18.2
11.2
20
0
1
0
2
24
48
96
(b)
100
0
100
100
75
92.5
(b)
1 cycle
2 cycle
80
60
40
20
0
Glp-Phe-Ala-Amc (1)
Glp-Phe-Ala-OH + Amc (2)
2
50
50
50
1
25
0
4
48
Time, h
Fig. 1. (a) A comparison of yields of a product of enzymatic
synthesis Glp-Phe-Ala-Amc in 20 : 80 DMF–MeCN cata-
lyzed by iChT and iSL preparations; (b) the efficiency of
Glp-Phe-Ala-Amc synthesis catalyzed by iChT in two cycle
mode. See the Experimental section for reaction conditions.
350
400
450
500
550
600
λem, nm
Fig. 2. Fluorescence spectra of (1) (a) Abz-Phe-Ala-pNA
(I) and (2) (b) Glp-Phe-Ala-Amc (II) and the products of
their enzymatic hydrolysis by papain in 0.1 M universal
buffer containing 1 mM dithiothreitol, 0.1 mM EDTA, and
2.5% DMF; concentration of substrate of 5 µM, concentra-
tion of papain 2.5 µg/ml.
The compounds synthesized are stable crystalline
substances. The resulting peptides were characterized
by amino acid analysis, mass spectrometry, chromato-
graphic mobility in several systems (TLC) and reten-
tion time (HPLC).
than that of bromelain and ficin, respectively. Note that
the activities of all proteases under study were essen-
tially higher toward Glp-Phe-Ala-Amc (II) than toward
Abz-Phe-Ala-pNA (I).
The minimum papain concentrations detected were
2.4 × 10–10 M for substrate (I) and 1.2 × 10–11 M for sub-
strate (II).
The compounds obtained were hydrolyzed with
papain, ficin and, bromelain. Unambiguity of the enzy-
matic hydrolysis of substrates after residue Ala was
shown by HPLC. Hydrolysis of substrate (I) was
accompanied by a 15-fold increase in fluorescence
(Fig. 2‡) due to the cleavage of quencher pNA.
The enzymatic hydrolysis of substrate (II) by cys-
teine proteases resulted in the elimination of fluoro-
genicAmc-group and to the shift of the spectrum of flu-
orescence to the more long-wave region (Fig. 2b).
Enzymatic cleavage was detected at λex = 380 nm and
λem = 460 nm; i.e. under the conditions when fluores-
cence of the starting substrate was not observed.
Specific activities of papain, bromelain, and ficin for sub-
strates Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II)
Specific activity, nmol/min OU280
Enzyme
(I)
(II)
The data on the specific activity of papain, brome-
lain, and ficin for hydrolysis of substrates (I) and (II)
are given in the table. Obviously, papain was found to
have the greatest activity toward both substrates. Its
activity was approximately 5-fold and 15-fold higher
Papain
40
1
338.5 0.8
63.1 0.5
23.1 0.1
Bromelain
Ficin
7.9 0.4
2.35 0.07
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 3 2008

342
SEMASHKO et al.
The special experiment was carried out to show that fluorimeter. Emission spectra were registered at λex
proteases of other classes, aspartyl (pepsin), serine 340 nm for Abz-containing derivatives as descried in
(chymotrypsin, subtilisin Carlsberg, trypsin), and met- [7]; at λex 330 nm for substrate Glp-Phe-Ala-Amc (II),
alloproteases (thermolysin) do not hydrolyze Glp-Phe- and at λex 355 nm for the products of enzymatic hydrol-
Ala-Amc. This fact confirms a high selectivity of cys- ysis of (II) as described in [10].
teine proteases toward this substrate, which allows its
Centrifugation was carried out on a Minispin
use for testing cysteine proteases in the presence of
Eppendorf (Germany) centrifuge at 13000 g.
other classes of enzymes.
Amino acid analyses were carried out on a Hitachi
835 (Japan) automatic amino acid analyzer after acidic
hydrolysis of samples with 5.7 M HCl at 105°ë in evac-
EXPERIMENTAL
uated ampoules for 48 h.
Papain (EC 3.4.22.2, M 23.4 kDa) was from Sigma
(United States), bromelain (EC 3.4.22.32, M 24 kDa)
was from Lawson Chemicals (UK), ficin (EC 3.4.22.3,
M 23.8 kDa) was from Calbiochem (United States),
subtilisin Carlsberg (EC 3.4.21.62, 27 kDa) was from
Sigma (United States), porcine pepsin (EC 3.4.23.1,
M 35 kDa) was purified by the method [14], bovine
trypsin (EC 3.4.21.4, M 23.8 kDa) was from Sigma
(United States), and thermolysin (EC 3.4.24.27,
M 37.5 kDa) and chymotrypsin (EC 3.4.21.1, M 25 kDa)
were from Fluka (Switzerland). Acetonitrile (Lekbio-
farm, Russia; special purity grade) containing less than
0.01% water was used for HPLC; DMF, (Reakhim,
Russia; analytical grade) was additionally purified by
the method [15]; TFA (analytical grade) was from
Fluka (Switzerland), acetic acid was from Reakhim
(Russia), CaCl2 was from Sigma (United States), and
Tris was from ISN Biomedicals (United States). TEA
(Reakhim, Russia; analytical grade) was additionally
purified by the method [15]. EDTA was from Sigma
(United States), dithiothreitol was from Aldrich; Abz-
OH was from Sigma (United States); and DCC, Glp-
OH, C6F5OH, and TFA·H-Ala-Amc were from Bachem
(Germany).
Mass spectra were taken on a Finnigan LCQ-IOn
Trap (Thermo Electron, United States) instrument
using electrospray ionization method.
Abz-Phe-OMe. Anhydrous TEA (0.7 ml, 5 mmol)
and Abz-OBt (1.90 g, 7.5 mmol) were added to a solu-
tion of HCl · H-Phe-OMe (1.07 g, 5 mmol) in anhy-
drous DMF (20 ml); the mixture was stirred for 24 h at
20°ë; the solution was cooled to 10°ë; and 0.5 N
NaHCO3 (100 ml) was added. The precipitate was dis-
solved in ethyl acetate and evaporated; the residue was
dissolved in 1 : 1 diethyl ether–petroleum ether (40 ml)
and kept for 24 h at 0°ë. The precipitate was filtered
and washed with petroleum ether (3 × 30 ml); yield:
0.63 g (42%); Rf 0.63 (B) and 0.64 (C).
Abz-Phe-Ala-pNA. N-Methylmorpholine (0.1 ml
of 1 M solution in DMF), DMF (0.3 ml), 0.25 ml of
0.2 M buffer (Tris-HCl, pH 7.2, or Tris-carbonate,
pH 9.9) and chymotrypsin (1 mg) were added to a mix-
ture of Abz-Phe-OMe (29.8 mg, 0.1 mmol) and HCl ·
H-Ala-pNA (24.5 mg, 0.1 mmol). The reaction mixture
was stirred for 1 h at 20°ë and kept for 12 h at 0°ë. The
precipitate was separated by centrifugation and washed
with 0.1 M HCl (2 × 0.3 ml), water (2 × 0.3 ml), 3%
NaHCO3 (2 × 0.3 ml), and water up to pH 7; yield:
32.2 g (70%); Rf 0.32 (C). Retention time for Abz-Phe-
Ala-pNA 23.5 min. Amino acid analysis (nmol): Ala
9.592; Phe 9.256 (1.04 : 1). MS, m/z: 475.1. Calculated:
475.1.
Abz-OBt, Glp-Phe-OMe, and Ala-pNA were syn-
thesized as described in [11, 12, 16], respectively. Prep-
arations of chymotrypsin and subtilisin Carlsberg
immobilized on PVA cryogel were obtained as
described in [13]. The amount of immobilized protein
was estimated by amino acid analysis.
Glp-Phe-Ala-Amc (analytical variant). Triethy-
lamine (12 µl of 1 M solution in DMF), Glp-Phe-OMe
(2.9 mg, 10 µmol) in 20 : 80 (vol/vol) DMF–MeCN
(50 µl), and MeCN (188 µl) were added to TFA · H-
Ala-Amc (3.6 mg, 10 µmol) in DMF (50 µl). The
resulting mixture was poured to a preparation of immo-
bilized chymotrypsin (20 mg, protein content 0.1 mg)
or to immobilized subtilisin Carlsberg (30 mg, protein
content 0.05 mg); the resulting mixture was shaken on
an orbital shaker at 20°ë, and 5-µl samples for HPLC
analysis were taken at regular intervals. Retention time
for Glp-Phe-Ala-Amc 15.9 min.
TLC of peptides was carried out on Silufol (Czech
Republic) using the following developing systems: (A)
15 : 12 : 10 : 3 n-butanol–water–pyridine–acetic acid,
(B) 3 : 1 : 1 n-butanol–acetic acid–water, (C) 100 : 25 : 4
benzene–acetone–acetic acid; and (D) 10 : 6 : 5 : 1 tol-
uene–chloroform–methylethyl ketone–isopropanol.
Peptides were analyzed by rpHPLC on an Altex
Model 110 A (United States) chromatogtaph using a
Nucleosil C18 (4.6 × 250 mm) column (Biokhimmak,
Russia) (elution with 0.1% TFA in a linear 10
70%
gradient of MeCN, flow rate 1 ml/min, 30 min). Pep-
tides were detected at 220 nm. The composition of the
reaction mixture was calculated without adjustment for
the difference in molar extinction coefficients.
To study the opportunity of repeated use of immobi-
lized chymotrypsin in Glp-Phe-Ala-Amc synthesis
after the first cycle, the biocatalyst granules were sepa-
Spectrophotometric measurements were carried out rated, washed with 20 : 80 (vol/vol) DMF–MeCN (2 ×
on a U-2800A (Hitachi, Japan) spectrophotometer; flu- 300 µl) for 20 min, then with 0.05 M Tris-HCl buffer
orescence was measured on a MPF-4 (Hitachi, Japan) (pH 8.0) containing 50 mM CaCl2 for 10 min, and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 3 2008

CHEMOENZYMATIC SYNTHESIS OF NEW FLUOROGENOUS SUBSTRATES
343
entered into the next cycle of synthesis, adding the solu- larly, using 0.05 M Tris-HCl buffer (pH 8.0) containing
tion of the starting components.
50 mM CaCl2 for α-chymotrypsin, subtilisin Carlsberg,
and thermolysin and 0.1 M acetate buffer (pH 4.2) for
pepsin.
Preparative synthesis of Glp-Phe-Ala-Amc. A
solution of TFA · H-Ala-Amc (36.0 mg, 100 µmol) in
DMF (0.62 ml), 120 µl of 1 M TEA in DMF, a solution
of Glp-Phe-OMe (29.0 mg, 100 µmol) in 20 : 80
(vol/vol) DMF–MeCN (0.5 ml), and MeCN (1.88 ml)
were added to the a preparation of chymotrypsin immo-
bilized on PVA cryogel (200 mg, protein content 1 mg).
The reaction mixture was stirred on an orbital shaker at
20°ë for 24 h, taking 5-µl samples at regular intervals
for HPLC analysis. After 24 h, the biocatalyst was sep-
arated, washed with 20 : 80 (vol/vol) DMF–MeCN (4 ×
3 ml). The reaction mixture and combined washings
were evaporated in a vacuum. Hydrochloric acid
(1.5 ml of 0.1 M solution) was added dropwise to the
residue, the mixture was stirred, cold water (5 ml) was
added in small portions, and the resulting mixture was
kept for 12 h at 0°ë. The precipitate was separated by
centrifugation and dried over NaOH in a vacuum desic-
cator; yield: 22.8 mg (46%); retention time for Glp Phe-
Ala-Amc 15.9 min; Rf 0.32 (C). Amino acid analysis
(nmol): Ala 1.142; Phe 1.491 (1 : 1.3). MS, m/z: 504.2.
Calculated 504.2.
ACKNOWLEDGMENTS
The work was supported by the Russian Foundation
for Basic Research (project nos. 06-03-33056-a and
07-04-12025-ofi_a).
REFERENCES
1. Rawlings, N.D.. and Barrett, A.J., Biochem. J., 1993,
vol. 290, pp. 215–218.
2. Otto, H.-H. and Schirmeister, T., Chem. Rev., 1997,
vol. 97, pp. 133–171.
3. Lecaille, F., Serveau, C., Gauthier, F., and Lalmanach, G.,
FEBS Lett., 1999, vol. 445, pp. 311–314.
4. Melo, R.L., Pozzo, R.C.B., Alves, L.C., Perissutti, E., Cali-
endo, G., Santagada, V., Juliano, L., and Juliano, M.A., Bio-
chim. Biophys. Acta, 2001, vol. 1547, pp. 82–94.
5. Stepanov, V.M., Lysogorskaya, E.N., Filippova, I.Yu.,
Oksenoit, E.S., and Lyublinskaya, L.A., USSR Inven-
tor’s Certificate No. 1 198 082, Byull. Izobret., 1985,
no. 46.
Measurements of the specific activity of papain,
bromelain, and ficin. A solution of enzyme (50 µl, the
starting concentration 20 µg/ml)) in the 0.1 M universal
buffer (phosphoric, acetic, and boric acids, NaOH, and
KCl, pH 6.0) [17] containing 1 mM dithiotreitol, 0.1
mM EDTA, and 2.5% DMF was added to 2 ml of 5 µl
solution of Abz-Phe-Ala-pNA or Glp-Phe-Ala-Amc in
the same buffer. The reaction mixture was stirred at
25°ë; the increase in fluorescence intensity with time
was measured at λex 340 nm and λem 415 nm for Abz-
Phe-Ala-pNA and at λex 380 nm and λem 460 nm for
Glp-Phe-Ala-Amc.
6. Vinokurov, K.S., Elpidina, E.N., Oppert, B., Prabhakar, S.,
Zhuzhikov, D.P., Dunaevsky, Y.E., and Belozersky, M.A.,
Comp. Biochem. Physiol., 2006, vol. 145B, pp. 126–137.
7. Filippova, I.Yu., Lysogorskaya, E.N., Anisimova, V.V.,
Suvorov, L.I., Oksenoit, E.S., and Stepanov, V.M., Anal.
Biochem., 1996, vol. 234, pp. 113–118.
8. Filippova, I.Yu., Lysogorskaya, E.N., Oksenoit, E.S.,
Troshchenkova, E.P., and Stepanov, V.M., Bioorg.
Khim., 1988, vol. 14, pp. 467–471.
9. Smith, R.E., J. Histochem. & Cytochem., 1983, vol. 31,
pp. 199–219.
The specific activity was computed for 1 éÖ280 of
an enzyme from the formula:
10. Weder, J.K.P. and Kaiser, K.-P., J. Chromatogr., A, 1995,
vol. 698, pp. 181–211.
11. Stewart, F.H.C., Aust. J. Chem., 1983, vol. 36, pp. 1629–
dI/dt[S]
--------------------------------------
A =
,
1638.
OU280(I100 – I0)
12. Filippova, I.Y., Lysogorskaya, E.N., Oksenoit, E.S.,
Rudenskaya, G.N., and Stepanov, V.M., Anal. Biochem.,
1984, vol. 143, pp. 293–297.
where dI/dt is an increase in fluorescence intensity with
time; [S] is the concentration of a substrate in a sample,
µM; I0 is the own fluorescence intensity of a substrate;
I100 is the fluorescence intensity upon 100% hydrolysis
of a substrate (it is measured in a separate experiment
by incubation of a substrate solution with the excess of
an enzyme).
13. Lozinskii, V.I., Plieva, F.M., and Zubov, A.L., Biotekh-
nol., 1995, pp. 32–37.
14. Solov’eva, T.A., Belyaev, S.V., and Stepanov, V.M.,
Khim. Prir. Soedin., 1977, no. 3, pp. 398–403.
15. Gordon, A.J. and Ford, R.A., A Handbook of Practical
Data, Techniques, and References, New York: Wiley,
1972.
Calculations were carried out by the least square
method using Microsoft Excel and OriginPro 7.5 pro-
grams.
16. Gershkovich, A.A. and Kibirev, V.K., Khimicheskii sin-
tez peptidov (Chemical Synthesis of Peptides), Kiev:
Naukova dumka, 1992.
Measurement of specific activity of proteases of
other classes for hydrolysis of substrates Abz-Phe-
Ala-pNA or Glp-Phe-Ala-Amc was carried out simi-
17. Frugoni, J., Gazz Chem. Ital., 1957, vol. 87, pp. 403–
407.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 3 2008