Stability of His-Phe-Arg-Trp-Pro-Gly-Pro to Leucine Aminopeptidase, Carboxypeptidase Y, and Rat Nasal Mucus, Blood, and Plasma

Article abstract of DOI:10.1134/S1068162016020126

The His-Phe-Arg-Trp-Pro-Gly-Pro [ACTH-(6-9)-PGP] peptide was synthesized. Proteolysis of ACTH-(6-9)-PGP and semax (Met-Glu-His-Phe-Pro-Gly-Pro) in the presence of leucine aminopeptidase and carboxypeptidase Y was studied. If the proteolysis of ACTH-(6-9)-PGP is mainly defined by aminopeptidases, the basic metabolite is Trp-Pro-Gly-Pro. Identification of major metabolites of ACTH-(6-9)PGP when incubated with the enzymes in vitro made it possible to evaluate proteolysis pathways of the peptide and prepare necessary amounts of its metabolites for using them as standards. Kinetics of ACTH-(6-9)PGP degradation in the presence of enzyme systems of nasal mucus, blood, and plasma were also explored. It was found that proteolysis of ACTH-(6-9)-PGP in rat blood and plasma occurs mainly under the effect of enzymes whose action is similar to leucine aminopeptidase.

Full text of DOI:10.1134/S1068162016020126

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2016, Vol. 42, No. 2, pp. 153–161. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © K.V. Shevchenko, S.A. Dulov, L.A. Andreeva, I.Yu. Nagaev, V.P. Shevchenko, A.S. Radilov, N.F. Myasoedov, 2016, published in Bioorganicheskaya
Khimiya, 2016, Vol. 42, No. 2, pp. 171–181.
Stability of HisꢀPheꢀArgꢀTrpꢀProꢀGlyꢀPro to Leucine
Aminopeptidase, Carboxypeptidase Y, and Rat Nasal Mucus, Blood,
and Plasma
a, 1
b
a
a
a
K. V. Shevchenko , S. A. Dulov , L. A. Andreeva , I. Yu. Nagaev , V. P. Shevchenko ,
b
a
A. S. Radilov , and N. F. Myasoedov
a
Institute of Molecular Genetics, Russian Academy of Sciences, plosh. Kurchatova 2, Moscow, 123182 Russia
b
Research Institute of Hygiene, Occupational Pathology, and Human Ecology, Federal Medical–Biological Agency,
st. Kapitolovo 93, Kuz’molovsky, 188663 Russia
Received April 20, 2015; in final form, September 16, 2015
Abstract—The HisꢀPheꢀArgꢀTrpꢀProꢀGlyꢀPro [ACTHꢀ(6–9)ꢀPGP] peptide was synthesized. Proteolysis
of ACTHꢀ(6–9)ꢀPGP and semax (MetꢀGluꢀHisꢀPheꢀProꢀGlyꢀPro) in the presence of leucine aminopeptiꢀ
dase and carboxypeptidase Y was studied. If the proteolysis of ACTHꢀ(6–9)ꢀPGP is mainly defined by amiꢀ
nopeptidases, the basic metabolite is TrpꢀProꢀGlyꢀPro. Identification of major metabolites of ACTHꢀ(6–9)ꢀ
PGP when incubated with the enzymes in vitro made it possible to evaluate proteolysis pathways of the pepꢀ
tide and prepare necessary amounts of its metabolites for using them as standards. Kinetics of ACTHꢀ(6–9)ꢀ
PGP degradation in the presence of enzyme systems of nasal mucus, blood, and plasma were also explored.
It was found that proteolysis of ACTHꢀ(6–9)ꢀPGP in rat blood and plasma occurs mainly under the effect of
enzymes whose action is similar to leucine aminopeptidase.
Keywords: semax, ACTHꢀ(6–9)ꢀPGP, leucine aminopeptidase, carboxypeptidase Y, degradation of peptides,
enzymes of nasal mucus, blood, and plasma
DOI: 10.1134/S1068162016020126
1
INTRODUCTION
such a study should be the investigation of resistance of
a substance to the effect of proteolytic enzymes [2, 3].
In this connection, resistance of ACTHꢀ(6–9)ꢀPGP
to proteolytic enzymes and its enzymatic hydrolysis
pathways should be determined and the intermediate
products of the peptide degradation should be accuꢀ
mulated to be used as standard substances.
Previously, various fragments of adrenocorticotroꢀ
pic hormone (ACTH) that exhibited nootropic and
neuroprotective activities comparable to those of
7
8
9
10
semax, including Phe ꢀArg ꢀTrp ꢀGly ꢀProꢀGlyꢀPro
4
5
6
7
8
(
ACTHꢀ(7–10)ꢀPGP), Met ꢀGlu ꢀHis ꢀPhe ꢀArg ꢀ
9
10
Trp ꢀGly ꢀProꢀGlyꢀPro
(ACTHꢀ(4–10)ꢀPGP),
6
7
8
9
His ꢀPhe ꢀArg ꢀTrp ꢀProꢀGlyꢀPro (ACTHꢀ(6–9)ꢀ
Different routes of administration of starting pepꢀ
5
6
7
PGP), and Glu ꢀHis ꢀPhe ꢀProꢀGlyꢀPro (ACTHꢀ(5– tides result in different sets of hydrolysis products,
7
)ꢀPGP), have been tested [1]. Experiments on aniꢀ while these shorter peptides might have biological
mals demonstrated that ACTHꢀ(6–9)ꢀPGP was the activities of their own. This fact should be taken into
most promising from the point of view of its biological account when choosing the route of administration.
activities. Not only did the peptide exhibit nootropic For example, data on ProꢀGlyꢀProꢀLeu metabolism
and anxiolytic activities, it also enhanced the viability indicate that different routes of administration result
of cultured glia cells collected at the cerebral cortex of in up to threeꢀfold differences in GlyꢀPro content [4].
rats having suffered ischemic brain stroke. Study of the Meanwhile, GlyꢀPro exhibits a pronounced protecꢀ
effect of ACTHꢀ(6–9)ꢀPGP on size of necrotic tive effect and prevents anxiety increase and decrease
lesions in rats demonstrated that, similarly to semax, of orientational–investigative activity [5]. Since
the peptide causes an approximately 50% decrease in ACTHꢀ(6–9)ꢀPGP is planned to be administered
the size of the lesions in the course of ischemic stroke. either intravenously or intranasally, the kinetics of
degradation was determined using the enzymes of
nasal mucus, blood, and plasma.
Substances that are to be used as drugs must be subꢀ
jected to a multifaceted study. One of the aspects of
Intranasal administration is noninvasive, painless,
simple, and convenient to use. On the other hand,
there is an enzymatic barrier designed to prevent forꢀ
eign substances from entry into the organism through
Abbreviations: ACTH, adrenocorticotropic hormone; PGP,
ProꢀGlyꢀPro.
1
Corresponding author: phone: (499) 196ꢀ02ꢀ16; fax: (499) 196ꢀ
0216; eꢀmail: ATRegister@mail.ru.
153

154
SHEVCHENKO et al.
HIS
PHE
ARG
TRP
PRO
GLY
OH
OH H
PRO
Boc OH
H
(
I)
Boc
Boc
H
OBzI
OBzI
OBzI
OBzI
OBzI
OBzI
OBzI
OBzI
OBzI
OBzI
OH
(II)
(
III)
IV)
V)
(VI)
Boc OH
(
Boc
H
(
Boc OH
NO2
Boc
NO2
H
(VII)
Boc OH
Boc
NO2
NO2
NO2
NO2
(VIII)
(IX)
Boc OH H
(X)
Boc
Boc
H
(XI)
(XII)
OH
Scheme 1. Scheme of ACTHꢀ(6–9)ꢀPGP heptapeptide synthesis.
the nasal cavity, which should be taken into account tion of 1ꢀhydroxybenzotriazole, and the method of
6–8]. Yet, in the case of low stability of a substance in activated esters. Protective groups were removed by
blood, intranasal administration may provide for hydrogenation with dry hydrogen over palladium
[
higher bioavailability of the drug.
black (in case of benzyl ether, or OBzl) or hydrolysis in
trifluoroacetic acid (in case of tretꢀbutoxycarbonyl, or
Boc). To turn NO2ꢀderivative of Arg into Arg, condiꢀ
tions similar to removal of OBzl were used. ACTHꢀ
The aims of the work were the synthesis of ACTHꢀ
6–9)ꢀPGP and the study of its resistance to the effects
(
of leucine aminopeptidase (EC 3.4.11.2) and carboxꢀ
ypeptidase Y (EC 3.4.16.1). Study of the kinetics of
ACTHꢀ(6–9)ꢀPGP degradation in the presence of
enzymes of nasal mucus, blood, or plasma and determiꢀ
nation of the set of proteolytic fragments thus formed will
provide the means to determine the direction of ACTHꢀ
(
6–9)ꢀPGP thus obtained, along with other subꢀ
stances, was characterized by LCꢀMS (Fig. 1).
Data on kinetics of ACTHꢀ(6–9)ꢀPGP and semax
degradation by leucine aminopeptidase and carboxꢀ
ypeptidase Y are presented in Fig. 2.
(6–9)ꢀPGP degradation in the body. Semax preparation
As expected, the resistance of ACTHꢀ(6–9)ꢀPGP
and semax to hydrolysis by leucine aminopeptidase was
different (due to the different Nꢀterminal amino acid resꢀ
idues, histidine and methionine; Fig. 2a); degradation in
the presence of carboxypeptidase Y proceeded at a simiꢀ
lar rate since the Cꢀterminal GlyꢀPro fragment is the
same in both peptides (Fig. 2b). After 60 min, the rate of
semax degradation by carboxypeptidase Y became lower
than that of ACTHꢀ(6–9)ꢀPGP. Supposedly, degradaꢀ
tion products of the peptides modify the enzyme, which
leads to greater slowdown of semax hydrolysis compared
to that of ACTHꢀ(6–9)ꢀPGP.
was used as a reference since its degradation pathways
have been studied thoroughly [9, 10].
RESULTS AND DISCUSSION
Hydrolysis of ACTHꢀ(6–9)ꢀPGP with commercial
enzymes was performed to find out which enzymes
determine the efficiency of degradation and which set
of intermediate peptides is formed in the reaction mixꢀ
ture. The shape of the kinetic curves of ACTHꢀ(6–9)ꢀ
PGP degradation in the presence of leucine amiꢀ
nopeptidase and carboxypeptidase Y answers the first
question. Analysis of the composition of reaction mixꢀ
ture formed upon ACTHꢀ(6–9)ꢀPGP hydrolysis by
either of the enzymes answers the second question.
Dynamics of ACTHꢀ(6–9)ꢀPGP hydrolysis with
leucine aminopeptidase and carboxypeptidase Y
assessed by LCꢀMS of the reaction mixtures allowed
the study of the kinetics of intermediate products genꢀ
eration in the course of the peptide degradation by
these two commercial exopeptidases and determinaꢀ
tion of major degradation pathways (Fig. 3).
Firstly, the starting ACTHꢀ(6–9)ꢀPGP peptide was
synthesized for further studies. The synthesis was perꢀ
formed with classical methods of peptide chemistry in
solution using both protected and free
Lꢀamino acids
according to Scheme 1. The peptides indicated in the
Analysis of the reaction mixtures afforded the folꢀ
scheme were synthesized with the method of mixed lowing conclusions. The maximum concentrations of
anhydrides, the carbodiimide method with the addiꢀ the intermediate products are reached in separated
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STABILITY OF HisꢀPheꢀArgꢀTrpꢀProꢀGlyꢀPro
155
759.44
100
448.89
90
80
70
60
50
40
30
20
10
0
8
96.49
4
41.37
3
00.34
4
62.33
599.38
627.39
753.52 781.53
2
14.12
304.26 424.34
391.35
467.92
595.45
35.91
918.45
957.30
2
57.23
814.46
159.23
662.38 696.44
863.63
5
200
300
400
500
600
700
800
900
1000
2
+
+ HꢀHis]+, 896.4 = + H]+).
[M
Fig. 1. LCꢀMS spectrum of the ACTHꢀ(6–9)ꢀPGP standard solution (448.9 =
[M
+ 2H] , 759.4 = [
M
The m/z ratio is plotted against the horizontal axis and the relative response, against the vertical axis.
(а)
(b)
1
00
100
90
80
70
60
50
40
30
20
10
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
1
2
1
2
0
30 60 90 120 150 180 210 240
Time, min
0
30 60 90 120 150 180 210 240
Time, min
Fig. 2. Hydrolysis of ACTHꢀ(6–9)ꢀPGP (
1) and semax (2) with (a) leucine aminopeptidase and (b) carboxypeptidase Y (in perꢀ
cent to the initial amount of the corresponding peptide).
time points. For example, when ACTHꢀ(6–9)ꢀPGP
The data evidence that TrpꢀProꢀGlyꢀPro is the
was hydrolyzed with leucine aminopeptidase, the most stable peptide; it is followed by PheꢀArgꢀTrpꢀ
maximum content of PheꢀArgꢀTrpꢀProꢀGlyꢀPro was
observed 90 min after the reaction start, while the
maximum content of TrpꢀProꢀGlyꢀPro and ArgꢀTrpꢀ
ProꢀGlyꢀPro was observed 180 min after the start. This
indicates that ArgꢀTrpꢀProꢀGlyꢀPro and TrpꢀProꢀ
GlyꢀPro are generated from PheꢀArgꢀTrpꢀProꢀGlyꢀ
Pro, and phenylalalnine cleavage proceeds much less
efficiently than further cleavage of arginine (Scheme 2).
ProꢀGlyꢀPro and ArgꢀTrpꢀProꢀGlyꢀPro. It should be
noted that under given conditions the strength of Hisꢀ
Phe, PheꢀArg, and TrpꢀPro bonds is higher than that
of ArgꢀTrp due to substrate specificity of leucine amiꢀ
nopeptidase. The ArgꢀTrpꢀProꢀGlyꢀPro peptide is the
most convenient substrate for the enzyme (both by
conformation and by charge), therefore, its hydrolysis
proceeds rapidly, while cleavage of HisꢀPhe and Pheꢀ
Arg bonds in initial and generated peptides is the limꢀ
iting stage for further hydrolysis (Figs. 2a and 3a).
Therefore, the maximum content of ArgꢀTrpꢀProꢀ
GlyꢀPro in the reaction medium does not even reach
HisPheArgTrpProGlyPro
Aminopeptidases
Carboxypeptidases
PheArgTrpProGlyPro HisPheArgTrpProGly
9
%, while the same values for the PheꢀArgꢀTrpꢀProꢀ
GlyꢀPro and TrpꢀProꢀGlyꢀPro peptides are 30 and
ArgTrpProGlyPro
TrpProGlyPro
HisPheArgTrp
Trp
40%, respectively.
In the study of the hydrolysis of Cꢀterminal peptide
bonds in ACTHꢀ(6–9)ꢀPGP with carboxypeptidase Y,
a single Cꢀterminal Pro was cleaved (Fig. 3b). The
HisꢀPheꢀArgꢀTrpꢀProꢀGly peptide was the most staꢀ
ble in the presence of carboxypeptidase Y.
Scheme 2. Generation of ACTHꢀ(6–9)ꢀPGP
hydrolysis products under the effect of proteases.
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156
SHEVCHENKO et al.
(а)
(b)
(c)
0
0
0
0
0
0
0
.50
.45
.40
.35
.30
.25
.20
1
00
45
40
35
30
25
20
15
10
5
1
2
3
4
9
8
7
6
5
4
3
2
0
5
0
0
0
0
0
0
5
0.15
0.10
0.05
10
0
60
120 180 240
150 210
0
60
120 180 240
150 210
0
60
120 180 240
90 150 210
30
90
30
90
30
Time, min
Time, min
Time, min
Fig. 3. Dynamics of the ACTHꢀ(6–9)ꢀPGP hydrolysis product content upon the hydrolysis with (a) leucine aminopeptidase and
(
b, c) carboxypeptidase Y:
1
, PheꢀArgꢀTrpꢀProꢀGlyꢀPro;
2
, TrpꢀProꢀGlyꢀPro;
3
, ArgꢀTrpꢀProꢀGlyꢀPro;
4, HisꢀPheꢀArgꢀTrpꢀ
ProꢀGly; , Trp (in percent to the initial amount of ACTHꢀ(6–9)ꢀPGP).
5
(а)
(b)
1
00
100
90
80
70
60
50
40
30
20
10
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
2
3
5
6
1
2
4
5
6
0
30 60 90 120 150 180 210 240
Time, min
0
30 60 90 120 150 180 210 240
Time, min
Fig. 4. Dynamics of the ACTHꢀ(6–9)ꢀPGP hydrolysis product content (in percent to the maximum amount of each of the indiꢀ
cated peptides formed in the experiment) upon different procedure of treatment with exopeptidases: (a) primary cleavage with
leucine aminopeptidase and hydrolysis of the formed peptides with carboxypeptidase Y and (b) primary hydrolysis with carboxꢀ
ypeptidase Y and hydrolysis of the products with leucine aminopeptidase.
1
, PheꢀArgꢀTrpꢀProꢀGlyꢀPro;
2, PheꢀArgꢀTrpꢀProꢀ
Gly; , HisꢀPheꢀArgꢀTrpꢀProꢀGly; , TrpꢀProꢀGlyꢀPro; , TrpꢀProꢀGLyꢀPro; 6
3
4
5
, Trp.
Therefore, one can expect that if ACTHꢀ(6–9)ꢀ carboxypeptidase Y. The second experimental setup
PGP hydrolysis is determined by leucine aminopeptiꢀ included initial treatment of ACTHꢀ(6–9)ꢀPGP with
dase, TrpꢀProꢀGlyꢀPro should be the main degradaꢀ carboxypeptidase Y and similar procedures of peptide
tion product. Similarly, hydrolysis of ACTHꢀ(6–9)ꢀ mixture purification and solubilization followed by
PGP with carboxypeptidase Y should lead to formaꢀ hydrolysis in the presence of leucine aminopeptidase.
tion of HisꢀPheꢀArgꢀTrpꢀProꢀGly (Scheme 2). To
Stability of the peptides generated in the two experꢀ
model degradation of ACTHꢀ(6–9)ꢀPGP in natural
imental setups described above is presented in Fig. 4.
media, an additional study was performed to produce
all possible degradation products of ACTHꢀ(6–9)ꢀ
The first setup results can be described as follows.
PGP enzymatic degradation. According to the first The percent concentration of PheꢀArgꢀTrpꢀProꢀGlyꢀ
experimental setup, ACTHꢀ(6–9)ꢀPGP was first subꢀ Pro and TrpꢀProꢀGlyꢀPro peptides formed under the
jected to hydrolysis with leucine aminopeptidase. effect of leucine aminopeptidase decreases, while the
Then, the enzyme was deactivated and the peptide concentration of tryptophan, TrpꢀProꢀGly, and Pheꢀ
mixture was separated with solidꢀphase extraction, ArgꢀTrpꢀProꢀGly in the reaction mixture grows (Fig. 4a).
resolubilized in an appropriate buffer, and treated with It should be noted that while PheꢀArgꢀTrpꢀProꢀGlyꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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STABILITY OF HisꢀPheꢀArgꢀTrpꢀProꢀGlyꢀPro
157
Table 1. Dynamics of ACTHꢀ(6–9)ꢀPGP and semax sum up this stage of the work, five products of progresꢀ
hydrolysis in rat mucus (in percent to the initial amount of
peptide; protein concentration 1.62 mg/mL)
sive cleavage of ACTHꢀ(6–9)ꢀPGP by leucine amiꢀ
nopeptidase and carboxypeptidase were registered.
Incubation time, min
Peptide
Generation of these products in the reaction mixꢀ
tures allowed for evaluation of the directions of
ACTHꢀ(6–9)ꢀPGP enzymatic hydrolysis. Besides, we
managed to accumulate enough of intermediate prodꢀ
ucts to be used as standard samples in the analysis of
the composition of the reaction mixtures produced
upon treatment of the peptide with more complex
enzymatic systems in vitro and identify these peptides
in biological samples obtained in vivo.
5
10 30 40 60 90 120
Semax
92 70 29 12
4
0.5
0
0
ACTHꢀ(6ꢀ9)ꢀPGP 81 64 52 45 31 14
Table 2. Dynamics of ACTHꢀ(6–9)ꢀPGP and semax
hydrolysis in rat blood (in percent to the initial amount of
peptide; protein concentration 9.35 mg/mL)
Data on the stability of ACTHꢀ(6–9)ꢀPGP in the
presence of rat nasal mucus and blood are presented in
Tables 1 and 2.
Incubation time, min
Peptide
2
5
10 20 30 40 60 90 120
At the initial stage, residual concentrations of
ACTHꢀ(6–9)ꢀPGP and semax in nasal mucus of rats
were approximately the same (Table 1). Then, semax
hydrolysis proceeded faster than that of ACTHꢀ(6–
Semax
ACTHꢀ(6ꢀ9)ꢀ 75 50 47 43 40 36 12
PGP
90 74 68 48 39 30
9
3
5
0
0
9
)ꢀPGP. Therefore, ACTHꢀ(6–9)ꢀPGP is somewhat
more resistant to nasal mucus than semax.
Comparison of ACTHꢀ(6–9)ꢀPGP and semax staꢀ
bilities in rat blood demonstrated that they were very
close (Table 2). During the first 20 min semax content
somewhat exceeded that of ACTHꢀ(6–9)ꢀPGP and
after 20 min ACTHꢀ(6–9)ꢀPGP content in the incuꢀ
bation medium was higher than that of semax.
Pro disappears completely after 90 min of the reacꢀ
tion, the TrpꢀProꢀGlyꢀPro content only drops to a
half. TrpꢀProꢀGly and PheꢀArgꢀTrpꢀProꢀGly are
highly stable; kinetic curves of generation of these
peptides reach a plateau. According to mass spectromꢀ
etry data, ProꢀGlyꢀPro, ProꢀGly, and GlyꢀPro are not
formed. In the second setup, the major product of
ACTHꢀ(6–9)ꢀPGP hydrolysis is completely cleaved
by carboxypeptidase within 40 min forming PheꢀArgꢀ
TrpꢀProꢀGly and TrpꢀProꢀGly. Concentration of
PheꢀArgꢀTrpꢀProꢀGly reaches its maximum after
Comparison of ACTHꢀ(6–9)ꢀPGP stabilities in
the presence of enzymatic systems of nasal mucus and
blood demonstrated that ACTHꢀ(6–9)ꢀPGP is more
stable in nasal mucus (see Tables 1 and 2).
Data on ACTHꢀ(6–9)ꢀPGP stability in blood
plasma of rats are presented in Table 3.
4
0 min of the reaction and the peptide disappears
completely 90 min after the reaction start. TrpꢀProꢀ
Gly is stable under the given conditions (Fig. 4b).
As follows form the presented data, ACTHꢀ(6–9)ꢀ
PGP stability in rat plasma is higher than that of
Therefore, products of ACTHꢀ(6–9)ꢀPGP hydrolꢀ semax. The trend persists also upon the increase in
ysis with leucine aminopeptidase being treated with plasma content in the incubation medium. It should
carboxipeptidase Y lose only the Cꢀterminal proline be noted that the difference in ACTHꢀ(6–9)ꢀPGP
residue; in the reverse experimental setup progressive and semax stabilities was the lowest when the amount
cleavage of Nꢀterminal amino acid residues from the of plasma added to the incubation mixture contained
product of ACTHꢀ(6–9)ꢀPGP hydrolysis with carꢀ twice as much protein as there was in rat blood in simꢀ
boxypeptidase Y results in TrpꢀProꢀGly peptide. To ilar experiments.
Table 3. Dynamics of ACTHꢀ(6–9)ꢀPGP and semax hydrolysis in rat blood plasma (in percent to the initial amount of
peptide)
Incubation time, min
Protein concentration
Peptide
according to Lowry, mg/mL
20
30
60
90
120
Semax
9.60
9.0
88
78
36
96
81
68
77
56
28
91
63
42
50
31
14
78
35
16
32
10
0
14
0
1
3
8.0
0
ACTHꢀ(6ꢀ9)ꢀPGP
9.60
9.0
67
14
56
5
1
3
8.0
0.8
0.2
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SHEVCHENKO et al.
Table 4. Dynamics of PheꢀArgꢀTrpꢀProꢀGlyꢀPro and TrpꢀProꢀGlyꢀPro content upon hydrolysis of ACTHꢀ(6–9)ꢀPGP in
rat blood (in percent to the initial amount of ACTHꢀ(6–9)ꢀPGP; protein concentration 9.35 mg/mL)
Incubation time, min
Peptide
2
5
10
20
30
40
60
90
120
PheꢀArgꢀTrpꢀProꢀGlyꢀPro
TrpꢀProꢀGlyꢀPro
23
2
46
4
48
5
51
6
47
12
42
13
18
10
3
9
0
0
Table 5. Dynamics of ACTHꢀ(6–9)ꢀPGP product content upon its hydrolysis in rat blood plasma (in percent to the initial
amount of ACTHꢀ(6–9)ꢀPGP)
Protein concentraꢀ
tion according to
Lowry, mg/mL
Incubation time, min
Hydrolysis product
2
20
30
60
90
120
PheꢀArgꢀTrpꢀProꢀGlyꢀPro
TrpꢀProꢀGlyꢀPro
Trp
0.0
0.0
0.3
0.0
0.0
0.2
0.0
0.0
0.4
10.0
0.9
13.4
2.2
23.7
7.2
26.8
12.5
0.6
28.7
15.3
0.6
9.60
19.0
38.0
0.3
0.3
0.3
PheꢀArgꢀTrpꢀProꢀGlyꢀPro
TrpꢀProꢀGlyꢀPro
Trp
15.3
4.0
20.3
5.9
30.2
17.9
0.7
18.2
20.0
0.9
2.8
13.9
1.4
0.5
0.5
PheꢀArgꢀTrpꢀProꢀGlyꢀPro
TrpꢀProꢀGlyꢀPro
Trp
23.7
7.6
27.8
10.8
0.6
24.7
22.1
1.0
2.7
0.2
18.2
1.8
6.5
0.6
2.5
Comparison of ACTHꢀ(6–9)ꢀPGP stabilities in GlyꢀPro and TrpꢀProꢀGlyꢀPro are reached decreased
blood and plasma demonstrated that degradation of and tryptophan content grew.
ACTHꢀ(6–9)ꢀPGP in blood and plasma containing
The data allowed the evaluation of ACTHꢀ(6–9)ꢀ
the same amount of protein differed considerably.
Incubation in the presence of blood for 30, 60, and PGP stability in the presence of individual enzymes
20 min resulted in 40, 12, and 0% peptide and when and enzymes of nasal mucus, blood, and plasma and
working with plasma, 91, 78, and 56%, respectively.
determination of the major ACTHꢀ(6–9)ꢀPGP
1
ACTHꢀ(6–9)ꢀPGP hydrolysis in blood generated hydrolysis products. These data will allow reliable
PheꢀArgꢀTrpꢀProꢀGlyꢀPro and TrpꢀProꢀGlyꢀPro evaluation of ACTHꢀ(6–9)ꢀPGP and its metabolites
(
Table 4). The maximum content of PheꢀArgꢀTrpꢀ in the analysis of biological samples obtained upon
ProꢀGlyꢀPro was observed 20 min after the reaction
start; as for TrpꢀProꢀGlyꢀPro, its maximum content
was observed after 40 min. These facts evidence that
the hydrolysis in blood mainly proceeds under the
effect of leucine aminopeptidaseꢀlike enzymes.
ACTHꢀ(6–9)ꢀPGP administration to animals. Thereꢀ
fore, in the work, approaches to ACTHꢀ(6–9)ꢀPGP
analysis in vivo upon intravenous and intranasal
administration to animals were elaborated.
As noted above, an increase in plasma content in
the reaction mixture resulted in faster hydrolysis of
ACTHꢀ(6–9)ꢀPGP (Table 3). One can suppose that a
change in the ACTHꢀ(6–9)ꢀPGP–plasma ratio may
result in the change of both the percentage content of
the initial peptide hydrolysis products and the time till
their maximum content in the incubation medium is
reached. Indeed, the increase in blood plasma content
resulted in an increase in the rate of ACTHꢀ(6–9)ꢀ
PGP and its hydrolysis products degradation
EXPERIMENTAL
Microsomal leucine aminopeptidase type IV from
pig kidney (EC 3.4.11.2; Sigma, United States;
.2 a.u./mg) and carboxypeptidase Y from baker’s
yeast (EC 3.4.16.1; Sigma; 17.0 a.u./mg), as well as
other necessary reagents, were commercial reagents.
Nasal mucus (5.2 mg protein/mL), blood (31 mg proꢀ
9
(
Table 5). The time till maximum concentrations of tein/mL), and blood plasma (63 mg protein/mL) were
intermediate hydrolysis products PheꢀArgꢀTrpꢀProꢀ obtained from Wistar rats (200 g).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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2016

STABILITY OF HisꢀPheꢀArgꢀTrpꢀProꢀGlyꢀPro
159
ACTHꢀ(6–9)ꢀPGP Synthesis
desiccator over KOH, Р2О5, and paraffin. The peptide
yield was 80%, mp 125–126
°C.
Synthesis of the initial peptide was performed with
classical methods of peptide chemistry in solution
using both protected and free Lꢀamino acids accordꢀ
ing to Scheme 1. First, ProꢀGlyꢀProꢀOBzl was
obtained; it was further elongated from Nꢀterminus
with appropriate amino acids.
II. ProꢀGlyꢀProꢀOBzl. To the solution of 5.4 g
(
11.7 mmol) BocꢀProꢀGlyꢀProꢀOBzl in 29 mL methꢀ
ylene chloride, trifluoroacetic acid (29 mL) was
added; the mixture was kept at room temperature for
5 min and coꢀevaporated progressively with absolute
ethanol, benzene, and ether, two times with each of
the solvents; then, the mixture was dissolved in benꢀ
zene and precipitated with hexane. Hexane was decanted
and the oil thus obtained was dried under vacuum in a
desiccator over KOH, P2O5, and paraffin. The yield of
4
I. BocꢀProꢀGly. To the solution of 10.8 g
50 mmol) BocꢀPro in acetonitrile cooled to –5°С,
(
triethylamine (TEA; 7.7 mL, 50 mmol) was added under
stirring, and the mixture was further cooled to –20 . To
the cooled solution, 6.8 mL (55 mmol) pivaloyl chloride
PivCl) was added during 20 min at –10 , and the
mixture was cooled to –30 . Then, solution of 4.5 g (16 mmol; 1.1ꢀfold excess) BocꢀTrp in 45 mL dimethꢀ
Gly (60 mmol; 1.2ꢀfold excess) in 35 mL water and ylformamide (DMF), carbonyldiimidazole (3.0 g,
0 mL acetonitrile supplemented with 8.4 mL 18 mmol) was added under stirring. When СО2 formaꢀ
60 mmol) TEA and cooled at –10 for 20 min was tion stopped, solution of 5.5 g (14.6 mmol) ProꢀGlyꢀ
added to the reaction mixture. Then, the reaction ProꢀOBzl in 30 mL DMF was added and the mixture
mixture was kept under stirring at –10
°С
TFA
⋅
ProꢀGlyꢀProꢀOBzl was 96%.
(
°С
TrpꢀProꢀGlyꢀProꢀOBzl. To the solution of 4.9 g
°
С
6
(
°С
°
С
for 1 h and at was left for one day at
0°C. The reaction mixture was
1
5
8–20°С, for 2 h, and evaporated. Approximately lyophilized. Approximately 300 mL EA was added to
0 mL water was added to the residual mixture. Aqueꢀ the residue. The solution was filtered and evaporated.
ous solution was acidified by the addition of 3ꢀfold The residue was dissolved in 30 mL absolute EA and
excess of NaHSO4 (24.84 g) to the final pH 3 and the product was precipitated with hexane. Hexane was
extracted with ethyl acetate (EA)
5 100 mL. Joint EA decanted and the product was dried. The peptide yield
×
extract was washed with water (50 mL), 10% KHSO4 after Boc removal was 70%.
(
(
50 mL), water (50 mL), and saturated NaCl solution
Arg(NO )ꢀTrpꢀProꢀGlyꢀProꢀOBzl. To the soluꢀ
2
50 mL). Then, EA extract was dried over MgSO4, filꢀ
tion of 3.1 g BocꢀArg(NO2) (9.7 mmol) in 50 mL tetꢀ
rahydrofuran (THF) and 10 mL DMF, N,Nꢀdicycloꢀ
hexylcarbodiimde (2.07 g, 10.7 mmol) was added. The
tered, and evaporated to the volume of 50 mL; then,
dry ether was added. This resulted in a precipitate,
which was filtered off and washed on the filter with dry
ether. The product thus obtained was dried under vacꢀ
uum in a desiccator over KOH, Р2О5, and paraffin.
mixture was cooled to
solution of 6.9 g TFA · TrpꢀProꢀGlyꢀProꢀOBzl
10.7 mmol) in 50 mL THF and 5.1 mL (11 mmol)
TEA was added. The reaction mixture was stirred for
days. The precipitated of dicyclohexylurea was
0°С, stirred for 40 min, and the
(
The peptide yield was 43%, mp 70°C.
3
BocꢀProꢀGlyꢀProꢀOBzl. To the solution of 6.0 g
21.7 mmol) BocꢀProꢀGly in 100 mL acetonitrile
filtered off, the solution was evaporated, and the resiꢀ
due was treated with 200 mL hexane. The oily target
product was isolated, dissolved in 500 mL EA, and
(
cooled to –5
excess) was added under stirring; then, the reaction
mixture was cooled to –20 . To the cooled solution,
.34 mL (23.9 mmol; 1.1ꢀfold excess) pivaloyl chloꢀ
ride (PivCl) was added during 20 min at –10 and
the mixture was cooled to –30 . Then, solution of
ProꢀOBzl (26.1 mmol; 1.2ꢀfold excess) in
0 mL acetonitrile supplemented with 4 mL
°С, TEA (3.35 mL, 23.9 mmol; 1.1ꢀfold
washed with 0.1 N HCl solution (
3
×
25 mL), water (3
×
°С
2
5 mL), and saturated NaCl solution (25 mL).
2
Organic phase was dried over MgSO , filtered off, and
°
С
4
evaporated. The residue was dissolved in EA and preꢀ
cipitated with dry ether. The precipitate thus formed
was filtered off and dried. The peptide yield after Boc
removal was 80%.
°С
6
.3 g HCl ·
5
(
28.7 mmol; 1.2ꢀfold excess) TEA and cooled at –10
for 20 min was added to the reaction mixture. Then,
the reaction mixture was kept under stirring at –10
for 1 h and at 18–20 C, for 2 h, and evaporated. Ethyl nitrile cooled to –5
acetate (300 mL) was added to the residual. Ethyl aceꢀ added and the mixture was further cooled to –20
tate extract was washed with water ( 25 mL), 10% the cooled solution, 1.2 mL (8.8 mmol) pivaloyl chloride
KHSO (3 25 mL), water (3 25 mL), 5% NaHCO3 (PivCl) was added under stirring during 20 min at
25 mL), water (3 25 mL), and saturated NaCl –10
, and the mixture was cooled to –30 . Then,
solution (25 mL). Then, EA extract was dried over solution of 6.1 g TFA
Arg(NO2)ꢀTrpꢀProꢀGlyꢀProꢀ
MgSO , filtered, and dried till the oilꢀlike residue was OBzl (7.3 mmol) in 27 mL of acetonitrile–TEA, 25 : 2
°
C
PheꢀArg(NO )ꢀTrpꢀProꢀGlyꢀProꢀOBzl. To
a
2
°
C
solution of 2.12 g (8 mmol) BocꢀPhe in 50 mL acetoꢀ
, TEA (1.2 mL, 8.8 mmol) was
. To
°
°C
°C
3
×
×
×
4
(
3
×
×
°С
°С
⋅
4
formed; then, approximately 100 mL dry ether was mixture, cooled at –10
°C for 20 min was added to the
added. This resulted in a precipitate, which was filꢀ reaction mixture. Then, the reaction mixture was kept
tered off and washed on the filter with dry ether. The under stirring at –10°C for 1 h and at 18–20°C, for
product thus obtained was dried under vacuum in a 2 h, and evaporated. Ethyl acetate (300 mL) was
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 2
2016

1
60
SHEVCHENKO et al.
added to the residue. EA solution was washed with
water (2 25 mL), 10% KHSO (2
25 mL), water (2 following solvent systems: butanol–acetic acid–water
25 mL), 5% NaHCO (2 25 mL), water (2
25 (4 : 1 : 1); ethyl acetate–acetone–50% acetic acid (2 :
Reaction mixtures were analyzed with TLC in the
×
×
4
×
×
×
3
mL), and saturated NaCl solution (12 mL). Then, EA 1 : 1); benzene–ethanol (8 : 2); chloroform–methaꢀ
solution was dried over MgSO , filtered, and evapoꢀ nol–ammonia (7 : 2.5 : 0.5); acetone–benzene–aceꢀ
4
rated. The residue was dissolved in 30 mL absolute EA tic acid (50 : 100 : 1); chloroform–methanol (9 : 1);
and the product was precipitated with hexane. Hexane hexane–acetone (3 : 2); methanol; butanol–acetic
was decanted and the product was dried. The peptide acid–water (20 : 5 : 1); and ethanol–ammonia (7 : 3).
yield after Boc removal was 85%.
Rf values in the systems varied in the range of 0.25–0.8.
BocꢀHisꢀPheꢀArg(NO )ꢀTrpꢀProꢀGlyꢀProꢀOBzl.
2
LCMS Analysis
To the solution of 1.3 g (5.1 mmol) BocꢀHis in 25 mL
acetonitrile cooled to –5
was added under stirring and the mixture was further
cooled to –20 C. Then, 0.7 mL (5 mmol) PivCl was
added under stirring during 20 min at –10 C and the
mixture was cooled to –30 C. Then, a solution of 4.0 g
4.1 mmol) TFA PheꢀArg(NO2)ꢀTrpꢀProꢀGlyꢀProꢀ
OBzl in 27 mL of acetonitrile–TEA, 25 : 2 mixture,
cooled to –10 was added during 20 min. Then, the
reaction mixture was kept under stirring at –10°C for
h and at 18–20 , for 2 h, and evaporated. To the
°
C, TEA (0.7 mL, 5 mmol)
Liquid chromatography was performed on a Surꢀ
veyor equipment with an LCQ Advantage MAX
Thermoelectron, United States) mass spectrometry
detector with the Reposil pur C18aq, 5.0 m, 2.0
00 mm (Dr. Maisch, Germany) column, flow rate of
0.2 mL/min, and linear gradient of eluent B in eluent
A from 0 to 80% in 20 min; eluent A, water–AcOH–
TFA (100 : 0.1 : 0.01), eluent B, methanol. Molecular
ion ArgꢀTrpꢀProꢀGlyꢀPro (4.18 min), 612.2; Trpꢀ
ProꢀGlyꢀPro (6.89 min), 456.3; ACTHꢀ(6–9)ꢀPGP
°
(
°
μ
×
°
1
.
(
°
C
1
°C
residue, 300 mL EA was added. The EA solution was
washed progressively with 25 mL of 10% KHSO4,
(
10.97 min), 896.4; PheꢀArgꢀTrpꢀProꢀGLyꢀPro
(13.4 min), 759.4; HisꢀPheꢀArgꢀTrpꢀProꢀGlyꢀPro
(16.1 min), 799.4; and TrpꢀProꢀGly (13.2 min), 359.1.
Н О, 5% NaHCO , Н О (two times each), and satuꢀ
2
3
2
rated NaCl (12 mL), dried over MgSO4, filtered, and
evaporated. The residue was dissolved in 30 mL absoꢀ
lute ethyl acetate and the product was precipitated
with hexane. Hexane was decanted and the product
was dried. The peptide yield was 75%.
Peptide Isolation with HPLC
Peptides were isolated with HPLC under the followꢀ
ing conditions: Reposil pur C18aq, 5
μm, 4 × 150 mm
(
Dr. Maisch) column; eluent A, methanol–25 mM
HisꢀPheꢀArgꢀTrpꢀProꢀGlyꢀPro. To the solution of
ammonium dihydrophosphate, pH 2.8 (30 : 70); eluꢀ
ent B, methanol; flow rate 1 mL/min in gradient of
eluent B (0, 50, 100, 0% B at 0, 15, 17, 30 min, respecꢀ
tively). Absorption was measured at 280 nm. The stanꢀ
dard solutions had the following retention times (min)
under the given conditions: Trp (3.42), ArgꢀTrpꢀProꢀ
GlyꢀPro (4.96), TrpꢀProꢀGlyꢀPro (6.58), ACTHꢀ(6–
1
.1 g (1.0 mmol) BocꢀHisꢀPheꢀArg(NO2)ꢀTrpꢀProꢀ
GlyꢀProꢀOBzl in 50 mL methanol, 0.5 mL 1 M HCl
and 0.85 g 10% Pd/Al O catalyst was added and the
2
3
hydrogenation proceeded in hydrogen flow at room
temperature and pressure of 1 atm for 6 h. Then the
catalyst was filtered off and the mixture was washed on
the filter with methanol. Joint filtrate was evaporated
to dryness. The residue was precipitated from absolute
methanol with ether and evaporated. Then, the resiꢀ
due was resuspended in 10 mL of 2 M HCl in dioxane
and kept at room temperature for 45 min. Hexane was
added to the reaction mixture. The precipitate was filꢀ
tered off with multiple washings with hexane to
remove excess HCl. The washed precipitate was dried
in vacuum and reprecipitated from absolute methanol
with dry ether. The precipitate was filtered off, dried in
vacuum, and analyzed with HPLC. The yield of
ACTHꢀ(6–9)ꢀPGP was 65%. The unprotected pepꢀ
9
)ꢀPGP (9.75), PheꢀArgꢀTrpꢀProꢀGlyꢀPro (11.84).
Hydrolysis of ACTHꢀ(6–9)ꢀPGP and Semax
with Leucine Aminopeptidase and Carboxypeptidase Y
Hydrolysis of ACTHꢀ(6–9)ꢀPGP and semax was perꢀ
formed at peptide–enzyme ratios of 10.53
leucine aminopeptidase and 0.043 mol/a.u. for carꢀ
boxypeptidase Y. Under these conditions, semax
degraded by over 90% within 90 min.
μmol/a.u. for
μ
To a solution of 0.19
μ
mol peptide in 260 L phosꢀ
μ
–
phateꢀbuffered saline (27.4 mM NaCl, 0.4 mM KCl,
and 2 mM Na PO4 in 100 mL H O, pH 7.4), 0.01804 a.u.
leucine aminopeptidase or 4.44 a.u. carboxypeptidase
Y was added in the same buffer. Incubation mixture
was stirred and kept at 30
collected at certain time periods. The reaction was terꢀ
minated by the addition of an equal volume of methaꢀ
nol to the collected samples.
tide was transformed into acetate (CH COO ) salt
3
3
2
with a cation exchange resin Amberlist Aꢀ21.
Individuality of the obtained compounds and
intermediate products was confirmed with TLC on
Silufol (Czech Republic) plates, HPLC, and mass
spectrometry. When TLC was used, the compounds
were detected using UV light, ninhidrin, Barton,
°С
; aliquots of 40 L were
μ
Pauli, and Reindel–Hoppe reagents, and
chlorine.
oꢀtolidine in
In the case of hydrolysis of the set of peptides
obtained upon ACTHꢀ(6–9)ꢀPGP leucine aminopepꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 2
2016

STABILITY OF HisꢀPheꢀArgꢀTrpꢀProꢀGlyꢀPro
161
tidase degradation with carboxypeptidase Y and vice
verse, the first enzyme was allowed to work for 120 min. with 40–50ꢀ
Then, it was deactivated with the addition of TFA points. The samples were purified by solidꢀphase
1.5%), and the peptide mixture was purified by solidꢀ extraction on a reverse phase as follows: the peptide
Incubation mixture was stirred and heated at 30
°С,
μ
L aliquots collected at preset time
(
phase extraction on a Lichroprep RPꢀ18 (100 mg) fraction was applied onto a Licroprep RPꢀ18 reverse
reverse phase. Phosphateꢀbuffered saline and deactiꢀ phase cartridge and the peptides were eluted with
vated enzyme were removed by washing of the reverse methanol–water (4 : 1) mixture containing 0.1% TFA.
phase with 1.5% TFA solution in water. The peptides Then, the mixture was evaporated and the residue was
were eluted with the methanol–water, 9 : 1, mixture dissolved in 200
supplemented with 0.1% TFA. Then, the peptide mixꢀ The mixture was further analyzed as described above.
ture was evaporated, dissolved in 170 L PBS and
μ
L methanol–water (10 : 90) mixture.
μ
treated with the second enzyme. The samples were
analyzed with HPLC.
ACKNOWLEDGMENTS
The work was performed with partial support from
the program of fundamental studies of the Presidium
of the Russian Academy of Sciences “Molecular and
Cell Biology” (project no. 01201353020) and a Scienꢀ
tific Schools grant no. 14.120.12.4222ꢀNSh under the
guidance of academician N.F. Myasoedov.
Preparative Accumulation of the Product
of ACTHꢀ(6–9)ꢀPGP Hydrolysis with Leucine
Aminopeptidase and Carboxypeptidase Y
To the solution of 26.23
in 3 mL PBS (27.3 mM NaCl, 0.4 mM KCl, 2 mM
Na PO in 100 mL H ), pH 7.4), 2.76 a.u. (300 g)
L) carboxꢀ
ypeptidase Y in the same buffer was added. Incubation
mixture was stirred and heated at 30 ; 3ꢀ L aliquots
were collected to control the hydrolysis process. Reacꢀ
tion mixtures were treated with 45 L TFA when all
μmol ACTHꢀ(6–9)ꢀPGP
μ
3
4
2
REFERENCES
leucine aminopeptidase or 20.4 a.u. (1200
μ
1
. Glazova, N.Yu., Levitskaya, N.G., Andreeva, L.A.,
Kamenskii, A.A., and Myasoedov, N.F., Dokl. Akad.
Nauk, 1999, vol. 367, pp. 137–140.
°C
μ
μ
2. V’yunova, T.V., Shevchenko, K.V., Shevchenko, V.P.,
Bezuglov, V.V., and Myasoedov, N.F., Radiokhimiya
009, vol. 51, no. 2, pp. 161–166.
degradation products were present in the incubation
medium at comparable amounts. The peptide mixꢀ
tures thus obtained were purified with solidꢀphase
extraction on a Lichroprep RPꢀ18 reverse phase as
described above. The extracts were evaporated and
dissolved in 2 mL ethanol. The samples were analyzed
and the peptides were isolated with HPLC.
,
2
3. V’yunova, T.V., Andreeva, L.A., Shevchenko, K.V.,
Shevchenko, V.P., Bobrov, M.Yu., Bezuglov, V.V., and
Myasoedov, N.F., Dokl. Biol. Sci., 2008, vol. 419,
pp. 95–96.
4
. Shevchenko, K.V., V’yunova, T.V., Andreeva, L.A.,
Nagaev, I.Yu., Shevchenko, V.P., and Myasoedov, N.F.,
Dokl. Biol. Sci., 2014, vol. 456, pp. 101–103.
Isolation of Biological Fluids Containing the Enzyme
Complexes of Nasal Mucus, Blood, and Plasma of Rats
5
. Kopylova, G.N., Umarova, B.A., Samonina, G.E.,
Guseva, A.A., and Platonova, R.D., in Tezisy nauchꢀ
nykh trudov I s”ezda fiziologov SNG. T. 2. Sochi. Dagoꢀ
mys. 19–21 sentyabrya 2005 (Abstr. 1st Congress of CIS
Physiologists, Sochi, Dagomys, September 19–21,
2005), Moscow: MeditsinaꢀZdorov’e, 2005, vol. 2,
p. 677.
Nasal mucus was washed off from rat nasal septum
bones with PBS. Blood was collected from rat body
into a tube containing 20 L heparin (5000 U/mL)
μ
and mixed carefully. Part of the blood was centrifuged
at room temperature at 2000 rpm for 15 min. Blood
plasma (the upper layer) was carefully collected,
mixed, and poured into iceꢀcold microtubes. Protein
concentration in samples of nasal mucus, blood, and
plasma was determined according to Hartree–Lowry.
Protein concentrations were 5.2, 31, and 63 mg/mL in
nasal mucus, blood, and plasma extracts, respectively.
6
. Pires, A., Fortuna, A., Alves, G., and Falcao, A.,
J. Pharm. Pharm. Sci., 2009, vol. 12, pp. 288–311.
7
. Furubayashi, T., Kamaguchi, A., Kawaharada, K.,
Masaoka, Y., Kataoka, M., Yamashita, S., Higashi, Y.,
and Sakane, T., Biol. Pharm. Bull., 2007, vol. 30,
pp. 1007–1010.
8
. Hashizume, R., Ozawa, T., Gryaznov, S.M.,
Bollen, A.W., Lamborn, K.R., Frey II, W.H., and
Deen, D.F., NeuroꢀOncology, 2008, vol. 10, pp. 112–120
ACTHꢀ(6–9)ꢀPGP Hydrolysis in Nasal Mucus, Blood,
and Plasma of Rats
9
. Shevchenko, K.V., V’yunova, T.V., Nagaev, I.Yu.,
Andreeva, L.A., Alfeeva, L.Yu., and Myasoedov, N.F.,
Russ. J. Bioorg. Chem., 2011, vol. 37, no. 4, pp. 421–427.
To a solution of 0.19
mucus extract (5.2 mg protein/mL), or 95
31 mg protein/mL), or 48 (95; 190) L (63 mg proꢀ
tein/mL) blood plasma (total volume of incubation
mixture was 315 L) were added.
μ
mol peptide, 98
μ
L of nasal
L blood
1
0. Shevchenko, K.V., Nagaev, I.Yu., Andreeva, L.A., Alfeꢀ
eva, L.Yu., and Myasoedov, N.F., Dokl. Biol. Sci., 2013,
vol. 450, pp. 146–148.
μ
(
μ
μ
Translated by N. Onishchenko
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 2
2016