Effect of the SH Group of Myelopeptide MP-3 on Its Macrophage Stimulating Activity

Article abstract of DOI:10.1023/A:1012901200640

Peptide Leu-Val-Cys-Tyr-Pro-Gln, identical to the bone marrow peptide MP-3, and its Val³ and Ser³ analogs, lacking SH group, were synthesized by conventional methods of peptide chemistry in solution and, along with the MP-3 S-S-dimer

Full text of DOI:10.1023/A:1012901200640

Russian Journal of Bioorganic Chemistry, Vol. 27, No. 6, 2001, pp. 357–361. Translated from Bioorganicheskaya Khimiya, Vol. 27, No. 6, 2001, pp. 403–407.
Original Russian Text Copyright © 2001 by Fonina, Baldin, Efremov, Gur’yanov, Belevskaya.
Effect of the SH Group of Myelopeptide MP-3
on Its Macrophage Stimulating Activity
1
L. A. Fonina*, M. I. Baldin**, M. A. Efremov*, S. A. Gur’yanov*, and R. G. Belevskaya*
*Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia
**Institute of Experimental Cardiology, Russian Cardiological Research and Production Complex,
Ministry of Health of Russian Federation, Moscow, Russia
Received January 26, 2001; in final form, March 11, 2001
Abstract—Peptide Leu-Val-Cys-Tyr-Pro-Gln, identical to the bone marrow peptide MP-3, and itsVal3 and Ser3
analogs, lacking SH group, were synthesized by conventional methods of peptide chemistry in solution and,
along with the MP-3 S–S-dimerization product, were studied with respect to their effect on the macrophage
phagocytic activity. It was shown that the activity was only enhanced by peptide MP-3, which demonstrated the
essential role of the SH group in this function. The dimer analog of MP-3, unlike dimer analogs of other mono-
cycteine-containing peptides, glutathione and HP5b, did not exhibit the inhibitory effect.
Key words: MP-3 analogs, cysteine, synthesis, phagocytosis
1
INTRODUCTION
different biological effects [3]. To reveal the reasons for
so diverse behavior, the peptides themselves and their
analogs are to be synthesized and their biological and
physicochemical properties studied. Here we per-
formed it with regard to peptide MP-3 and its analogs.
Cells of bone marrow, one of the key organs of the
immune system, produce a group of biologically active
peptides, myelopeptides, influencing various popula-
2
tions of immunocompetent cells [1]. Recently, several
individual MPs were isolated and identified from the
supernatant of the porcine bone marrow cell culture
[2]. Their functional study showed them to be biologi-
cally active and specifically act on a certain type of
RESULTS AND DISCUSSION
Peptide MP-3, capable of promoting the functional
immunocompetent cells [3]. Thus, peptide MP-1 (Phe- activity of mononuclear macrophage system [7], con-
Leu-Gly-Phe-Pro-Thr) possesses an immunocorrect- tains a cysteine residue not involved in the disulfide
ing effect in immunodeficiencies of diverse etiologies bond, which is extremely rare for endogenous bioregu-
[4, 5]. Peptide MP-2 (Leu-Val-Val-Tyr-Pro-Trp) can latory peptides. Two endogenous peptides are known
restore the functional activity of human T-lymphocytes that contain each a cysteine residue, namely, glu-
and possesses an antitumor effect [6]. Peptide MP-3 tathione [8] and HP5b [9]. Both of them are involved in
(Leu-Val-Cys-Tyr-Pro-Gln) promotes macrophage regulation of granulocytosis: free SH-group-containing
phagocytosis to display a pronounced protective effect monomers inhibit granulocytosis whereas their oxi-
at animal infection with pathogenic strains of microor- dized forms, dimers, promote this process by increas-
ganisms [7].
MP-1–MP-3 are hexapeptides preferentially con-
ing the production of the granulocyte–macrophage col-
ony stimulating factor [8, 10]. Obviously, SH-group
plays the crucial role in realization of functional activ-
ity of these peptides. It is also known that a number of
peptides lacking cysteine (taftsin, substance P, neuro-
tensin) also display phagocytosis-stimulating effect
but, unlike MP-3, this effect is only observed on the
activated, e.g., thioglycolate-treated, peritoneal mac-
rophage. It should be noted that specific receptors for
these peptides have been found on the macrophage sur-
face [11].
taining hydrophobic amino acid residues and a proline
residue in position 5. Peptides MP-2 and MP-3 only
differ by two amino acid residues (in positions 3 and 6).
While being structurally rather close, peptides MP1–
MP-3 display a specific immunoregulatory activity, act-
ing on different immunity links and causing basically
1
To whom correspondence should be addressed; phone: +7 (095)
330-7256; fax: +7 (095) 330-7210; e-mail: stas@ibch.ru.)
2
Abbreviations: DCC, N,N'-dicyclohexylcarbodiimide; HOBT,
N-hydroxybenzotriazol; HOSu, N-hydroxysuccinimide; ONp,
p-nitrophenoxy; and SRBC, sheep red blood cells.
To reveal the influence of the SH group of peptide
MP-3 on its biological activity, we synthesized the orig-
1068-1620/01/2706-0357$25.00 © 2001 MAIK “Nauka /Interperiodica”

358
FONINA et al.
inal peptide (I) and its dimer (IV) as well as two MP-3 rophage or upon addition of SRBC. The experiments
analogs with the cysteine in position 3 replaced by were carried out in a peptide wide concentration range
(1 × 10–7–1 × 10–13 g/ml). The natural macrophage
phagocytosis (without addition of peptides) served as
control. The statistical processing of the data was per-
formed with the Statgraphics program package.
other amino acid residues: Val (II) or Ser (III).
Leu-Val-Cys-Tyr-Pro-Gln (I), MP-3
Leu-Val-Ser-Tyr-Pro-Gln (II)
Leu-Val-Val-Tyr-Pro-Gln (III)
Leu-Val-Cys-Tyr-Pro-Gln
It was shown (figure) that the original peptide MP-3
(I) in concentrations of 1 × 10–8–1 × 10–7 g/ml stimu-
lated the functional activity of the mouse peritoneal
macrophage, causing an increase in phagocytosis of
SRBC by 180%. For the rest of the analogs studied, the
phagocytosis level in a similar concentration range did
not exceed the control value. The dimer analog (IV) did
not inhibit the macrophage functional activity either,
that is, was devoid of the opposite effect characteristic
of the glutathione dimer analogs [8] and hemoregula-
tory peptide HP5b [10].
Leu-Val-Cys-Tyr-Pro-Gln
(IV)
Leu-Val-Cys-Tyr-Pro-Gln
The peptides were synthesized by the conventional
methods of peptide chemistry. The peptide chain was
extended from the N-end by the carbodiimide or active
ester methods. Amino groups were protected with the
Z- and Boc-groups, the cysteine SH group was Acm-
blocked, and the serine OH-group was converted to
But-ether. The dimer analog (IV) was obtained by oxi-
dation of SH groups with hydrogen peroxide, monitor-
ing the reaction by the Ellman method and analytical
HPLC. The intermediate stages of the synthesis were
monitored using thin-layer chromatography on silica
gel. The final and intermediate peptides were isolated
by preparative reverse-phase HPLC. The target pep-
tides (I)–(IV) were characterized by the analytical
HPLC data and their structure was confirmed by
sequencing.
Thus, the study of peptide MP-3 and its analogs
lacking SH groups has revealed that peptide’s phagocy-
tosis-stimulating activity strongly depends on the pres-
ence in its molecule of a free SH group.
EXPERIMENTAL
Commercial amino acids and their derivatives
obtained from Reanal (Hungary) and Fluka (Switzer-
land) or prepared according to the standard procedures
were used. The homogeneity of the compounds synthe-
sized was checked by TLC on chromatographic plates
Kieselgel 60 (Merck, Germany) in the following sol-
vent systems: 60 : 45 : 20 chloroform–methanol–32%
acetic acid (A); 5 : 3 : 1 chloroform–methanol–32%
acetic acid (B); 15 : 4 : 1 chloroform–methanol–32%
acetic acid (C); 3 : 1 : 1 n-butanol–acetic acid–water
(D); 18 : 2 :1 chloroform–methanol–acetic acid (E); 9 : 1
chloroform–methanol (F).
Effect of MP-3 and its analogs on the functional
activity of mouse peritoneal macrophage was studied
using TNBT (Tetranitro Blue Tetrazolium) test [12],
with opsonized SRBC as a corpuscular antigen. The
peptides studied were added either together with mac-
Phagocytosis level, %
300
Analytic HPLC was performed using an CL-10Avp
chromatographic system (Shimadzu, Japan) on an
Ultrasphere C18 column (4.6 × 250 mm) in an acetoni-
trile gradient in 0.1% trifluoroacetic acid (0–80%,
32 min). Flow rate 1.6 ml/min, detection at 214 nm.
Preparative HPLC of the final and intermediate
peptides was performed using a Diasorb C 16-T col-
umn (50 × 250 mm) in a gradient of buffer B in buffer
A (10–50%, 120 min; buffer A, 0.01 N CH3COONH4;
buffer B, 80% acetonitrile in buffer A). Flow rate
50 ml/min, detection at 226 nm. Fractions correspond-
ing to the major peak were pooled, acetonitrile was dis-
tilled off, and the resulting solution was diluted with
water and lyophilized.
Control
(III)
(I)
(II)
250
200
150
100
50
(IV)
0
H-Leu-Val-Cys-Tyr-Pro-Gln-OH (I)
–13
–12
–11
–10
–9
–8
–7
Z-Pro-Gln-OBut. To a stirred solution of 20.23 g
(100 mmol) of H-Gln-OBut, 24.93 g (100 mmol) of
Z-Pro-OH, and 4.05 g (30 mmol) of HOBT in 500 ml
of ethyl acetate cooled down to –20°ë was added
21.63 g (105 mmol) of DCC. The reaction mixture was
log([peptide], g/ml)
The level of SRBC phagocytosis by mouse peritoneal mac-
rophage in the presence or absence (control) of peptides (I)–
(IV).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001

EFFECT OF THE SH GROUP OF MYELOPEPTIDE MP-3
359
stirred for 12 h at +4°ë. The resulting precipitate of 200 ml) and water to the neutral reaction. The organic
dicyclohexylurea was filtered off; and the filtrate was layer was evaporated, the oily residue was triturated
sequentially washed with saturated NaHCO3 solution with ether, the solid formed was filtered and air dried at
20°ë. After HPLC the yield of the peptide was 18.17 g
(79.9%). Rf 0.75 (D), 0.65 (B), 0.2 (C).
(2 × 200 ml), water (1 × 200 ml), 5% H2SO4 (1 ×
200 ml), and water to the neutral reaction of the wash-
ings. The organic layer was evaporated, the oily residue
was triturated with ether, and the solid formed was fil-
trated and air dried at 20°ë. After HPLC the yield was
19.9 g (66.5%). Rf 0.77 (C), 0.52 (E), 0.48 (F).
H-Val-Cys(Acm)-Tyr-Pro-Gln-OH ¥ TFA.
A
solution of 18.17 g (23 mmol) of Boc-Val-Cys(Acm)-
Tyr-Pro-Gln-OH in 150 ml of TFA was kept at 20°ë for
1 h. The reaction mixture was concentrated, the oily
residue was triturated with ether, and the solid formed
was filtered and dried in desiccator over KOH. After
HPLC the yield was 17.5 g (95.6%). Rf 0.45 (A), 0.45
(D), 0.3 (B).
Z-Tyr-Pro-Gln-OBut. A solution of 19.9 g
(66.5 mmol) of Z-Pro-Gln-OBut in 300 ml of ethanol
was kept under hydrogen over 10% Pd/C until the ini-
tial peptide disappeared (TLC monitoring); the catalyst
was filtered off and the filtrate evaporated. The oily res-
idue was dissolved in 200 ml of DMF, and 20.95 g
(66.5 mmol) of Z-Tyr-OH and 3.45 g (30 mmol) of
HONSu were added. The resulting solution was cooled
down to –20°ë and 14.38 g (69.8 mmol) of DCC was
added. The reaction mixture was stirred for 12 h at
+4°ë, the dicyclohexylurea was filtered off, and the fil-
trate was evaporated. The oily residue was dissolved in
500 ml of ethyl acetate and sequentially washed with
saturated NaHCO3 solution (2 × 200 ml), water (1 ×
200 ml), 5% H2SO4 (1 × 200 ml), and water to the neu-
tral reaction of the washings. The organic layer was
evaporated, the oily residue was triturated with ether,
the resulting powder was filtrated and air dried at 20°ë.
After HPLC the yield was 33.12 g (83%). Rf 0.47 (E),
0.75 (C), 0.42 (F).
Boc-Cys(Acm)-Tyr-Pro-Gln-OBut. A solution of
33.12 g (55.3 mmol) of Z-Tyr-Pro-Gln-OBut in 300 ml
of ethanol was kept under hydrogen over 10% Pd/C
until the initial peptide disappeared. The catalyst was
filtered off and the filtrate was evaporated in vacuum.
The oily residue was dissolved in 200 ml of DMF, and
20.65 g (50 mmol) of Boc-Cys(Acm)-ONp was added.
The reaction mixture was stirred for 12 h at 20°ë; the
resulting suspension was diluted with 300 ml of ether
and the precipitate formed was filtered, washed with
ether, and air dried at 20°ë. After HPLC the yield of the
peptide was 26.51 g (64.5%). Rf 0.3 (E), 0.3 (C), 0.2
(F).
Boc-Leu-Val-Cys(Acm)-Tyr-Pro-Gln-OH. To a
solution of 17.5 g (22 mmol) of H-Val-Cys(Acm)-Tyr-
Pro-Gln-OH × TFA in 200 ml of DMF were added
0.94 g (7 mmol) of HOBT, 4.9 ml (44 mmol) of
N-methylmorpholine, and 7.22 g (22 mmol) of Boc-
Leu-ONSu. The reaction mixture was stirred for 12 h at
20°ë and concentrated. The oily residue was dissolved
in 300 ml of n-butanol and washed with 5% H2SO4 (1 ×
200 ml) and water to the neutral reaction. The organic
layer was evaporated, the oily residue was triturated
with ether, the solid formed was filtered and air dried at
20°ë. After HPLC the yield was 17.5 g (95%). Rf 0.65
(B), 0.25 (C), 0.8 (D).
H-Leu-Val-Cys(Acm)-Tyr-Pro-Gln-OH ¥ TFA.
A solution of 17.5 g (21 mmol) of Boc-Leu-Val-
Cys(Acm)-Tyr-Pro-Gln-OH in 150 ml of TFA was kept
for 1 h at 20°ë. The reaction mixture was evaporated,
the oily residue was triturated with ether, and the solid
formed was filtered and dried in desiccator over KOH.
After HPLC the yield was 17.9 g (94.3%). Rf 0.55 (A),
0.4 (B), 0.6 (D).
H-Leu-Val-Cys-Tyr-Pro-Gln-OH (I). To a solu-
tion of 17.9 g (19.8 mmol) of H-Leu-Val-Cys(Acm)-
Tyr-Pro-Gln-OH × TFA in 200 ml of 30% acetic acid
was added 12.72 g (40 mmol) of Hg(OAc)2. The reac-
tion mixture was stirred for 1.5 h at 20°ë, and gaseous
H2S was then passed through the solution for 20 min.
The resulting precipitate was filtered off and the solu-
tion was concentrated in vacuum. After HPLC the yield
was 7.3 g (51%). Rf 0.6 (A), 0.45 (B), 0.65 (D). The
retention time in the analytical HPLC was 9.1 min.
H-Cys(Acm)-Tyr-Pro-Gln-OH ¥ TFA. A solution
of 26.51 g (32.2 mmol) of Boc-Cys(Acm)-Tyr-Pro-
Gln-OBut in 150 ml of TFA was kept for 1 h at 20°ë.
The reaction mixture was evaporated, the oily residue
was triturated with ether, the solid formed was filtered
and dried in desiccator over KOH. After HPLC the
yield was 20.15 g (89%). Rf 0.45 (A), 0.45 (D), 0.3 (B).
H-Leu-Val-Ser-Tyr-Pro-Gln-OH ¥ TFA (II)
Z-Ser(But)-Tyr-Pro-Gln-OBut. A solution of
2.99 g (5 mmol) of Z-Tyr-Pro-Gln-OBut in 25 ml of
Boc-Val-Cys(Acm)-Tyr-Pro-Gln-OH. N-Methyl- ethanol was kept under hydrogen over 10% Pd/C until
morpholine (6.66 ml, 60 mmol) and Boc-Val-ONSu the initial compound disappeared (TLC monitoring).
(9.07 g, 28.8 mmol) were added to a stirred solution of The catalyst was filtered off and the filtrate was concen-
20.15 g (28.8 mmol) of H-Cys(Acm)-Tyr-Pro-Gln- trated in vacuum. The oily residue was dissolved in
25 ml of DMF, and 2.28 g (5 mmol) of Z-Ser(But)-ONB
was added. The reaction mixture was stirred for 12 h at
20°ë and DMF was evaporated. The oily residue was
dissolved in 50 ml of ethyl acetate, the solution was
OH × TFA and 1.35 g (10 mmol) of HOBT in 200 ml
of DMF. The reaction mixture was stirred for 12 h at
20°ë and evaporated. The oily residue was dissolved in
300 ml of n-butanol and washed with 5% H2SO4 (1 ×
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001

360
FONINA et al.
sequentially washed with saturated NaHCO3 solution tial compound disappeared (TLC monitoring). The cat-
alyst was filtered off and the filtrate was evaporated.
The oily residue was dissolved in 25 ml of DMF, and
1.75 g (5 mmol) of Z-Val-ONSu and 0.135 g (1 mmol)
of HOBT were added to the solution. The reaction mix-
ture was stirred for 12 h at 20°ë, and DMF was evapo-
rated. The oily residue was dissolved in 50 ml of ethyl
acetate; the solution was sequentially washed with sat-
urated NaHCO3 solution (2 × 50 ml), water (1 × 50 ml),
5% H2SO4 (1 × 50 ml), and water to the neutral reac-
tion. The organic layer was evaporated, the oily residue
was triturated with ether, the solid formed was filtered
and air dried at 20°ë. After HPLC the yield was 3.02 g
(87%). Rf 0.63 (C), 0.42 (E), 0.65 (F).
Z-Val-Val-Tyr-Pro-Gln-OBut. A solution of 3.02 g
(4.35 mmol) of Z-Val-Tyr-Pro-Gln-OBut in 25 ml of
ethanol was kept under hydrogen over 10% Pd/C until
the initial compound disappeared (TLC monitoring).
The catalyst was filtered off and the filtrate was evapo-
rated. The oily residue was dissolved in 25 ml of DMF,
and 1.52 g (4.35 mmol) of Z-Val-ONSu and 0.135 g
(1 mmol) of HOBT were added to the solution. The
reaction mixture was stirred for 12 h at 20°ë, and DMF
was evaporated. The oily residue was dissolved in
50 ml of ethyl acetate; the solution was sequentially
washed with saturated NaHCO3 solution (2 × 50 ml),
water (1 × 50 ml), 5% H2SO4 (1 × 50 ml), and water to
the neutral reaction. The organic layer was evaporated,
the oily residue was triturated with ether, the solid
formed was filtered and air dried at 20°ë. After HPLC
the yield was 2.94 g (85%). Rf 0.6 (C), 0.35 (E), 0.61 (F).
Boc-Leu-Val-Val-Tyr-Pro-Gln-OBut. A solution
of 2.94 g (3.7 mmol) of Z-Val-Val-Tyr-Pro-Gln-OBut in
25 ml of ethanol was kept under hydrogen over 10%
Pd/C until the initial compound disappeared (TLC
monitoring). The catalyst was filtered off and the fil-
trate was evaporated. The oily residue was dissolved in
25 ml of DMF, and 1.21 g (3.7 mmol) of Boc-Leu-
ONSu and 0.135 g (1 mmol) of HOBT were added to
the solution. The reaction mixture was stirred for 12 h
at 20°ë, and DMF was evaporated. The oily residue
was dissolved in 50 ml of ethyl acetate, the solution was
sequentially washed with saturated NaHCO3 solution
(2 × 50 ml), water (1 × 50 ml), 5% H2SO4 (1 × 50 ml),
and water to the neutral reaction. The organic layer was
evaporated, the oily residue was triturated with ether,
and the solid formed was filtered and air dried at 20°ë.
After HPLC the yield was 2.9 g (90%). Rf 0.58 (C), 0.31
(E), 0.58 (F).
(2 × 50 ml), water (1 × 50 ml), 5% H2SO4 (1 × 50 ml),
and water to the neutral reaction. The organic layer was
evaporated, the oily residue was triturated with ether,
and the solid formed was filtered and air dried at 20°ë.
After HPLC the yield was 2.4 g (65%). Rf 0.72 (C), 0.48
(E), 0.71 (F).
Z-Val-Ser(But)-Tyr-Pro-Gln-OBut. A solution of
2.4 g (3.2 mmol) of Z-Ser(But)-Tyr-Pro-Gln-OBut in
25 ml of ethanol was kept under hydrogen over 10%
Pd/C until the initial compound disappeared (TLC
monitoring). The catalyst was filtered off and the fil-
trate was concentrated in vacuum. The oily residue was
dissolved in 25 ml of DMF, and 1.15 g (3.2 mmol) of Z-
Val-ONSu and 0.27 g (2 mmol) of HOBT were added
to the solution. The reaction mixture was stirred for
12 h at 20°ë, and DMF was evaporated. The oily resi-
due was dissolved in 50 ml of ethyl acetate, and the
solution was sequentially washed with saturated
NaHCO3 solution (2 × 50 ml), water (1 × 50 ml), 5%
H2SO4 (1 × 50 ml), and water to the neutral reaction.
The organic layer was evaporated, the oily residue was
triturated with ether, and the solid formed was filtered
and air dried at 20°ë. After HPLC the yield was 2.7 g
(93%). Rf 0.7 (C), 0.4 (E), 0.68 (F).
Boc-Leu-Val-Ser(But)-Tyr-Pro-Gln-OBut.
A
solution of 2.7 g (3 mmol) of Z-Val-Ser(But)-Tyr-Pro-
Gln-OBut in 25 ml of ethanol was kept under hydrogen
over 10% Pd/C until the initial compound disappeared
(TLC monitoring). The catalyst was filtered off and the
filtrate was concentrated in vacuum. The oily residue
was dissolved in 25 ml of DMF, and 0.99 g (3 mmol) of
Boc-Leu-ONSu and 0.135 g (1 mmol) of HOBT were
added to the solution. The reaction mixture was stirred
for 12 h at 20°ë, and DMF was evaporated. The oily
residue was dissolved in 50 ml of ethyl acetate; the
solution was sequentially washed with saturated
NaHCO3 solution (2 × 50 ml), water (1 × 50 ml), 5%
H2SO4 (1 × 50 ml), and water to the neutral reaction.
The organic layer was evaporated, the oily residue was
triturated with ether, and the solid formed was filtered
and air dried at 20°ë. After HPLC the yield was 2.46 g
(89%). Rf 0.55 (A), 0.4 (E), 0.68 (F).
H-Leu-Val-Ser-Tyr-Pro-Gln-OH × TFA. A solu-
tion of 2.46 g (2.68 mmol) of Boc-Leu-Val-Ser(But)-
Tyr-Pro-Gln-OBut in 50 ml of TFA was kept for 1 h at
20°ë. The reaction mixture was evaporated, the oily
residue was triturated with ether, and the solid formed
was filtered and dried in desiccator over KOH. After
HPLC the yield was 1.87 g (85%). Rf 0.55 (A), 0.22 (D),
0.22 (E). Retention time (analytical HPLC) 8.05 min.
H-Leu-Val-Val-Tyr-Pro-Gln-OH ¥ TFA (III). A
solution of 2.9 g (3.33 mmol) of Boc-Leu-Val-Val-Tyr-
Pro-Gln-OBut in 50 ml of TFA was kept for 1 h at 20°ë
and evaporated. The oily residue was triturated with
ether, the solid formed was filtered and dried in desic-
cator over KOH. After HPLC the yield was 2.54 g
(92%). Rf 0.55 (A), 0.4 (B), 0.6 (D). Retention time
H-Leu-Val-Val-Tyr-Pro-Gln-OH ¥ TFA (III)
Z-Val-Tyr-Pro-Gln-OBut. A solution of 2.99 g
(5 mmol) of Z-Tyr-Pro-Gln-OBut in 25 ml of ethanol
was kept under hydrogen over 10% Pd/C until the ini- (analytical HPLC) 9.325 min.
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EFFECT OF THE SH GROUP OF MYELOPEPTIDE MP-3
361
H-Leu-Val-Cys-Tyr-Pro-Gln-OH H-Leu-Val-Cys-Tyr-Pro-Gln-OH (IV). A solution of 1 g (1.39 mmol)
of H-Leu-Val-Cys-Tyr-Pro-Gln-OH in 100 ml of water was adjusted with ammonia to pH 8 and a 10-fold excess
of H2O2 was then added at vigorous stirring. The dimerization process was monitored using the Ellman reagent
and HPLC. After reaction completion, 30% ëç3ëééç was added to the reaction mixture to pH 4, and the result-
ing solution was concentrated in vacuum. The product was purified by HPLC. The yield was 0.53 g (53%).
Rf 0.6 (A), 0.25 (B), 0.65 (D). Retention time (analytical HPLC) 11.133 min.
6. Strelkov, L.A., Mikhailova, A.A., Fonina, L.A., and
Petrov, R.V., Dokl. Akad. Nauk, 1994, vol. 338, pp. 125–
126.
Phagocytic activity of peritoneal macrophage
was determined as described in [7].
7. Belevskaya, R.G., Mikhailova, A.A., Fonina, L.A., Efre-
mov, M.A., and Petrov, R.V., Dokl. Akad. Nauk, 1998,
vol. 358, pp. 847–849.
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