methods ofstabilizingtheactivityofhuman chorionic gonadotropin with

methods ofstabilizingtheactivityofhuman chorionic gonadotropin with

S ł u p s k i e P r a c e B i o l o g i c z n e 10 • 2013
METHODS OF STABILIZING THE ACTIVITY OF HUMAN
CHORIONIC GONADOTROPIN WITH THE USE
OF ORGANIC COMPOUNDS
METODY STABILIZACJI AKTYWNOŚCI LUDZKIEJ
GONADOTROPINY KOSMÓWKOWEJ ZA POMOCĄ
ZWIĄZKÓW ORGANICZNYCH
Yurij Slyvchuk1
Ivan Hevkan1
Iryna Matiukha1
Vasyl Syrvatka1
Halyna Tkachenko2
Natalia Kurhaluk2
1
National Academy of Agrarian Science
Institute of Animal Biology
V. Stusa Str. 38, 79034 Lviv, Ukraine
e-mail: [email protected]
2
Akademia Pomorska w Słupsku
Instytut Biologii i Ochrony Środowiska
Zakład Zoologii i Fizjologii Zwierząt
ul. Arciszewskigo 22b, 76-200 Słupsk
e-mail: [email protected]
[email protected]
STRESZCZENIE
Celem naszej pracy było znalezienie optymalnego jakościowego i ilościowego
składu związków organicznych niezbędnych dla stabilizacji ludzkiej gonadotropiny
kosmówkowej (hCG). W wyniku przeprowadzonych badań wykazano, że w próbkach gonadotropiny kosmówkowej inkubowanej w temperaturze 40°C przez 8 tygodni najbardziej stabilne stężenia hormonu stwierdzono w suspensji zawierającej
aminokwas L-lizynę w stężeniu 10 mg/mL. Stężenie hCG w suspensji z L-lizyną
wynosiło ponad 54% początkowego teoretycznego stężenia hormonu. W warunkach
przedłużonej inkubacji gonadotropiny w próbkach zawierających aminokwas L-glicynę zmiany stężenia natywnej hCG zanotowano we wszystkich seriach próbek.
Jednak ostateczne wyniki pokazują, że dodanie do suspensji L-glicyny w ilości od
0,2 mg/mL ma najbardziej wyraźny efekt stabilizujący. Przechowywanie próbki za133
wierającej aminokwas L-metioninę w stężeniu 0,2 mg/mL zapewnia stosunkowo
dużą i trwałą aktywność gonadotropiny przez 6 tygodni. Stężenie natywnej hCG
w tej serii próbek wynosiło około 50%.
Słowa kluczowe: gonadotropina kosmówkowa, aminokwasy, stabilizacja, L-lizyna,
L-glicyna, L-metionina, testy immunochemiluminescentne
Key words: chorionic gonadotropin, amino acids, stabilization, L-lysine, L-glycine,
L-methionine, immunochemiluminescent assay
INTRODUCTION
Human chorionic gonadotropin (hCG) is a placental glycoprotein hormone that
acts through binding to a G-protein-coupled receptor, leading to increased adenylatecyclase activity (Lapthorn et al. 1994, Erbel et al. 2002). The increase in cAMP
level stimulates the corpus luteum to produce progesterone until the placenta itself
acquires the ability to produce this pregnancy-sustaining steroid.
After dissociation of hCG into its subunits, the fully bioactive hormone can be
regained by recombination of the subunits (Lapthorn et al. 1994, Erbel et al. 1999).
Native hCG has four N-linked carbohydrate chains, two in the a-subunit at Asn-52
and Asn-78 (ahCG[glycan52,78]) and two in the b-subunit at Asn-13 and Asn-30
(bhCG). The mono- and di-antennary glycan structures are mainly sialylated, representing 10% of the total weight of hCG (Lapthorn et al. 1994, Erbel et al. 1999).
Fig. 1. Crystal structure of human chorionic gonadotropin hCG (according to Lapthorn et al.
1994)
Ryc. 1. Struktura krystaliczna ludzkiej gonadotropiny kosmówkowej (według Lapthorn i in.
1994)
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The three-dimensional structure of human chorionic gonadotropin (hCG) shows
that each of its two different subunits has a similar topology, with three disulphide
bonds forming a cystine knot. The same folding motif is found in some protein
growth factors. The heterodimer is stabilized by a segment of the β-subunit which
wraps around the α-subunit and is covalently linked like a seat belt by the disulphide
Cys 26–Cys 110. This extraordinary feature appears to be essential not only for the
association of these heterodimers but also for receptor binding by the glyco-protein
hormones (Lapthorn et al. 1994).
Lapthorn et al. (1994) described the crystal structure of hCG (Fig. 1). In Fig. 1
black lines represent the β-subunit of hCG and dark gray lines the α-subunit of hCG.
Light gray shaded areas show pertinent epitopes (Lapthorn et al. 1994).
During the recent years, the structure of gonadotropic hormones has been decoded for different species of fishes, animals and human (Wide and Wide 1984,
Fiddes and Goodman 1981, Fiddes and Talmadge 1984, Baenziger et al. 1992,
Moyle et al. 1994, Zambrano et al. 1995, Howles 1996, de Leeuw et al. 1996, Nozaki et al. 2013). The molecules of hCG and of chorionic gonadotropin (CG) of various animal species being considerably homologous are not identical. The α-hCG
subunit is identical to α-subunit of luteinizing hormone (LH), follicle-stimulating
hormone (FSH), as well as thyroid-stimulating hormone (TSH), and consists of 92
amino acid residues. α-CG contains two oligosaccharide chains attached to the polypeptide chain by a N-glycosidic bond between N-acetylglucosamine and an amid
group of two asparagine residues. The β-hCG subunit, the polypeptide chain consists
of 145 amino acid residues, is specific of this hormone, but displays a high level of
structural homology of about 80% with the β-subunit of luteinizing hormone, differing from the latter by its C-terminal section prolonged by 24 amino acid residues.
β-CG contains 6 oligosaccharide chains, 2 of which are joined by an N-glycosidic
bond at asparagine residues, while 4 are linked by an O-glycosidic bond between
N-acetylgalactosamine residues and the OH group of serine residues of the C-terminal section of the polypeptide chain. Specific biological properties of the hCG are
determined by the β-subunit. Structural affinity between the β-subunit of the hCG
and that of the LH is manifested by the similarity of their biological and immunological properties (Dimitrov 1979, Pierce and Parsons 1981, Fiddes and Talmadge
1984, Lempiäinen et al. 2012).
The carbohydrate part, characterized by a considerable heterogeneity, constitutes
about 30% of the CG molecular weight. It includes sialic acid, L-fructose, D-galactose,
D-mannose, N-acetylglucosamine and N-acetylgalactosamine. Carbohydrate components of the CG are needed for binding subunits, maintaining the conformation of
the molecule and protecting polypeptide chains of subunits from degradation under
the action of proteolytic enzymes. Elimination of carbohydrate residues causes
a considerable decrease of the half-life of CG in the organism (Chemistry Encyclopedia 1998).
The CG molecule is relatively easily dissociated into subunits, for instance, under the effect of urea or propionic acid. Isolated subunits are deprived of biological
activity, but can recombine producing a biologically active CG molecule (Smith
Waddell 2006).
It has been suggest that CG and its β-subunit are produced not only in the
135
chorion of trophoblast and placenta, but also in the tissues of the fetus (and even to
a greater degree than in the placenta), in many tissues of both sexes, i.e., during the
entire ontogenesis of mammals; it can also be produced by some tumors related by
their origin to placenta trophoblast cells. The synthesis of α- and β-subunits occurs
independently. Both dimeric (intact) hormone molecules and free (unbound) CG
subunits are released into the blood (Cole 2009).
High level of chorionic hormone are synthesized by placenta and extracted with
urea, from which it can be isolated and purified. Particularly, purified gonadotropins
are obtained by means of lyophilisation and preserved dry. Lyophilized preparations are
stable enough at conservation, however, lyophilisation is an expensive and laborconsuming stage in the process of obtaining gonadotropins, while their solutions are
unstable, which represents a disadvantage in their use. According to the researchers
study, both inorganic (ZnCl2 or AlCl3) and organic (methionine, glycine, mannitol,
arginine, lactose, sucrose) compounds are used for stabilizing gonadotropic preparations (Cameo et al. 2004, Maiti et al. 2011). Research in this way can result in considerable advantages consisting in the decrease of cost of preparation production and
ensuring the sufficient stability of liquid forms of gonadotropic preparations with
their prolonged activity (American Society for Reproductive Medicine 2008, Cameo
et al. 2004, Maiti et al. 2011). Therefore, the aim of our study was a comprehensive
search of an optimum quantitative and qualitative composition of organic compounds needed for the stabilization of the hCG.
MATERIALS AND METHODS
According to the general pattern of investigation presented in Table 1, three experiments were conducted.
The experiments differed between themselves by the amino acid added to the
solvent (L-lysine, L-glycine and L-methionine) and their concentrations. Each experiment consisted of three series of samples (control, experimental 1 and experimental 2 (Table 1)). The human chorionic gonadotropin (hCG) was obtained in the
Institute of Animal Biology, National Academy of Agrarian Science of Ukraine (Director Prof. Vasyl Vlizlo) from the urine of pregnant women (12-16 weeks of gestation) by means of filtering and precipitation by alcohol, acetone and ammonium acetate. The concentration of intact gonadotropin was established by means of electroand immuno-chemiluminescent method based on difference between total and free
hCG (Frimell 1987). The initial concentration of total hCG was 30,175 mIU per mL
(international units), while the concentration of free hCG was 375 mIU per mL. Theoretically, the activity of hCG was 29,800 mIU per mL (Table 1).
The obtained gonadotropin was solved in phosphate buffer (pH 7.34) and
aliquoted by 2,500 mIU per mL. L-lysine, L-glycine and L-methionine were added
to the aliquoted samples, in the amount of 10 and 0.2 mg per cm3 according to the
pattern of analysis, and the volume was brought to 1 cm3 with phosphate buffer with
pH 7.34. The samples were placed into a thermostat for incubation at the temperature of 40°C. After each 2 weeks during two months, the concentration of total
(hCG+β-hCG) and free (β-hCG) gonadotropin was measured. The hCG concentra136
tion was determined by the difference between (hCG+β-hCG) and (β-hCG)
(McPherson and Pincus 2006).
Table 1
Pattern of studies of the dynamics of gonadotropins activity with addition of amino acids
to the solvent
Tabela 1
Schemat badania dynamiki aktywności gonadotropiny z dodatkiem różnych aminokwasów
do roztworu
Groups
Characteristic of groups
Assay
Solved preparation of gonadotropin was stored at 40°C, and the
activity of hormone was determined every two weeks for two
Experimental 1
months by immuno-electrochemiGonadotropin solved in phosphate buffer (pH 7.34) with
adding 10 mg per cm3 of L-lysine/ L-glycine/ L-methionine luminiscentic methods
Control
Gonadotropin solved in phosphate buffer (pH 7.34)
Experimental 2
Gonadotropin solved in phosphate buffer (pH 7.34) with
adding 0.2 mg per cm3 of L-lysine/ L-glycine/ L-methionine
RESULTS AND DISCUSSION
Dynamics of gonadotropin activity with addition of L-lysine to the solvent is presented in the Table 2 and Figure 2.
Differences in activity of chorionic hormone preserved in a thermostat at the
temperature of 40°С with added L-lysine were studied in all groups. After 2 weeks
of conservation the concentration of intact hCG decreased by 58% in the control
group and by 63.56% in the 2nd experimental group. In the 1st experimental series,
where 10 mg per cm3 of L-lysine were inserted, the concentration of gonadotropin
was 51.72% of the initial concentration of hCG (Table 2, Fig. 2).
By the 4th week of samples conservation, a tendency to further decrease of gonadotropin concentration in all series of samples was demonstrated. However, by the
6th week of conservation, the hCG concentration in all experimental and control series increased and exceeded the respective values obtained at the 4th week of incubation (Fig. 2). The highest concentration (51.93% of the initial one) in the 1st experimental series with 10 mg per cm3 of L-lysine in the sample was noted. By the 8th
week of samples conservation in the thermostat, no changes of concentration of intact hCG were found, as compared with the 6th week conservation in the control and
2nd experimental series. A tendency to the increase of concentration of intact gonadotropin in the 1st experimental group was demonstrated (Fig. 2).
Dynamics of gonadotropin activity with addition of L-glycine to the solvent is
presented in the Table 3 and Figure 3. Adding of 0.2 mg per cm3 of L-glycine on the
2nd week of conservation resulted in the 53% preservation of the intact hCG concentration, as compared to the initial level. The concentration of hCG in the control se137
Тable 2
Dynamics of gonadotropin activity with addition of L-lysine to the solvent
Tabela 2
Dynamika aktywności gonadotropiny z dodatkiem L-lizyny do roztworu
Time
of incubation
2 weeks
4 weeks
6 weeks
8 weeks
Samples
Parameter
control
experimental 1
experimental 2
hCG+β-hCG
1168.5
1398.7
1003.58
β-hCG
96.98
104.9
92.23
hCG
1071.52
1293.38
911.35
hCG+β-hCG
1072.0
1330.3
1000.25
β-hCG
73.58
108.34
221.06
hCG
998.42
1221.9
779.19
hCG+β-hCG
1291.3
1426.1
1009.82
β-hCG
109.4
127.8
78.15
hCG
1181.9
1298.3
931.67
hCG+β-hCG
1167.9
1409.2
1003.84
β-hCG
246.9
53.8
66.66
hCG
921.0
1355.4
937.18
Fig. 2. Dynamics of gonadotropin activity with addition of L-lysine to the solvent
X axis presents the time of incubation of hCG with addition of L-lysine to the solvent
Y axis presents the changes of hormone activity in the % from initial concentration
Ryc. 2. Dynamika aktywności gonadotropiny z dodatkiem L-lizyny do roztworu
Oś X przedstawia czas inkubacji gonadotropiny hCG z dodatkiem L-lizyny do roztworu
Oś Y przedstawia zmiany aktywności gonadotropiny hCG w % od początkowego stężenia
ries of samples and in the experimental one, where 10 mg per cm3 of L-glycine were
added, decreased by 57% and 62%, respectively (Fig. 3). On the 4th week of conservation, a further decrease of the concentration of intact gonadotropin was observed
in the control and 2nd experimental series of samples, while in the series of samples
containing 10 mg per cm3 of L-glycine, an increase of gonadotropin concentration
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by almost 16%, as compared with the 2nd week of conservation of samples, was noted (Table 3, Fig. 3).
Table 3
Dynamics of gonadotropin activity with addition of L-glycine to the solvent
Tabela 3
Dynamika aktywności gonadotropiny z dodatkiem L-glicyny do roztworu
Time
of incubation
2 weeks
4 weeks
6 weeks
8 weeks
Samples
Parameter
control
experimental 1
experimental 2
hCG+β-hCG
1168.5
1041.73
1247.5
β-hCG
96.98
99.85
92.74
hCG
1071.52
941.88
1154.76
hCG+β-hCG
1072.0
1387.7
1022.09
β-hCG
73.58
62.9
93.46
hCG
998.42
1324.8
928.63
hCG+β-hCG
1291.3
1121.8
1183.0
β-hCG
109.4
79.3
84.0
hCG
1181.9
1042.5
1099.0
hCG+β-hCG
1167.9
1046.52
1342.2
β-hCG
246.9
90.2
8.35
hCG
921.0
956.32
1333.45
Fig. 3. Dynamics of gonadotropin activity with addition of L-glycine to the solvent
In the X axis is presented the time of incubation of hCG with addition of L-glycine to the
solvent
In the Y axis are presented the changes of hormone activity in the % from initial concentration
Ryc. 3. Dynamika aktywności gonadotropiny z dodatkiem L-glicyny do roztworu
Oś X przedstawia czas inkubacji gonadotropiny hCG z dodatkiem L-glicyny do roztworu
Oś Y przedstawia zmiany aktywności gonadotropiny hCG w % od początkowego stężenia
139
Table 4
Dynamic of gonadotropin activity with addition of L-methionine to the solvent
Tabela 4
Dynamika aktywności gonadotropiny z dodatkiem L-metioniny do roztworu
Time
of incubation
2 weeks
4 weeks
6 weeks
8 weeks
Samples
Parameter
control
experimental 1
experimental 2
hCG+β-hCG
1168.5
1008.58
1384.3
β-hCG
96.98
48.19
97.10
hCG
1071.52
960.39
1287.2
hCG+β-hCG
1072.0
1011.74
1398.2
β-hCG
73.58
45.24
79.00
hCG
998.42
966.5
1319.2
hCG+β-hCG
1291.3
1011.64
1312.6
β-hCG
109.4
88.65
105.3
hCG
1181.9
922.99
1207.3
hCG+β-hCG
1167.9
1019.98
1053.94
β-hCG
246.9
475.4
117.23
hCG
921.0
544.58
936.71
Fig. 4. Dynamics of gonadotropin activity with addition of L-methionine to the solvent
In the X axis is presented the time of incubation of hCG with addition of L-methionine to the
solvent
In the Y axis are presented the changes of hormone activity in the % from initial concentration
Ryc. 4. Dynamika aktywności gonadotropiny z dodatkiem do roztworu L-metioniny
Oś X przedstawia czas inkubacji gonadotropiny hCG z dodatkiem do roztworu L-metioniny
Oś Y przedstawia zmiany aktywności gonadotropiny hCG w % od początkowego stężenia
140
On the 6th week of conservation, a tendency to the increase of gonadotropin concentration in the control and 2nd experimental group was observed. In the first experimental group this value sharply decreased almost by 12%. On the 8th week of incubation, the content of intact gonadotropin decreased in the control and 1st experimental series of sample remaining on the same level, while in the 2nd experimental
series of samples it increased almost by 10% in respect to the previous determination of the intact gonadotropin concentration (Fig. 3).
The dynamics of gonadotropin activity with L-methionine added to the solvent
are presented in the Table 4 and Figure 4.
The analysis of data regarding the changes of intact gonadotropin activity in the
conditions of prolonged conservation while adding various doses of L-methionine
into the experimental series of samples showed that in the 2nd experimental series of
samples containing 0.2 mg per cm3 of L-methionine its activity was 52% of the initial concentration on the 2nd week of incubation. These values in this series of samples remained on the same level also on the 4th week of conservation; on the 6th
week of conservation, however, the hCG concentration decreased by 4%. The activity of gonadotropin in the 1st experimental series of samples containing 10 mg per
cm3 of L-methionine after two weeks of cultivation was the lowest and constituted
only 38.41% of the initial concentration (Fig. 4). Gonadotropin activity remained on
the same level during 6 weeks of incubation. On the 8th week of conservation, the
hCG concentration in this series of samples continued to decrease and constituted
Fig. 5. Dynamics of gonadotropin activity with addition of amino acids in concentration of 10
mg per cm3 (A) and 0.2 mg per cm3 (B) to the solvent
X axis presents the time of incubation of hCG with addition of amino acids in different concentration to the solvent
Y axis presents the changes of hormone activity in the % from initial concentration
Ryc. 5. Dynamika aktywności gonadotropiny z dodatkiem do roztworu aminokwasów w stężeniu 10 mg/cm3 i 0,2 mg/cm3 (B)
Oś X przedstawia czas inkubacji gonadotropiny hCG z dodatkiem do roztworu aminokwasów
w różnym stężeniu
Oś Y przedstawia zmiany aktywności gonadotropiny w % od początkowego stężenia
141
about 22% of the initial concentration. On the 8th week of conservation of samples in
a thermostat, the concentration of hCG in the control and 2nd experimental series of
samples was approximately on the same level (Fig. 4).
As a result of the performed comparative analysis regarding the effects of adding
the studied concentrations of amino acids during conservation of hCG samples, the
highest activity of the hormone during the entire period of incubation in the presence
of 10 mg per cm3 of L-lysine was demonstrated (Fig. 5).
Variations of the concentration of intact gonadotropin in all series of samples in
the conditions of a prolonged conservation of the gonadotropin in the samples containing L-glycine were demonstrated. The final results that L-glycine added in the
quantity of 0.2 mg per cm3 has the most pronounced stabilizing effect were demonstrated (Fig. 5B).
The extensive analysis of the data from literature testifies to the fact that the research of optimum methods of stabilization of gonadotropic preparations for their
use both in human and veterinary medicine is urgent and is being performed by
many researchers (van Zuylen et al. 1997, Tsivou et al. 2010). Moreover, the spectrum of gonadotropin-releasing drugs using constantly expanding, they find their
application in different areas: the creation of test systems in medicine, sport (doping
control), hormone therapy of reproductive abnormalities, etc. (van Zuylen et al.
1997, Tsivou et al. 2010). Given this demand and interest to the gonadotropic preparations, researchers continue to search the optimal stabilizer of hormones activity
during their incubation. It has been established that the use, for instance, of mannite
as a stabilizing agent does not cause a visible decrease of the activity of the hormone
after 24 weeks of conservation (Geigert 1999).
The US patent No 5270057 describes a lyophilized composition containing gonadotropin (for example, LH, TBG, FSH and hCG) stabilized with polybasic carboxylic acid or its salt. The US patent No 5650390 reveals a lyophilized composition
containing FSH, LH or hCG stabilized with a combination of sucrose and glycine
(Samaritani and Natale 1995, Jang and Sung 2008). However, lyophilized products
are inconvenient due to the fact that they have to be dissolved in water for injection
(WFI) before their use. Moreover, the process of production of lyophilized products
includes a stage of freezing-drying, and requires, thus, a serious financing. As an alternative for overcoming these limitations, the stability of the protein can be improved by adding a stabilizer to the dissolved protein. Examples of the used protein
stabilizers include surfactants, plasma albumin, polysaccharides, amino acids, polymers, etc. (Wang 1999).
The most suitable stabilizer should be chosen and used taking into consideration
unique physical and chemical properties of individual proteins. The combined use of
different protein stabilizers may cause unfavorable side reactions leading to an effect
opposed to the desirable one, due to a competitive interaction and side reactions between concrete stabilizers. Besides, the successful stabilization of proteins in the solution requires a great attention and many efforts, since individual protein stabilizers
have a certain range of concentrations required for stabilizing respective proteins
(Wang 1999). Glycine solution favours the accumulation of a greater number of
molecules of water around FSH, making thus more stable the remotest hydrophobous aminoacids among the multitude of amino acids constituting hFSH and in
142
this way stabilizing the latter. Some compositions include methionine, which stabilizes the FSH preventing its oxidation in water solution (Wang 1999).
CONCLUSIONS
It has been demonstrated that the addition of various doses of L-lysine, L-glycine
and L-methionine into the solvent for gonadotropin causes various levels of stabilizing effects, in particular:
1. Conservation of samples of chorionic hormone supplemented with L-lysine in
the amount of 10 mg per cm3 yielded the most stable concentration of gonadotropin during 8 weeks – over 52-54% of the initial concentration of the hCG;
2. Under the conditions of a prolonged conservation of the gonadotropin in the
samples containing L-glycine, variations of the concentration of intact gonadotropin are observed in all series of samples; yet the most pronounced stabilizing
effect is obtained with L-glycine added in the quantity of 0.2 mg per cm3.
3. Conservation of samples containing 0.2 mg per cm3 of L-methionine provides
a relatively high and stable activity of gonadotropin during 6 weeks of conservation with the concentration of intact hCG of about 50%.
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