Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 1 pp. 41ñ51, 2017
ISSN 0001-6837
Polish Pharmaceutical Society
ANALYSIS
PRELIMINARY HIGH PERFORMANCE CAPILLARY ELECTROPHORESIS
(HPCE) STUDIES OF ENZYMATIC DEGRADATION OF HYALURONIC ACID
BY HYALURONIDASE IN THE PRESENCE OF POLYVALENT METAL IONS
BARTOSZ URBANIAK, SZYMON PLEWA and ZENON J”ZEF KOKOT*
Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences,
Grunwaldzka 6 St., 60-780 PoznaÒ, Poland
Abstract: The aim of this study was, at first, to examine the influence of metal ions on digestion process of
hyaluronic acid by hyaluronidase (HAse) using high performance capillary electrophoresis (HPCE) method.
The influence of copper(II), zinc(II), manganese(II) ions on enzymatic degradation of HA by hyaluronidase
enzyme (HA-se) were investigated. Secondly, the kinetic parameters, Vmax, Km, kcat and kcat/Km, were determined
to estimate the impact of these metal ions (Me) on digestion process of hyaluronic acid (HA). The two different HA-Me mole ratios were analyzed. The examined data were always compared to the digestion process of
pure HA solution by hyaluronidase, to exhibit the differences in the digestion process of pure hyaluronan as
well as the hyaluronan in the presence of metal ions. It was observed that all of the investigated metal ions have
influenced the hyaluronic acid degradation process. The most important conclusion was a decrease of the kinetic parameters both Km and Vmax. In the result, it can be assumed that in all of the studied samples with metal ions
addition, the uncompetitive mechanism of enzyme inhibition occurred. The results of this study may give new
insight into foregoing knowledge about hyaluronic acid behavior. Due to the fact that our study was carried out
only for three different metal ions in two concentrations, it is necessary to continue further research comprising wider range of metal ions and their concentrations.
Keywords: hyaluronic acid (HA), hyaluronidase enzyme, high performance capillary electrophoresis (HPCE),
metal ions
aqueous solutions. During dissolution in water,
hyaluronan forms viscoelastic gels. Because of
these viscous properties, presence of HA in synovial fluid (SF) allows to decrease the friction in
joint cavity and enable movement without pain.
The average concentration of HA in human synovial fluid varies from 1400 to 3600 µg/mL, but the
higher concentration is observed in umbilical cord
(about 4100 µg/mL). The next are: dermis
(500ñ200 µg/mL), epidermis (100 µg/mL), vitreous
body (340ñ140 µg/mL), but if we want to evaluate
solid mass of HA in body tissue the total amount in
skin is the highest (1, 2). Another important features of HA are the osmotic properties, as a consequence of two processes. The first: formation of
hydrogen bonds between polymer molecule and
solvent. The second: ionization of carboxyl groups
of hyaluronic acid chain in extracellular environment. It causes migration and retention of water
because of increasing osmotic pressure. It allows to
Hyaluronan belongs to glycosaminoglycans
(GAGs) and widely occurs in human body. This
group of linear biopolymers including heparins,
dermatan, keratan and hyaluronan, plays various
and important physiological roles. For example:
heparins are involved in coagulation process, while
dermatan and keratan are compounds used for
building the cartilage tissue. Single chain of HA
polymer is composed of 2000ñ25000 disaccharide
units. Each basic unit is built of the dimer of D-glucuronic acid and N-acetyl-D-glucosamine. These
units can form hydrogen bond bridges between
each other as well as between polymer and solvent.
When compared with other GAGs, the hyaluronic
acid exhibits higher mass because of its significantly longer polysaccharide chain and in origin
does not contain any sulfate groups. These differences reflects its physicochemical properties and
physiological functions (1). The structure of this
polymer determines rheological properties of its
* Corresponding author: e-mail: [email protected]
41
42
BARTOSZ URBANIAK et al.
regulate hydrodynamic balance, and it is useful in
preparing skin-care products (moisturizers, and
anti-wrinkles creams), based on retention of water
in skin layer (1, 2). There are plenty of additional
physiological and biochemical functions of
hyaluronan in human body, for example in ovulation and fertilization, inflammatory processes,
angiogenesis, embryogenesis and also cancerogenesis. HA biopolymer molecules influence these
processes through signaling pathways using
hyaluronan binding proteins like CD44 and
RHAMM. The lower molecular weight HA (e.g.,
degradation products of native HA chain) exhibit
quite different biological properties in signal transduction processes than the high molecular weight
native hyaluronan (3). As it was stated above, HA
is most abundant in the connective tissue, and
plays two important roles - moisturize function in
skin and lubrication of the joint cavity. The synovium (synovial fluid, SF) consists of water in
70%, plasma proteins, γ-globulins, albumins, and
also polysaccharides (1, 4). Another functions of
SF are: nutrition of cartilage, reducing shocks during movements and reduction of friction force that
enables smooth gliding of two bone surfaces.
However, in the case of inflammation process of
the joint (rheumatoid arthritis, RA or osteoarthritis,
OA), high molecular weight hyaluronic acid molecule is degradated to low molecular weight
hyaluronan. This degradation process is resulting
in loss of the viscoelastic properties of SF, and is
changing its rheological properties. This pathological process is caused by two reasons. The first
based on higher free radicals and reactive oxygen
species (ROS) levels which are strongly connected
with inflammatory processes. The second results
from the activity of hyaluronidases (2).
Degenerative joint disease affects about 60% population above 60 years old, and about 85% above
75 years old (2). These degenerative diseases
develop with an acute pain and joint deformation.
The changes ongoing in infected joint are indissolubly connected with degradation of natural occurring hyaluronic acid. The results of this process
are: pain, stiffness and reduced mobility of patients
suffering from degenerative joint diseases.
Treatment usually starts from weight loss, and
sport activities. Pharmacological treatment based
on analgesics and non-steroid anti-inflammatory
drugs (NSAIDs), and intra-articular corticosteroid
injections. As a last resort surgical intervention is
used (5). Viscosupplementation is a relatively new
method of noninvasive treatment of joint disease,
introduced on the medical market in 1987 in Japan,
1995 in Europe and in 1997 in USA (5). The great
advantage of this therapeutic method is the pain
relief (5, 6). The first studies about the injections of
hyaluronan as a viscosupplement were published in
the seventies of the past century. Balazs and coworkers, tried to establish benefits after injecting
hyaluronan into affected knees (5). The pain relief
(analgesic) effect after viscosupplementation was
observed up to 8-12 weeks after injection, despite
the fact that the half-time of hyaluronan in healthy
joints equals only 13-20 h (6) or up to 12 h in
inflammated joints (5). Previous trials described
only local side effects connected with injections
(pain, redness, swelling) (5, 7-11). In contrast to
studies mentioned above, another retrospective trials described good effects of intraarticular treatment, even better than NSAID (12, 13) or corticosteroid injections (14, 15). Important is to continue
study and clinical tests with accurate collecting
informations from patients and health care team
about side effects. In available literature there are
plenty of clinical trials about viscosupplements,
but their interpretations are difficult, because of
various methodologies of these studies.
Hyaluronic acid can be degraded according to
two different pathways: 1) by hyaluronidase enzyme
or 2) can be induced by ROS (reactive oxygen
species) or free radicals. These ROS are produced as
a result of the biochemical reactions of the inflammation process located in joints. ROS accelerate the
degradation of hyaluronan through the attack on the
D-glucuronate/D-glucuronic acid and N-acetyl-Dglucosamine (16). Some authors have found that the
polyvalent metal ions in the presence of hyaluronan
influence the HA degradation process by changing
the oxidation/reduction potential of ROS produced
during inflammation. Moreover, from the therapeutical and clinical point of view, the most important
are the transition (d-block/orbital) metal ions,
because of its significant redox potential (17, 18).
äoltÈs and co-workers (19) had investigated degradation of high molecular weight hyaluronan in the
presence of copper(II) ions. They proved that even
small amount of copper(II) in hyaluronic acid solution can accelerate process of its degradation. It
must be emphasized that the influence of copper(II)
ions was not observed on the elimination of hydrogen peroxide from the examined mixture. äoltÈs and
Kogan (20) have studied dynamic viscosity in the
presence of copper(II) ions. In both situations addition of metal ions resulted in reduction of viscosity.
Similar studies had been conducted by Valachova et
al. (17). They studied influence of manganese(II)
ions on hyaluronan degradation. The decreased
Preliminary high performance capillary electrophoresis (HPCE) studies of...
reduction rate of dynamic viscosity was observed in
the mixtures containing manganese(II) ions. They
have concluded that intraarticular preparations of
hyaluronic acid should be enriched by addition of
manganese(II) ions. Pasqualicchio et al. (21) examinated role of zinc(II) and copper(II) ions on proteoglycan metabolism in articular cartilage. In vitro
investigation of porcine cell cultures have showed
that copper(II) exhibits some cartilage cells protection effects in contrast to zinc(II), but the mechanism of this action remains unknown. Balogh et al.
(22) have investigated the anti-oxidative role of dblock metal ions (copper(II), zinc(II), manganese(II), cobalt(II)) and tried to distinguish antioxidative role of pure hyaluronan from antioxidative role
of hyaluronan with addition of metal ions.
Interestingly, it was concluded again that manganese(II) had the strongest antioxidative properties.
As a result of search of the scientific literature concerning the degradation of the hyaluronan, it is difficult to draw out any unambiguous conclusion
about role of polyvalent metal ions on degradation
process of HA, and moreover, its influence on the
extension on the durability of the viscosupplementation agents after intraarticular application. However,
further research should be conducted to improve the
rheological behavior and, in the future, increase
properties of intraarticular drugs based on hyaluronic acid.
In available literature data, authors did not find
any studies describing the influence of polyvalent
metal ions on decomposition process of hyaluronic
acid. Moreover, most of the publications describing
the degradation studies of HA and influence of
polyvalent metal ions on it, were only focused on the
measuring the changes of dynamic viscosity of
hyaluronan mixture (17, 19, 20, 23).
The aim of presented studies was to develop a
high performance capillary electrophoresis method
suitable for monitoring the digestion process of
hyaluronan as well as for evaluation of the influence
of metals on this process. In this paper, authors for
the first time have used the HPCE method for evaluation of kinetic parameters of hyaluronan enzymatic degradation by hyaluronidase in the presence of
metal ions. The following parameters were calculated: Vmax, Km, kcat and kcat/Km, respectively. The influence of copper(II), zinc(II), manganese(II) ions on
enzymatic degradation of hyaluronic acid (HA) by
hyaluronidase enzyme (HA-se) were investigated at
different concentrations. Based on the experimental
data and calculated kinetic parameters the estimation of the influence of metal ions on digestion
process of HA by HAse was described.
43
EXPERIMENTAL
Materials
Hyaluronic acid (from Streptococcus equi) and
hyaluronidase enzyme (from bovine testes, type I-S)
were purchased from Sigma-Aldrich (USA). Zinc(II)
sulfate (ZnSO4∑7H2O; M.W. 287.54 g/mol), copper(II) sulfate (CuSO4∑5H2O; M.W. 249.69 g/mol)
from P.P.H. STANDARD, manganese (II) chloride
(MnCl2∑4H2O; M.W. = 197.92 g/mol) from P.P.H.
Polskie Odczynniki Chemiczne S.A., sodium tetraborate decahydrate (B4Na2O7∑10H2O; M.W. 381.37
g/mol) from Fluka, sodium dihydrogen phosphate
(NaH2PO4∑2H2O; M.W. 156.01 g/mol) from Riedel-de
HaÎn, sodium dodecyl sulfate (SDS; C12H25NaO4S;
M.W. 288.4 g/mol) from Sigma-Aldrich, sodium
hydroxide, solution 1.0 mol/L; 1.041 g/mL and 0.1
mol/L; 1.003 g/mL and water ultra pure for HPCE
from Agilent Technologies, methanol (M.W. 32.04
g mol-1) from J.T. Baker.
Preparation of standards
Stock solution of hyaluronic acid in water
(2.5∑10-5 mol/L), was prepared by dissolving an
appropriate amount of HA salt in distilled water.
The mixture was extensively mixed on vortex shaker by about 1 min. Stock solution of hyaluronidase
enzyme (HAse) was obtained by dissolving of
0.0020 g of hyaluronidase in 10.00 mL of distilled
water. After that, solution was ultrasonicated for 10
min, and finally filtered through 0.45 µm pore size
filters. The stock solutions of metal ions were prepared in two different concentrations: 5.0∑10-5
mmol/L and 2.5∑10-4 mmol/L. In both cases, an
appropriate amounts of zinc(II), copper(II), and
manganese(II) salts were dissolved in distilled water
and ultrasonicated for 5 min. The running buffer was
prepared by dissolving and mixing an appropriate
weights of sodium dodecyl sulfate (SDS) in 25.0 mL
of phosphate buffer (pH 8.11). After that, running
buffer was put in ultrasonic bath for 5 min. Then, it
was filtered by syringe filter (pore size 0.45 µm).
Preparation of samples
The enzymatic digestion process of HA by
HAse was analyzed using following concentrations
of HA: 3.125∑10-6 mol/L, 4.375∑10-6 mol/L, 6.25∑10-6
mol/L, 8.125∑10-6 mol/L, 1.00∑10-5 mol/L and
1.1875∑10-5 mol/L, and the solution of HAse 200
µg/mL was always added to the HA solution. The
total volume of the sample was 1.0 mL. The sample
preparation of the HA with the addition of metal ions
was prepared according to the following schedule: to
the series of HA solutions, an appropriate volume of
44
BARTOSZ URBANIAK et al.
metal solution was added due to keep the constant
mole ratios (HA-Me) 1 : 1 and 1 : 5. Finally, the solution of HAse of 200 µg/mL was added to the mixture.
Total volume of 1.0 mL was always kept. The samples were incubated in 37OC for 30 min, and analyzed
by HPCE method. The mixture of pure HA and
HAse was set as a reference system.
Instrumentation and methods
The HPCE separation was performed using
Agilent G1600 instrument (Agilent Technologies,
Germany) equipped with a UV-DAD detector (190600 nm), and the ChemStation software (Agilent
Technologies) Separation conditions: silica capillary - total length 64.5 cm (effective length 56.0
cm), diameter 75 µm and length of optic way 150
µm, running temperature: 25OC, voltage 20 kV, sample injection 7.0 s, under pressure 50.0 mbar; precondition flush: NaOH (1 mol/L) 1 min, MeOH 1
min, NaOH (0.1 mol/L) 1 min, H2O 1 min, running
buffer 1.5 min ñ post-condition flush: HCl (0.1
mol/L) 0.5 min, MeOH 0.5 min, H2O 1 min.
Validation procedure of determination of
hyaluronic acid
The electrophoretic method useful for investigation of enzymatic digestion process of hyaluronic
acid was developed by us earlier (24). The application of this method, allows for identification of
hyaluronic acid and its degradation products. Our
additional experiments with the use of products of
hyaluronic acid degradation (hyaluronan hexa- and
tetra- oligosaccharides) confirms eventual possibility to distinguish hyaluronic acid and its degradation
products. The well separated peaks of analyzed substances were obtained what corresponds with good
selectivity of the analytical method used.
In the present work, according to the ICH
Guideline 2Q - Validation of Analytical Procedures,
the following parameters were calculated: precision
of an analytical procedure expressed as the closeness of agreement between a series of measurements
obtained from multiple sampling of the same homogeneous sample under the prescribed conditions and
usually expressed as the standard deviation (SD) or
relative standard deviation (RSD [%]) of a series of
measurements; linearity and range of the analytical
method as well as the detection limit (LOD) and
quantitation limit (LOQ) of the hyaluronic acid
(Table 1.)
Michaelis-Menten kinetics
The Michaelis-Menten kinetics can be
described by the following equation:
Table 1. The calibration curve parameters (R2 coefficient of determination, a slope and b intercept) and calculated values of standard deviation (SD), relative standard deviation (RSD [%]), limit of determination (LOD)
and limit of quantitation (LOQ) for hyaluronic acid in the analyzed concentration range.
Hyaluronic acid
concentration
[mol/L]
Standard
deviation
(SD)
RSD
[%]
6.250∑10-7
4.18∑10-8
7.014
1.250∑10-6
3.74∑10-8
3.015
3.125∑10
7.05∑10
-8
2.260
6.300∑10-6
4.17∑10-8
0.663
8.125∑10
3.05∑10
-7
3.408
1.250∑10-5
1.47∑10-7
1.174
3.150∑10
1.48∑10
-7
0.474
1.04∑10-6
1.649
-6
-6
-5
6.300∑10-5
Calibration curve parameters
R2
a ± SD
b ± SD
1.999 ± 0.006
-0.090 ± 1.187
Detection limit LOD
= (3.3 σb)/a
Quantification limit
LOQ = (10 σb)/a
2.4∑10-7 ± 1.5∑10-7 [mol/L]
7.4∑10-7 ± 4.5∑10-7 [mol/L]
0.9999
where: SD denotes the standard deviation; RSD [%] - relative standard deviation; R2 - coefficient of determination; a ñ slope of the calibration curve, b ñ intercept of the calibration curve; LOD - detection limit and LOQ
- limit of quantitation.
Preliminary high performance capillary electrophoresis (HPCE) studies of...
45
Table 2. The calculated kinetic parameters (Vmax, Km, kcat, Kcat/Km) of digestion process of hyaluronic acid in the presence of copper(II),
zinc(II) and manganese(II) ions.
Analyzed systems
Kinetic
parameters
HA
Vmax [mol s-1]
1.03 ◊ 10-9
9.76 ◊ 10-10
6.25 ∑◊ 10-10
7.91 ◊ 10-10
6.45 ∑◊ 10-10 6.88 ◊ 10-10 6.87 ◊∑ 10-10
Km [mol/L]
1.68 ◊ 10
1.65 ◊ 10
9.83 ◊ 10
1.01 ◊ 10
1.12 ◊ 10-6
R2 = 0.9366
R2 = 0.9916
R2 = 0.8845 R2 = 0.9962 R2 = 0.9952
Lineweaver-Burk
R2 ñ coefficient of
determination
HA-Cu
1:1
-6
HA-Cu
1:5
-6
R2 = 0.9938 R2 = 0.9948
HA-Zn
1:1
-7
HA-Zn
1:5
-6
HA-Mn
1:1
1.11 ◊ 10-6
HA-Mn
1:5
1.14 ◊ 10-6
kcat [1∑s-1]
0.00248
0.00234
0.00150
0.00190
0.00155
0.00165
0.00165
kcat/Km
[mol-1∑L∑s-1]
1477.09
1419.11
1524.22
1884.57
1377.65
1493.21
1451.17
k1
k2
E+S→
ES →
E+P
(1)
k-1
where: E ñ represents enzyme; S ñ substrate; ES ñ
enzyme-substrate complex; P ñ product; k1, k-1, k2 ñ
reaction rate constant in particular direction.
Based on this equation the velocity rate is
equal to:
v = -d[S]/dt = d[P]/dt = [ES]k2 and [ES] =
[E]0[S]/([S] + Km)
(2)
where, k2 can be also called as kcat and it means
turnover number of an enzyme. It is the number of
catalytic cycles of active site of enzyme (when it is
saturated by substrate) in particular time (usually per
second). Michaelis-Menten constant can be
described as Km = (k-1 + kcat)/k1. To sum up, equations mentioned above can be rearranged to:
V = Vmax [S]/(Km + [S])
(3)
When [S] >> Km, [S]/(Km + [S]) is approximately equal 1, so Vmax = kcat[E]0. This equation
shows strong correlation between kcat and the
enzyme concentration.
To comply with Michaelis-Menten law, the
following conditions must be applied: constant concentration of enzyme and constant concentration of
enzyme-substrate complex in comparison to
changes of concentration of substrate or product.
Michaelis-Menten kinetic model enable to
determine two parameters Km, Vmax, both of them are
calculated based on Lineweaver-Burk plot which is
a reciprocal of Michaelis-Menten kinetic equation.
First parameter (Km) describes affinity of the
enzyme to the substrate and the second (Vmax) velocity of the enzymatic reaction, respectively. In other
words, the Vmax represents the maximum rate of the
analyzed system, at maximum (saturating) substrate
concentrations, while the Michaelis constant Km is
the substrate concentration at which the reaction rate
is a half of Vmax (25).
Comparing the catalytic efficiency (η-parameter) for different substrate-enzyme-metal ions samples, it can be concluded that higher η values reflect
better efficacy of enzyme toward examined substrate (26-28).
RESULTS AND DISCUSSION
The HPCE investigations of influence of polyvalent metal ions on enzymatic digestion of
hyaluronic acid was the aim of the presented studies.
This enzymatic degradation process of hyaluronan
by hyaluronidase was analyzed due to the decomposition of HA by hyaluronidase in the presence of
three different metal ions: zinc(II), copper(II) and
manganese(II) ions. The analyzed mixtures of
hyaluronan and metal ions were prepared in two different mole ratios (HA-Me): 1 : 1 and 1 : 5. The
enzyme concentration was always constant (see:
Preparation of samples). The obtained HPCE data
were further analyzed to calculate the MichaelisMenten kinetics parameters: Vmax and Km (Table 2).
The analysis of the electrophoretic data was
sophisticated owing to multistage degradation
process of HA by HAse.
The hyaluronidase randomly hydrolyze 1,4linkages between N-acetyl-β-D-glucosamine and Dglucuronate residues in hyaluronate, and decomposes polymer systematically to the low molecular
hyaluronan chains, then oligosaccharides and finally to the single dimer units of HA. Although the
place of degradation of hyaluronan by hyaluronidase
enzyme is well defined, it must be emphasized that
the hyaluronidase degrades HA polymer regardless
of the length and mass (29). Generation of different
products of hyaluronan degradation by HAse was
impeding factor during interpretation of the electrophoretic data due to the fact of creation of great
46
BARTOSZ URBANIAK et al.
amount of products, by the enzyme, during short
time.
The correct establishing of the substrate and
product concentration is crucial for the proper calculation of Michaelis-Menten kinetic parameters.
Due to the number of degradation products obtained
by HPCE technique, an increment of all products
was calculated as a difference between peak area of
sum of peaks areas of products and pure hyaluronan,
separated after 30 min of incubations. This approach
enabled both: precise determination of concentra-
tions of enzymatic digestion products and hyaluronan concentration, respectively (Fig. 1).
The linearity of the analytical method was
proved by estimation of the calibration curve.
Coefficient of determination (R2) confirms high linearity of the method. The low values of calculated
SD and RSD parameters of electrophoretic method
of determination of hyaluronic acid confirm the
good precision of the analytical method used (Table
1). Limit of detection (LOD) and limit of quantitation (LOQ) presented in Table 1 indicate that the
Figure 1. The comparison of electropherograms of: pure hyaluronic acid (upper); hyaluronic acid and hyaluronidase mixture, 30 min of incubation, 37OC (lower). Concentration of HA 8.125∑10-6 mol/L in both cases. Electrophoretic conditions (see: Instrumentation and methods)
Preliminary high performance capillary electrophoresis (HPCE) studies of...
47
Figure 2. The variation of the enzyme-catalyzed reaction rate against the substrate concentration HA-Me 1 : 1 (upper) and 1 : 5 (lower),
treated by hyaluronidase
method exhibits good sensitivity; the LOD was
lower than the lowest hyaluronan concentration used
for the calibration of the method. All these parameters gathered and presented in Table 1, confirm the
reliability of the electrophoretic data obtained.
The seven different compositions of hyaluronic acid (HA) and hyaluronidase (HA-se) in the presence of polyvalent metal ions were studied. The
decomposition of hyaluronan by hyaluronidase with
addition of metal ions in a given mole ratio was
investigated up to the 30 min in the 37OC. It was
proved, that with the passing of time, the new peaks
were appeared as a products of HA degradation.
Based on the observations mentioned above,
enzyme kinetic parameters of decomposition reac-
tion of hyaluronic acid by hyaluronidase in the presence of three different metal ions were calculated.
To describe and compare digestion process of
hyaluronan with and without the presence of metal
ions MichaelisñMenten kinetics model was used.
This model is based on the theory of bidirectional
reaction according to which substrate reacts with
enzyme, to produce a temporary complex enzymesubstrate. At the end, the product and unchanged
enzyme are released. As it was mentioned earlier,
the Michaelis constant (Km) is the substrate concentration at which the velocity of reaction is equal a
half of maximum velocity (Vmax) of this enzyme
reaction. The kinetic parameters were calculated
according to Lineweaver-Burk function f(1/S) =
48
BARTOSZ URBANIAK et al.
1/V, where 1/S is the inverse of product concentration, while 1/V is the reciprocal value of the reaction
velocity. The parameters a and b, obtained from the
Lineweaver-Burk plot allowed to calculate the Vmax
and Km. Calculations were based on the analysis of
decomposition product concentration changes (30).
The results of digestion reaction of HA by
HAse in the presence of divalent metal ions is shown
in Table 2. It is clear, that addition of metal ions have
influenced the maximum velocity rate (Vmax) and
Michaelis-Menten constant (Km) of decomposition of
hyaluronan in comparison with pure HA degradation
profile, in all cases both parameters were decreased
with addition of metal ions.
Interestingly, comparing digestion process for
1 : 1 and 1 : 5 mole ratios (HA : Me) samples it can
be observed some dissimilarity. The Vmax parameter
was decreased stronger for higher concentration of
metal ions with exception of the case of manganese(II) ions. For the contrast, the Km parameter was
decreased at the same level for both mole ratios with
exceptions the copper ions, where difference
between samples was higher (Table 2).
The kinetic parameters calculated for the
degradation reaction of hyaluronic acid in the presence of metal ions by hyaluronidase (Table 2), were
always analyzed due to the reference of Vmax and Km
of HA-HAse sample (Vmax = 1.03∑10-9 [mol/s] and Km
Figure 3. Lineweaver-Burk plot for the analysis of hyaluronic acid degradation HA-Me 1 : 1 (upper) and 1 : 5 (lower)
Preliminary high performance capillary electrophoresis (HPCE) studies of...
= 1.68∑10-6 [mol/L]). As it was assumed earlier, the
Km parameter refers to the affinity of the enzyme to
the substrate. The affinity of enzyme to the substrate
increases with the decreasing value of the Km parameter.
Based on obtained data (Table 2), it was stated,
that the HA-Zn and HA-Mn slightly decreased the
Km values notwithstanding the metal ions concentration, in comparison to pure HA degradation by
HAse. The analysis of data for copper (II) ions led to
quite different statements: in the lower concentration (HA-Me, 1 : 1) the Km values were not significantly changed when compared with the reference
HA-HAse sample. However, in the higher concentration of copper ions (HA-Me, 1 : 5), the Km values
were decreased, as for zinc or manganese ions. To
sum up, all of investigated metal ions decreased the
Km values. The graphical presentation of experimental data is shown in Figure 2.
It represents the Michaelis-Menten plots of
pure hyaluronan and hyaluronan-metal ions mixture
digested by hyaluronidase enzyme. These plots
show the changes of velocity rate of reaction. All the
curves go to plateau (maximum velocity, Vmax). It
was shown that in the case of manganese the curve
is radically different. The velocity is increasing most
slowly in both concentration (HA-Mn, 1 : 1 and 1 :
5).
The Lineweaver-Burk plots of degradation
process of hyaluronan in the presence of metal ions
are shown in Figure 3.
The decrease of the maximum velocity of the
enzymatic reaction with the simultaneous increase
of the affinity of the enzyme to the substrate, suggests that during digestion process of the hyaluronic
acid by hyaluronidase enzyme, in the presence of
examined ions, the uncompetitive inhibition mechanism of digestion was observed (31). This hypothesis requires further and detailed studies.
An additional attempt was made to establish
the catalytic properties of metal ions for the digestion process of HA by HAse. The specific parameters kcat and kcat/Km were calculated.
The first of these parameters refers the
turnover frequency or catalytic constant of an
enzyme. It is defined as the number of chemical conversions (catalytic cycles) performed by active site
of an enzyme per unit of time. Entity of turnover frequency is reciprocal of time, and it can be calculated from the maximum velocity Vmax and total
enzyme concentration, as follows:
kcat = Vmax / [E]0
(4)
where, [E]0 denotes the total concentration of the
enzyme. Calculated rate of the kcat (Table 2.) param-
49
eters are equal to rate of releasing of product from
the enzyme-substrate complex (26). The advantage
of the analysis of the kcat parameter in comparison to
the Vmax constant is due to the fact, that this turnover
frequency is independent of the concentration of
enzyme. Thus it is more appropriate to use kcat to
compare enzymes performance. Analysing the calculated kcat parameters for samples with addition of
manganese ions, with pure HA and HAse as a reference one, it can be assumed that the presence of
metal ions causes the decrease of catalytic ability of
hyaluronidase to the hyaluronan in both mole ratios.
On the other hand, the addition of zinc (II) ions and
copper (II) ions decrease kcat parameters stronger in
higher ions concentration (HA-Me 1 : 5). In the HAMe 1 : 1 system, the zinc (II) ions decrease the catalytic constant stronger than copper (II) ions.
The second parameter η describes the catalytic
efficiency or specificity constant, in means of the kcat
ratio to Km parameter, and can be calculated, by the
following equation:
η = kcat / Km
(5)
Parameter η exemplifies the preference of an
enzyme to different substrates (27). It connects both
Km parameter, which describes affinity of enzyme to
the substrate and kcat which informs about ratio of
converting substrate into product. It should be
emphasized that hypothetical - maximal possible values of catalytic efficiency can reach 108-109 [dm3
mol-1 s-1] (26). The ratio kcat/Km is useful parameter for
comparing the relative rates of an enzyme toward
different substrates. In the present studies, it was
assumed to analyze the digestion process of
hyaluronidase on different substrates: pure hyaluronic acid, hyaluronic acid mixture with the copper(II),
zinc(II) and manganese(II) ions, respectively. The
analysis of the kcat/Km index revealed that the rate of
action of the hyaluronidase enzyme on the analyzed
HA and HA-Me systems, was rather at the similar
level, with one exception, observed for the zinc (II)
ions (mole ratio HA-Zn, 1 : 1). This finding seems to
correspond with the Michaelis-Menten plots, where
it can be observed that the presence of zinc(II) ions
(mole ratio HA-Zn, 1 : 1) and copper(II) ions (mole
ratio HA-Cu, 1 : 5) exhibit the differences between
them and HA-HAse sample as a reference one.
CONCLUSIONS
The growing popularity of treatment of joint
diseases based on various hyaluronan preparations
entails necessity of examining behavior of hyaluronan in various conditions. Knowing the processes
affecting the enzymatic digestion of hyaluronan it
50
BARTOSZ URBANIAK et al.
will be possible to project safer formulation of drugs
containing hyaluronic acid. It is important to provide high purity of viscosupplements, especially if
they would be administered directly into infected
joints.
In presented studies, the influence of divalent
metal ions on the process of decomposition of
hyaluronic acid by hyaluronidase using high performance capillary electrophoresis was described.
It was proved that all of the analyzed polyvalent metal ions, have altered the digestion process of
the hyaluronan by hyaluronidase when compared
with the digestion of the pure hyaluronan without
addition of any metals (Table 2.). Moreover, after
analysis of obtained experimental data, and calculated kinetic parameters it was established, that the
HA-Me system inhibits the digestion reaction
according to the uncompetitive mechanism of
enzyme inhibition. It is an important statement with
a great cognitive and practical value. Due to the fact
that our studies were carried out based on only three
metal ions in two different concentrations, it is necessary to continue this research on other metal ions
in a wider concentrations range. Moreover, using
reference product of hyaluronic acid degradation
may allow to distinguish other depolymerization
product and as the result could help to clarify
enzyme kinetics mechanism.
In available literature data we did not find any
studies describing kinetic parameters of hyaluronic
acid decomposition by hyaluronidase. Some
authors have investigated the influence of various
metal ions on HA degradation (17, 19, 20, 22, 32,
33). Vercruysse et al. have studied enzymatic digestion of hyaluronan by HAse and the effect of the
preincubation of HAse with different chloride salts.
They confirmed that all of these ions enhance the
degradation process of HA (32). Other researchers
focus on the influence of certain reactive oxygen
species (ROS) on hyaluronan degradation, and the
role of metal ions with altering oxidative status in
protecting this glycosaminoglycan against ROSinduced depolymerization (22). äoltÈs et al. extensively studied degradation of hyaluronic acid by
copper (II) chloride and ascorbate system. Using
various analytical methods they proved that even
small amount of copper ions, in the presence of
ascorbate, distinctively intensifies the degradation
process (33).
To sum up, from the literature research it
comes that the majority of authors focus on ROSinduced depolymerization of hyaluronic acid. This
work is probably the first describing influence of the
metal ions on the kinetic parameters (Vmax, Km, kcat
and kcat/Km) of degradation of hyaluronan by
hyaluronidase. Nevertheless, our preliminary study
needs the further research, as it was mentioned earlier.
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Received: 8. 01. 2016