1. 1
ABSTRACT
This Work is focused on interactions of hyaluronan (HA) and bovine serum albumin
(BSA). These interactions were studied by the SEC-MALLS method, where molar mass, root
mean square (rms) radii, hydrodynamic radius and intrinsic viscosity of particles of the
system were defined. Additionally, interactions were examined by measurement of DLS and
zeta potential, where hydrodynamic radius and a value of the zeta potential were defined.
The interactions were confirmed at any of the used media, but with different efficiency.
Furthermore it was found out, that the high ionic strength minimizes the range of the
interactions, magnifies the radii of the complexes, their intrinsic viscosity and zeta potential. It
was proved that complexes become smaller in their radii and viscosity with growing BSA
concentration.
KEY WORDS
Interactions, hyaluronan, albumin, SEC-MALLS, DLS, zeta potential, molar mass, root
mean square (rms) radii, hydrodynamic radii, intrinsic viscosity
2. 2
SEREDA, A. Study of interactions of hyaluronan and albumin by the SEC-MALLS method (in
English refers to: Study of interactions of hyaluronan and albumin by SEC-MALLS
method) Brno: Vysoké učení technické v Brně (in English refers to: Brno University of
Technology), Fakulta chemická (in English refers to: Faculty of Chemistry), 2014. 56 s., Head
of Bachelor´s Work Ing. Martin Chytil, Ph.D..
DECLARATION
I hereby make the declaration that I have prepared the Bachelor´s Work independently
and I have quoted correctly and completely all used literary sources. The Bachelor´s Work is
in terms of content the property of the Faculty of Chemistry at the Brno University of
Technology (VUT) and it can be used for commercial purposes only with consent from the
head of the Bachelor´s Work and the Dean of the Faculty of Chemistry at the Brno University
of Technology.
…………………
Student´ssignature
3. 3
ACKNOWLEDGEMENT
I would like to hereby express my thanks to the Head of my Bachelor´s Work, Ing. Martin
Chytil, Ph.D, for professional advices, help and patience. Further I would like to express my
thanks to Ing. Michal Kalina for cooperation and professional advices, which were necessary
to understand the theoretic mechanisms.
4. 4
CONTENT
1. Úvod.................................................................................................................................. 5
2. Teoretická část.................................................................................................................. 6
2.1. Hyaluronan 6
2.2. Albumin 8
2.3. Interakce molekul hyaluronanu a hovězího sérového albuminu 9
2.4. SEC 12
2.4.1. Teorie vylučovací chromatografie 13
2.4.2. Detektory 15
2.5. MALLS 16
2.6. DLS 21
3. Experimentální část..........................................................................................................25
3.1. Materiály 25
3.2. Metody 25
3.2.1. Příprava vzorků 25
3.2.2. DLS měření vzorků 27
3.2.3. SEC-MALLS měření vzorků 27
4. Výsledky a Diskuze k DLS................................................................................................27
4.1. DLS sady nízkomolekulové HA v Milli-Q a 0,1M NaNO3 27
4.2. DLS sady vysokomolekulové HA v Milli-Q a 0,1M NaNO3 32
5. Výsledky a Diskuze k SEC-MALLS...................................................................................35
5.1. SEC-MALLS sady nízkomolekulové HA v Milli-Q a 0,1M NaNO3 35
5.2. SEC-MALLS sady vysokomolekulové HA v 0,1M roztoku NaNO3 38
5.3. Porovnání sad nízkomolekulové HA 40
5.4. Porovnání sad vysokomolekulové HA 45
6. Závěr................................................................................................................................48
7. Seznam použitých zkratek................................................................................................49
8. Reference.........................................................................................................................50
5. 5
INTRODUCTION
Interactions of hyaluronan and albumin is the topic of many actual and also previous
scientific works and researches. It is due to excellent properties of both the hyaluronic acid
and albumin. They are completely bio-compatible and occur naturally in the human body.
Hyaluronic acid occurs most at joints and in vitreous body and in extracellular matrix. It is a
signal molecule in many receptors and thereby it becomes an important part in many
significant processes in organism. Hyaluronic acid has also exceptional viscoelastic
properties. Albumin is important due to its ability to bind different substances, including
pharmaceuticals. Interactions of hyaluronan and albumin could provide us with an excellent
carrier of pharmaceuticals. It becomes an actual target of scientific researches. The target of
this Work is to investigate interactions of hyaluronan and albumin by means of the SEC-
MALLS method. SEC-MALLS represents an up-to-date, high-efficient and high-precise
chromatographic method for determination of important parameters of bio-polymers as they
are molar mass and root mean square (rms) radius. Interactions by means of measuring of
dynamic light scattering DLS will be investigated additionally in this Work. Both SEC-MALLS
and DLS are equipped with the optical detection method. Results should provide information
on forming complexes, their mass weight, root mean square radius and intrinsic viscosity.
These data should serve for review, whether these interactions occur and what is the
character of possible forming complexes of hyaluronic acid and albumin.
6. 6
THEORETICAL PART
2.1. Hyaluronan
Hyaluronic acid was firstly separated by K.Mayer in 1934 from vitreous body of bovine
cattle. It is a galactan (muco-galactan) naturally occurring in vertebrates.
Due to carboxyl groups it is a linear polyanion consisting of repeated disaccharide units.
Each such unit consists of N-acetyl-D-glucosamine and D-glucoronic acid bound with β-1,3-
glycosidic bond. Disaccharide units are mutually bound with β-1,4-glycosidic bond. One
disaccharide unit has weight about 400 Da and length about 1 nm. One molecule of
hyaluronan can contain up to 10 000 such units (4 MDa, 10 µm).
Picture 1. Structure of hyaluronic acid
Hydrogens occur in axial position and form the hydrophobic part of molecule. Extensive
groups, as hydroxyl groups and carboxyl groups, are in equatorial positions and they are
hydrophobic. Hydrogen bridges cause molecule stabilization. They are replaced in water with
the so-called "aqueous bridges", when molecules of water build in the hydrogen bond.
Picture 2. Hydrogen bridges in molecule HA
Picture 3. "Aqueous bridges" in molecule HA
7. 7
Due to hydrogen bridges and amphiphilic character (hydrophobic/hydrophilic part) HA
molecule decants to helix, which than decants to an accidental ball, which is the resultant
structure of molecule. Hyaluronic acid due to such structure and high content of OHgroups is
highly hygroscopic. It is capable to absorb water with up to 1000x higher total weight than the
molecule itself[1].
Its hydroxyl groups have the value 9,2apK [2] and that is why they occur under
physiological conditions (pH 7.2-7,4) in form of anion, as sodium salt (hyaluronan sodium).
It is represented the most in extracellular matrix (ECM) of cells of synovial liquid, chorda
umbilicalis and vitreous body of mammals. However, it also occurs in lungs, kidneys and
muscles. Synthesis takes place on plasmatic membrane, enzymes of synthesis participate in
it, which are integral membrane proteins (HAS1, HAS23, HAS3) [3]. These enzymes use as
substrates nucleotide precursors, UDP-glucoronic acid and UDP-N-acetylglucosamine.
Synthesis takes place from a reducing end[4].
Industrial synthesis takes place by means of bacterias Streptococcus equi (low-molecule
HA), andStreptococcus zooepidemicus (high-molecule up to 2 MDa). It is also separated in
lower extent from cocks combs, chorda umbilicalis and other soft tissues of mammals.
HA is completely bio-compatible and it does not cause any negative immune response, be
specific HA itself and its degradation metabolites.
HA degradation is caused by enzymes of hyaluronidase, β-D-glucoronidase and β-N-
acetyl-hexosaminidase. These enzymes are mainly intracellular. Hyaluronidase splits high-
molecule HA to oligosaccharide HA, when β-D-glucoronidase and β-N-acetyl-
hexosaminidase degrade oligosaccharides [5]. Enzymes of hyaluronidase and thereby the
property to split chains of hyaluronic acid were found in bacterias, as they are
Staphylococcus aureus, Streptococcus pyogenes et pneumonia andClostridium perfringens.
HA life cycle in organism is relatively short (1-3 days), but it is compensated with its high
circulation in bloodstream and lymphatic system. It eliminates from blood in livers and partly
in kidneys.
It is known that depending on its molar massHA can perform different functions [6]. For
example low-molecule HA has angiogenesis ability (creation of new capillaries)[7], but it
simultaneously causes gene expression at macrophages, which results in inflammatory
processes. On the contrary high-molecule HA has anti-inflammatory effects and due to its
ability it creates more viscous environment, which prevents against move of macrophages
and leucocytes. We can summarize in general that cells or tissues in normal undamaged
conditions in its surrounding extracellular matrix contain mainly high-molecule HA. However
when damaged they split to fragments of low-molecule HA, which bind with certain receptors
of cell membrane, initiate immune response and cause inflammation[8]. Low-molecule HA is
also known as an inhibitor of cancer[9].
Hyaluronan has unique viscoelastic properties and it acts as pseudoplastic liquid, which
makes of it an ideal biological lubricant. Under quiescent state it is highly viscous liquid,
however due to pressure and action of force it reduces its viscosity (Newton´s behaviour).
This is caused by mutual interwining of chains of high-molecule HA, which in case of physical
load (for example slip speed) untangle and thereby the resultant viscosity is reduced. Low-
molecule HA with a certain extent of molar masss acts in a solution as completely new
Newton´s liquid, interwining of chains do not occur.
An important function of hyaluronan in organism is hydration of tissues and water
transport due to its extreme ability to bind it in its structure.
Another function of HA (low-molecule) is regenerative, because it is a substrate for many
extracellular enzymes. So, it regulates bond of cellars among themselves, their migration,
differentiation, proliferation, mitosis and also growth of cancerous tissue and inflammatory
8. 8
processes[Ошибка! Источник ссылки не найден.,Ошибка! Источник ссылки не
найден.]. It is also essential during embryogenesis..
Some of receptors binding HA are: CD44, TSG6, GHAP, LYVE-1, RHAMM. Two of them,
CD44 and RHAMM,are not essential during cancer development[10]. These receptors have
extremely high affinity to HA.
Use of hyaluronic acid is quite extensive. It is used for example during injecting to synovial
liquid of joint, due to increase of viscoelasticity, during eye surgery, healing of injuries
(scaffolds). Scientists recently try to form from hyaluronan a medium for targeted distribution
of pharmaceuticals.
2.2. Albumin
Albumin is the most represented protein in blood plasma of mammals, of which content is
within the range 52-60 % of plasma[22], and its concentration is 0.6 mM [21]. It synthesis
takes place in parenchymal cells of livers and it goes to the bloodstream in form of
neglycosylated protein. Due to its relatively high concentration it participates in osmotic
pressure. Life cycle of albumin in blood is approx 36 days[17].
Albumin is relatively large molecule with approx molecule weight of 66 kDa. Its primary
structure is a chain containing 585 aminoacids (foralbumin of human serum, 583 for
BovineSA), secondary and tertiary structure is stabilized by means of 17 disulphidic bridges,
molecule also contains a free thiolic SH group, by means of which it is able to form dimers
with other molecules of albumin. Its 3D tertiary structure is global, which consists of 3 helical
domains. Each domain consists of two sub-domains, which represent 4 up to 6 connected
helixes[12,13]. -helical structure forms 67% of entire molecule[15].
Picture4. Structure BSA (Bovine Serum Albumin)
9. 9
Iso-electric point (pI) BSA (Bovin Serum Albumin) is 4.7. Charge of protein with
physiological pH is negative and equals to HSA - 18 and BSA - 15 [12,14,15]. Structure of
protein is naturally dependant on pH environment[12,16]. For example with pH from 4.5 up to
7 it is globular, with 4.0-4.5 it is partly unrolled (accidental ball).
Albumin in general contents high content of Cysteine and low content of Tryptophan.
Albumin from serum of bovine cattle is identical with albumin in human blood plasm from
76% [13]. Difference of BSA is mainly in additional content of 1 Tryptophan[12].
BSA binds non-specifically 3 cations Ca2+.
through 3 bonds in domain I (from 3). Although
this bond is weak, it maintains 45% of total calcium content bound in plasm to albumin. With
lower affinity albumin also bonds ions Mg2+
, be specific also 45% from the total volume[18].
Albumin is a multi-functional protein with large capacity of bond of ligands. It is capable to
bind and transport a wide range of substances, as they are different metabolites, nutrients,
pharmaceuticals, bio-macromolecules, fatty acids, amino acids, bilirubin and others. Bond of
different ligands causes change in protein conformation, more often partial unroll, loss in
content of helical structures and creation of a structure of accidental ball and in some cases
even denaturation.
Pharmaceuticals are mostly bound through IIA domain or IIIA domain[19]. IIA domain
mediates rather hydrophobic interactions, IIIA – hydrophobic, hydrogen bonds or electrostatic
interactions[20]. When interaction is hydrophobic, in most cases minimal changes in protein
conformation take place, at electrostatic interactions conformations are changed.
2.3. Interaction of molecules of hyaluronan and bovine serum albumin
It was proven that interactions between BSA and HA are mainly of electrostatic
character[23,24,25]. Hydrophobic interactions and hydrogen bridges apply in small extent.
Interactions of hyaluronan with albumin result in aggregates. Creation of aggregates is
regulated by drop of Gibbs´s energy 0G . As we know, Gibbs´s energy consists of
enthalpy and entrophy component STHG . Hydrogen bridges and hydrophobic
interactions contribute to H (reduce bond energy), electrostatic – k S (release of ions with
free entrophy;higher irregularity). At electrostatic interaction molecule structure is mainly
changed, or possibly also building-in of water molecule to macro-molecule structure and
release of free ions, so 0H and 0S , endothermal process. At side non-covalent
interactions release of bond enthalphy occurs 0H and higher system regularity 0S ,
process is exothermic. Since we consider the most electrostatic interactions, change of
entrophy is a driving force. Interactions with strongly charged polyelectrolytes are
endothermic, with lightly charged exothermic[24].
During interaction complexes or coacervates are produced. This is determined by such
factors as they are ratio of albumin concentration to hyaluronan, ion force, pH, length of
chain HA.
Three types of complexes exist[23]:
1. Insoluble complexes without charge.
2. Little soluble complexes with small charge, which can sediment during centrifuging.
3. Extremely soluble complexes with high charge.
At electrostatic interaction of albumin and hyaluronic acid a negatively charged carboxy
group HA and positively charged protonated amino groups BSA interact. In total, certain
quantity of amino groups react with carboxyl groups of one molecule HA.
It was proved that insoluble complexes with surplus of free charge up to 5% (surplus of
charge),less-soluble with surplus up to 10% and extremely soluble are with surplus of charge
more than 30%[26].
10. 10
When structure of complexes dependant on length of chain of hyaluronan is concerned, it
is assumed that:
A number of HA with very short chain (up to approx. 2400 g/mol) interact with one
molecule of albumin,
HA with medium length(15 kDa) will wind around 1 molecule BSA,
A number of albumin molecules will build in the structure of ball of native high-
molecule HA [26]
Picture 5. Structure of HA-BSA complexes dependent on length of chain
Ion force has the strongest impact on electro-static interactions[27], which are greatest
with weak, but no zero ion force[28]. Salts of low-molecule substances shadow chain of
hyaluronic acid before interactions with albumin. So, the ion force is higher, the less BSA
molecules will be bound to HA molecule, and thereby lower number of carboxyl groups will
be bound to albumin. Ion force also affects the size and charge of resultant complexes. The
Lenormanda spol.studio[23]discovered that complexes were less compact without the ion
force, with surplus of positive charge (comes from albumin). With ion force 5-80 mmol.l-1
the
complexes are neutralized and sedimenting (large coacervates). With ion force higher than
80 mmol.l-1
the complexes are negatively charged, and with 300 mmol.l-1
– large and also
negatively charged (comes from low-molecular electrolyte). We can deduct from it that the
higher ion force is, the larger complexes are and the more negative charge (due to
shadowing of chains). Size of charge and complexes is last but not least affected by molar
mass or as the case may be length of chain of hyaluronic acid and properties of used low-
molecular electrolyte.
Fundamental effect on creation of complexes between HA and BSA has also pH. It is
known that with lower pH better protonization of amino groups of protein is achieved. The
better albumin is protonized, the better it will interact with carboxyl groups HA. Optimal pH is
about 4, with respect to values of iso-electric point of albumin 2,5pI [29] and dissociation
constants of carboxyl groups of hyaluronic acid 9,2apK [2]. With higher pH than 4
protonization of albumin is worsened, and thereby interactions will not be very
effective[25].The Grymonpre a spol.group studied combination of ion force and pH [30], their
discovery was that with increase of ion force it is necessary to have lower pH for
protonization of protein. The Xu a spol.group studied effect of pH on solubility of complexes.
The result was that pH, with which precipitating of complexes occurred, was dependant on
concentration of additional albumin. The higher concentration was, the higher pH value was
necessary for phase separations.[32].
The Lenomard a spol.group determined in its research the theoretical and practical ratio of
the number of BSA molecules to HA in complexes at the phase of separation to the value
11. 11
14,3
2,0
628,0
HA
BSA
. This calculation was performed for hyaluronic acid with the molar
mass0.9 MDa and for BSAwith pH 4with the requirement of molar mass 67 kDa [23].
The ratio affects shape and naturally also charge of complexes. With low concentration
BSA and less than the value of phase separation the complexes are large due to the fact
that “accidental ball” HA starts to unwind with interactions with BSA molecules, complex
carries negative charge (surplus of free COO-
groups). With concentration BSA
corresponding with ratio of phase separation of complexes HA and BSA, complexes take
neutral charge and are not driven one from another, they agglomerate to aggregates and
sediment. With concentration corresponding with ratio higher than at phase separation
complexes have surplus of positive charge (surplus of amino groups of albumin),they are
small due to “shrinkage” of chain HA saturated with BSA molecules. Contraction of fibre
takes place due to electrostatic repelling in the complex structure, and due to the fact that
free positive charges BSAare located in the most favourable manner, be specific that “rolled
up chain“ HA remains inside the structure and free amino groups will be on the surface
(Picture6). Repelling of BSA molecules due to electrostatic repelling of positive charges
occur completely due to existence of negatively charged groups in albumin molecule, which
will partly interact with free amino groups. High-molecular HA is expected in this derivation.
[23].
Picture6. Structure of HA-BSA complexes dependant on volume of BSA
12. 12
Picture 7. Model of creation of complexes HA-BSA [32]
Theory of protein stabilization due to high temperatures is expected as useful utilization of
the mechanism of electrostatic interactions of protein-polyelectrolyte, be specific due to
increased electrostatic repelling of complexes with polyelectrolyte against complexes of pure
protein[24].
2.4. SEC
SEC – size exclusion chromatography – is the chromatographic method based on
division of particles of analyte by their sizes. First separation by size was performed by
Wheton and Bauman[31]. Separation of bio-macromolecules (proteins) was performed by
Lindqvist and Storgards, when they used starch as a charge[33].
Different stationary phases were used during development of this method (fillers of
columns– colony chromatography).We distinguish between types of columns on dextran
base, polyacrylamide and silicagel base, with ball-type and monolithic filler. The ball-type
fillers include dextransmeshed with epichlorhydrinewith the commercial name Sephadex,
which show good mechanical resistance and minimum interactions with proteins[34].
Polyacrylamide gels with commercial nameBio-Gel[35]are also very popular. In the
seventies, the porous silicagelwas predominant due to its mechanical resistance, non-
swelling properties and inert property againstanalyte under wide range of conditions.
Furthermore the Lia a spol.group prepared the first monolithic column from poly(ethylene
glycol methyl ether acrylate-co-polyethylene glycol diacrilate). Monolithic columns showed
excellent separation properties even though high range of molar masss.
Mobil phase is used as aquatic, and in this case it is gel filtration chromatography, or
organic, for which the term gel permeate chromatography is used.
The SEC principle consists in the fact that the surface of stationary phase is reach of
pores and different large notches. Thereby the stationary phase is a system of channels with
different size, through which particles of analyte in mobile phase go. The smaller particle is,
the higher number of chains it goes through, and thereby rests for a longer period (retention
time). Medium-size particles go through a smaller number of channels and thereby rest for
shorter time, large-size particles go through a minimum number of channels and pores in the
stationary phase and leave the column as first. In this manner division of analyte on the basis
of size of particles takes place (Stokes´s ratio), from which distribution of molar mass and
radius can be calculated (for example by means of calibration curve composed according to
elutions of macro-molecules with known dimensions). This effect is called “molecular
mesh“.[36]
Molecules of analyteare separated in SEC according to hydrodynamic (RH)radius, which
can be further converted through calibration curve (relative method) or with connection of
different detectors (LS - light scattering, absolute method) to molar mass.
13. 13
Such behaviour of analyte during separation is however ideal, but it does not always
occur. Undesirable interactions of analyte with stationary phase occur more or less, which
cause mainly its absorption on surface and thereby distortion of retention times. These
interactions are in most cases of electrostatic or hydrophobic character[37]. With
electrostatic interactions the same charged particles of analyte and stationary phase repel
and access of analyte to pores is limited. We can observe early elution. If they are oppositely
charged, they draw one another, and adsorption occurs, which causes longer retention time.
Hydrophobic interaction acts similarly (hydrophobic networks of stationary phase) – particle
is adsorbed. Ion interactions also cause extension and asymmetry of peaks and last but not
least 3D conformation of bio-macromolecules (they disturb secondary, tertiary and higher
molecule structures) [38].
However, these unwanted interactions can be minimized. The most usual manner is
increase of ion force with adding salt to mobile phase[38]. This mechanism can be explained
with shadowing of charged groups of stationary phase with salt ions. However, too high salt
concentration causes agglomeration of particles of analyte and effect of elimination of
ion.[39].Additive of organic substances, as for example arginine,also prevents to secondary
interactions[40Ошибка! Источник ссылки не найден.]. Another method is change of pH of
mobile phase, which mainly affects 3D conformation of bio-macromolecules and thereby
possibility of interaction with stationary phase[38].
Picture8. Structure of stationary phase and retention principle
2.4.1. Theory of elimination chromatography
2.4.1.1. Thermodynamics
Chromatographic processes can be described by the primary thermodynamic equation:
kRTSTHG ln000
(1)
Where 0
G is a difference of standard free energy, 0
H divide of enthalpy, 0
S divide of
entrophy, R is gas constant, T absolute value, k partition coefficient.
Most chromatographic separation models are controlled mainly by change in free Gibbs´s
energy due to enthalphy of adsorption. SEC is unique, because it is controlled mainly by
entropy processes, and adsorption does not occur in an ideal case, and that is why 00
H
and equation (1) is simplified and adjusted:
RSKD
0
ln (2)
14. 14
DK is athermodynamic retention factorin SEC
00
H (elimination of possibilities of interactions of analytewith stationary phase in an
ideal case)and thereby temperature should not affect retention, but it applies indirectly and in
small extent with change of conformation of molecule, viscosity of mobile phase and diffusion
of analyte.
Retention factor in SECcan be also defined as volume of pores accessible to analyte:
i
R
D
V
VV
K 0
(3)
RV is retention volume of analyte, 0V is volume around particles of stationary phase (limiting),
iV is volume inside pore.
DK can acquire values from 0, when analyte is completely eliminated from pores of
stationary phase, up to 1,when analyte has full access to pores inside the stationary phase.
When compiling calibration curve(relative method with use of standard as for example
polystyrene PS: a calibration curve will be compile with measuring polystyrene macro-
molecules with the defined molar mass) a value of molar masslog M will be entered into
graph against the retention volume VR.Resultant relation is in form of polynomial of third
degree, containing the linear sector. This sector is then used for calculation Mw (molar
mass)of samples. It shows the extent of molar masss, for which this column is suitable.
Slope of curve, which shows selectivity of column, is dependent on size of pores of column.
Complications during calculation and distortion occur firstly due to secondary interactions of
analyte at stationary phase of electrostatic and hydrophobic character, and secondly due to
the fact that bio-macromolecules differ in their shape (global, linear etc.), and therefore the
measured Stokes´s (hydrodynamic) radiuses, which is always approximated on a ball, does
not correspond with exact molar mass, because the shape of molecule sometimes more or
less differs from shape of ball.
Selectivity of stationary phase is defined by the equation:
bKmM D log (4)
The more close distribution of size of pores of the stationary phase is, the smaller is slope
m, and thereby the selectivity for extension of the range of molar masss is.
Proportion of volume of solutions inside pores and around particles SP (stationary
phase)is defined by the constant ´´k [41]:
0
´´
V
V
Kk i
D (5)
When we substitute equations(5)to equation(4), we can be sure that selectivity of column
can be increased with decreasing V0or increasing Vi. We can achieve this with higher density
of location of SP particles (stationary phase)in column and/or with use of particles with higher
total volume of pores. However, certain limits exist in this. Volume between particles of the
stationary phase cannot be so simply reduced under 35% from the total volume. Due to high
pressures pores must evince a minimum mechanic resistance (mainly against high pressures
HPLC - high performance liquid chromatography).
15. 15
There are certain optimal conditions for performance of analysis by means of SEC. Some
of them are very important for correct analysis of a sample. Concentration of salt in mobile
phase must not be very high, otherwise the risk of agglomeration of molecules is increased
and their molar mass is then increased. High share of water in mobile phase can cause on
the other side contamination by bacterias, which are capable to decompose some bio-
molecules and they are hardly removable from column[42]. Volume of sprayed sample as
opposed to other chromatographic methods should not exceed volume of column. It
predominantly amount 5-10% of volume of column[43].High volumes of spraying cause
asymmetry and extended width of peaks. Flow rate of the mobile phase should be quite low,
the reason is clear from the Van Deemter´s equitation:
mppmpmp DcudadDcudubDadH 22
where higher flow rate corresponds with increased theoretical pact, and thereby worse
resolution (length of column is constant and column is divided to a different number of
theoretical levels. The higher number of levels is, the better resolution of analytes is) [44].
Scattering of analyte occurs in HP-SEC (high performance),and thereby width of peak is
extended. The solution is increase of inner diameter of column, which suppresses scattering
significantly. In HP-SEC systems, column with diameter of 7.5 mm [45]is usually used. In
case of low-scattering systems, as UHPLC (ultra-high performance liquid chromatography),
diameter of 4.6 mm is sufficient.Length of column must be also long, optimal separation is
achieved with length of 30 cm.In order to improve separation, it is possible to connect a
number of columns in series (in tandem) one after another[46], but we must naturally
consider a longer time of analysis. Size of particles of the stationary phase must be small as
much as possible, the optimal diameter is 1-2 µm [47].
2.4.2. Detectors
UV-detectoris used mainly for proteins, which can absorb electromagnetic radiation in the
UV area due to aromatic amino acids. It can be regulated by means of wave length of
radiation. With low wave lengthsλsensitivity is increased and thereby it is possible to detect
samples with lower concentration. With higher wave lengths we achieve higher linear
range[48].
Fluorescent detectoris much more sensitive and more selective method than UV, so it is
suitable rather for detection of certain analytes in a sample, and be specific again proteins
(due to absorption).
The most important detector in SEC is a detector of light scattering (LS - light
scattering). LALS – Low Angle Light Scattering – is a detection method, when one detector
is used, located under angle 0°,and it can be changed within the range 3° - 5°. RALS –
RightAngle Light Scattering – detector is located under 90°to incident light. MALS detector
uses a higher number of detectors, located under different angles. It is the most sensitive for
analysis of aggregates with high molar mass. Just LS detectors can eliminate different
distortions of the molar mass, which are determined only by means of SEC (effects of
interactions, shape etc.).
Weight spectrometryis also used, but it is the most problematic, since dissolved salts in
mobile phase cause contamination of detector.
Viscosiometeris often linked to refractometer.By means of Mark-Houwink´s equation
can be calculated molar mass of polymer from the viscosity measured and derived intrinsic
viscosity:
16. 16
a
MK
Where is intrinsic viscosity, K is a constant, M is molar mass, ais a constant
characterizing a couple of polymer-dissolvent.
2.5. MALLS
Another detection method is MALLS – Multi Angle (Laser) Light Scattering. Incident
primary light ray on a particle scattering to different directions take place. The detector
records intensity of this dispersed light under certain angle. At static light scattering, on which
the MALLS detector is based, the time average of intensity is measured. Light as an
electromagnetic wave consists of two oscillating and perpendicular one to another fields:
electric and magnetic. When such wave hits a particle, the oscillating electric field will cause
polarization of this particle. In this manner an oscillating induced dipole is produced, in which
surroundings a periodic electric field with the same frequency as incident light is produced.
Thereby a particle becomes a source of disperse light. Rayleigh´s equationfor light
scattering with molecules of dilute gas was expressed as relation[49]:
242
0
22
0 r
F
I
i pp
(6)
pi is intensity of light dispersed by one particle under angle θ,
0I is the total intensity of primary radiation,
p ability to polarize of a particle (moment of induced dipole),
scattering angle (angle of observation),
0 permittivity of vacuum,
wave length of primary dispersed radiation (I0),
r distance of detector.
With use of relation for the ability to polarize and in muchdiluted environment (gas) the
equation 6 is adapted to relation:
wM
w
n
rN
F
I
i
A
v
2
24
2
0
4
(7)
where
w
n
is increment of fracture index.
Indiluted systemswith particles of size smaller than λ/20the equation (7) is applied,it is
only multiplied on the right side
2
0n (index of fracture of the scattering environment).
Dispersed light is very dependent on wave length, reductionλwill initiate twice increase in
intensity up to 15 times. We always take elastic light scattering into consideration, where
frequency and wave length of dispersed light are identical.
For elimination of geometric arrangement, the Rayleigh´s ratio is applied:
0
2
I
i
F
r
R v
(8)
17. 17
Where 1F at vertically polarized light, 2
cosF at horizontally polarized light and
2/cos1 2
F at non-polarized light. Vertically polarized light is mainly used in the
experiment, where .
This measured intensity converts to a unit distance from the detector and angle 0 . In
case of small particles the Rayleigh´s ration is not dependant on the angle of observation,
which however does not apply on large particles.
Should the considered system is perfectly homogenous, different volume element with ray
intensity at opposite phase applies to each volume element, and thereby dispersed waves
would cancel each other out and we would not observe any scattering to side. However, no
system is perfectly homogenous, fluctuation of density at liquids and in particular fluctuation
of concentration in scattering systems cause change in the ability to polarize volume
elements of the system. The resultant intensity of dispersed light is given by share of waves,
which are not disturbed by interference, and it is dependent on immediate value .
With rearrangement of equitation (7) with the expression of the ability to polarize through
fluctuation of concentration and virial development of osmotic pressure we will get the
Enstein-Debye´s relation:
2
2
24
2
0
2
0
...2
1
41
w
n
wA
M
rN
wFn
VI
i
A
e
v
(9)
Presence of the second virial coefficient means that the intensity of dispersed light
depends also on thermodynamic behaviour of the system. This means that concentration
fluctuations are more important with the lower local increase of Gibb´s energy they cause.
The second virial coefficient characterizes interactions or mutual effect between particle and
dissolvent. It acquires negative values, when attracting forces between particles
predominant, and thereby particles show tendency to agglomerate, then the intensity of
scattering is higher. In case of positive values they repeal, local fluctuations are minimal, the
intensity is then lower.
In case of particles of medium size (higher thanλ/20) we must count with interference
of light coming from individual points of one particle. Waves coming to the detector from one
particle are not at the same phase and resultant intensity is lower. However, attenuation is
dependent on angle of observation θ.With 0 intensity is not changed,with 180 the
intensity has the highest attenuation. Therefore the scattering factor P is applied, which
characterizes the inference attenuation of scattering on larger particles:
0R
R
P
(10)
R is the Rayleigh´s ratio
0R is the Rayleigh´s ratio with absence of attenuation
1F
18. 18
Picture9. Difference of route of rays between individual points
The picture 9shows the vector of connecting line between points P1 and P2r,axis of
symmetry of angle 180 - wave (scattering) vectorq. The picture given shows a
symmetrical case, when directions of vectorsq andr are identical. The track difference is
2222 QPPR .
Phase difference is then:
2
sin42
rl
(11)
Size of wave vector:
2
sin
4
q (12)
rq (13)
In case when connecting line of points and scattering vector have identical direction (hold
a certain angle ψ) scalar product of these vectors applies:
cos rqrq (14)
Furthermore it is necessary to express for calculation of scattering vector P difference
of phases in form of goniometric function, total up functions of waves coming from all points
of particle. Thereby we get a resultant wave coming to detector. Then resultant light intensity
is proportional to square of amplitude. We thereby get the intensity of dispersed light of
particles, which has a certain positional orientation against wave vector and which is not
constant against the thermal rotation move, and thereby we must calculate a diameter
despite all conceivable orientations. P represents the ratio of such calculated intensity to
the value pi from the Rayleigh´s equation (6)
General equation for the scattering factor,which was derived by Debye,is:
19. 19
n
j
n
i ij
ij
qr
qr
n
P
1 1
2
sin1
(15)
nis the number of point sources, by which large particle is substituted,
ijr is the distance between i-th and j-th point source.
After rearrangement of equation (15) with the use of virial development with small angle θ
or ratio
d
and also equations (11) and (13) we will get ratio for the scattering factor:
...
2
sin
3
16
1...
3
1
1 2
2
22
22
s
sqP (16)
2
s is a root mean square (rms) radius (medium quadratic radius), which characterizes
weights in a particle, can be expressed by means of relation:
n
j
n
i
ijr
n
s
1 1
2
2
2
2
1
(17)
We can deduct from these equation that the scattering factor is decreasing together with
increasing size (product ijqr ), and since
2
q is proportional
2
sin2
(wave vector), P
decreases with growth of angle of observation θfrom 0 to 180°, where it acquires its
minimum.
For experimental determination of the intensity of dispersed light through particles
lower than λ/20the Einstein-Debyeov´s equation was derived:
...2
1
2
wA
MR
wK
(18)
0RRR (19)
2
4
2
0
2
4
w
n
N
n
K
A
(20)
The constant K includes index of fracture of pure dissolvent 0n , wave length of primary
radiation and increment of index of fracture wn / .
We include into graph
R
wK
against w and with extra-polation to zero concentration we
will get straight line, from the section we calculate on axis y the value M and from direction
2A .Root mean square (rms) radius cannot be calculated for small particles.
For particles higher thanλ/20,we must calculate scattering factor in the previous
equation P
...
21 2
P
wA
PMR
wK
(21)
20. 20
PMR
wK
1
with 0w (22)
Picture 10. Zimm´s diagram
Zimm´sdiagram simultaneously allows for inclusion of extrapolation to 0 and 0w .
On axis y,
R
wK
is given and on axis xthe linear combination of independent variables,
where 'k is a voluntary constant. After extrapolation we will get two intersecting lines on the
axis of ordinates (at point
M
1
) with equations:
wk
k
A
MR
wK
'
'
2
1 2
for 0 (23)
2
sin
3
161 2
2
22
s
MMR
wK
for 0w (24)
We calculate from the tangent of first equation the second virial coefficient 2A , from the
tangent of second equation the root mean square (rms) radius s .
With using MALLS the measuring is performed with a number of angles, and achieved
data are extrapoled to zero value of angle 0 and at the same time to zero
concentration 0w . When we connect SEC chromatography and MALLS detector we can
determine values nM (numeric average), wM (weight average), zM (dimensional average),
root mean square (rms) radius s and second virial coefficient 2A .With the use of viscosimeter
we also determine the intrinsic viscosity [η].
The theory of light scattering in more detailed interpretations can be found in the used
sources: [49,50,51,55].
21. 21
2.6. DLS
DLS and/or dynamic light scattering, photon correlation spectroscopy or quasi-elastic light
scattering is based like MALLS also on light scattering with particles. Fundamental difference
compared to the static light scattering is that fluctuation of intensity of dispersed light is
measured in time instead of measuring of time average of intensity.
Picture 11Basic arrangement DLS
The Brown´s movement of particles is applied in the scattering system. Particles of
different size move with different speed. When a luminous ray falls on such particle, it will
show scattering only during time, when it will be located in the area of incident radiation.
Since a lot of particles are within the system, interference of dispersed rays falling on the
detector from different particles occurs. We will get in a particular moment of record a
particular interference picture of intensity, which is changed in time (in order
7
10
up to s10 9
),since dispersing particles move due to thermal move of particles. DLS compares these
interference records, how much they correlate one among another. In the time intervalΔt = 0,
this correlation equals to 1 (intensity is the same), but with increasing time interval
Δtcorrelation is decreased up to zero, when the interference pictures are completely different.
Decrease in correlation is given apart from the higher time interval by increasing size of
particles. Thereby the correlation determines, what size the particle has and how rapidly it
moves within the system, by means of the diffusion coefficient calculated from the Enstein –
Stokes´s equation:
H
B
R
Tk
D
6
(25)
Where Bk is the Boltzman´s constant, T is absolute temperature, is dynamic viscosity of
scattering environment, HR is the hydrodynamic radius.
This equation can be used only for particles of spherical (ball) shape. If it is not the case,
particles are extrapoled to ball shape. Hydrodynamicradius HR is the radius of ball particle
also with solvation pack, which shows the same diffusion coefficient as measured particle.
The larger the particle is, the lower diffusion coefficient it has and moves more slowly.
22. 22
Distribution of sizes of particles is calculated from the auto-correlation function (ACF):
ii
N
i
ii tItItItI
N
tG
1
)2( 1
(26)
Where index )2(
indicates that radiation intensity is concerned, but not the intensity of electric
field, is time interval between records.
Picture 12. Auto-correlation function
Since is selected much shorter than the fluctuation time, we can write:
tItItI ii
2
0
lim
(27)
For very long intervals, when correlation is zero:
2
lim tItItI ii
(28)
So ii tItI it should decrease within the range of values from tI2
to 2
tI
The auto-correlation function ACF is independent on initial time, so we can write:
IItItI ii 0 (29)
Then the normalized ACF is:
2
)2(
0
I
II
g
(30)
We can write it through the auto-correlation function ACF of electric field of radiation:
23. 23
2)1()2(
1 gg (31)
Where
2
)1(
0
E
EE
g
(32)
is an instrumental constant, characterizing the number of coherence areas recorded by the
detector, they must be targeted to the value 1 (the more coherence areas are recorded, the
better overlap of these areas is and the weaker signal is).
It is valid for mono-scattering systems:
t
eg
)1(
(33)
2
1
Dq
(34)
Whereqis the Bragg´s wave and/or scattering vector:
2
sin
4 0
n
q (35)
Where 0n is the index of fracture of the scattering environment, is the length of primary light
in vacuum, is the angle of observation (Picture 11).
We can calculate from equations(31),(33),(34),(35) the diffusion coefficient Df, and from it
according to equitation (25) the hydrodynamic radiusRH.
In case of heteroscattering systemsan integral calculation is performed:
t
eAg
0
)1(
Where A bears information on distribution of sizes of particles.
24. 24
Picture 13. Distribution of particles´sizes. Output from DLS
Most of up-to-date apparatuses perform measuring with one fixed angle, and with laser
ray with the wave length 675 nmas a source of primary light. The unwanted effect of multiple
ray scattering must be eliminated during measuring, and that is why the small as possible
coherence area is chosen (scattering to 1 particle without interference is an ideal case),
sufficiently low concentration of sample, the small as possible optical track, but it must be
also prevented to fall of primary ray on the detector (90°). As requirements for a sample are
concerned, it must be prevented against contamination by particles of dust, which due to
extreme sensitivity of apparatuses they would disturb signal, and that is why the sample must
be filtrated. Adequate ion force and adequate temperature (constant and exactly defined)
must be used, since viscosity of the environment and also thermal move of particles and/or
diffusion coefficient, are extremely dependant on it).
A photo-multiplier is used as a signal detector, which together with correlator, or possibly
attenuator and amplifier of rays transforms the light intensity to electric intensity, which is
further computer-processed.
25. 25
Picture No. 14. Instrumentation DLS
More detailed explanation of theory can be found in the references used[52 - 58].
EXPERIMENTAL PART
3.1. Materials
Sodium hyaluronate was used for preparation of a sample, supplied by the Contipro
Biotech s. r. o company (Dolní Dobrouč, the Czech Republic) within the range of molar
masss 90-130 kDa, 110-130 kDa and 130-150 kDa, and 1500-1750 kDa.All used samples of
hyaluronan were marked as Technical Grade. Bovine serum albumin (BSA) was supplied by
the Sigma-Aldrich company in form of lyophilized powder free from essential fatty acids and
purity˃98%.From other chemicals, sodium nitrate (NaNO3) (Sigma-Aldrich) and sodium
hydroxide (Sigma-Aldrich) were used. Citric acid monohydrate with the content of 99.8%
(PENTA) and hydrogen phosphate disulphate with the content of 99.0% (PENTA) are used
for preparation of buffer. Commercially supplied buffers with pH 2; 4,01; 7 (Mettler Toledo)
were used for calibration of pH meter. All solutions and spare solutions were prepared in
deionized water Milli-Q, prepared by means of the Purelab Option ELGA instrument.
3.2. Methods
3.2.1. Preparation of samples
Firstly spare solutions of sodium hyaluronate and bovine serum albumin were prepared in
Milli-Q water. Supply of Hyaluronan was firstly dried at the drying plant under 90°C for the
maximal time of 30 minutes. Exact quantities of hyaluronan and albumin were gradually
mixed with water under constant mixing on the magnetic mixing machine. Spare solutions
26. 26
prepared in this manner were mixed for the minimum time of 24 hours under the laboratory
temperature. Solutions of hyaluronan and albumin were prepared from these reserve
samples according to the table given below (see the Table 1). One set contained 8 samples
with the constant concentration of hyaluronan and variable concentration of albumin. In total,
12 series of samples were prepared, which differed one from another with the environment
and molar mass. Samples prepared in this manner were mixed at the magnetic mixing plant
for a minimum period of 12 hours. After this time NaNO3was added and its resultant
concentration in a sample was 0.1 M. Then samples were again mixed approximately 3 h.
Sodium hydroxide with the concentration 1.5 m was used for neutralization of the set of
samples prepared in buffer with pH 4to the resultant pH 7. Addition to each sample
amounted to approximately 550 µl.Approximately 2 ml were taken from the samples and
measured by SEC-MALL before neutralization.
Before neutralization of the last two sets of samples, pH-meterwas calibrated by means of
buffers with known pH values (pH 2; 4,01 and 7). Neutralization was performed in the
following manner:value of pH sample was firstly measured and sodium hydroxide was
gradually added up to reaching the required value of samples pH 7.
Preparation of buffer with pH 4 and 7 was performed in Milli-Q water. Firstly solutions of
citric acid with concentration of 0.05 M and hydrogen phosphate disulphate with the
concentration 0.1 M were prepared. After calibration of pH-meter the pH value of solution of
citric acid (hydrogen phosphate disulphate) was measured, and then hydrogen phosphate
disulphate (citric acid) was added under constant mixing up to reaching the required value of
pH buffer. Then the resultant buffer was mixed for half an hour to stabilize pH, and control
measuring of pH value was performed. In case of buffer with pH 4 the hydrogen phosphate
disulphate was added dose by dose to citric acid. In case of pH 7 it was the opposite
manner.
More detailed composition of resultant solutions is given in the table below (table 1)
Abbreviations will be used for convenience further in the text:
NMHyafor low-molecular hyaluronic acid
VMHyafor high-molecular hyaluronic acid
Table 1. Preparation of solutions
Set NM/VMHya Environment c(Hya) [g/L] c(BSA) [g/L] c(NaNO3) [mol/L] VTOTAL [ml]
1
NMHya
Milli-Q
5
0
0,25
0,5
1
2
5
10
0 7
2 Milli-Q 0,1 7
3 Pufr pH 4 0 7
4 Pufr pH 4 0,1 7
5 Pufr pH 7 0,1 7
6 Pufr pH 4* 0,1 12
7
VMHya
Milli-Q
1
0 7
8 Milli-Q 0,1 7
9 Pufr pH 4 0 7
10 Pufr pH 4 0,1 7
11 Pufr pH 7 0,1 7
12 Pufr pH 4* 0,1 12
*Samples marked with asterisk were then neutralized to the resultant pH 7
27. 27
3.2.2. DLS measuring of samples
DLS measuring was performed by means of the Malvern Zetasizer Nano ZS apparatuses
with the use of disposable plastic cuvettes for measuring of size of particles and submersible
cells zetasizer nanoseries Malvern for measuring of zeta potential.
Measuring of pH buffers and samples was performed by means of the SevenMulti Metler
toledo apparatus with the use of Mettler Toledo InLab Routine Pro pH 0-14 electrode.
Measuring of size of samples was performed under angle 173° and under the temperature
25°C. He-Ne laser with the wave length 632.8 nm was used as a light source. Time for
tempering of samples was set for 60 seconds. Setting was performed for the protein material
with the refractive index RI 1.450 and absorption A 0.001. Dissolvent was set as water with
viscosity 0.8872 cP, refractive index 1.330. Viscosity of dissolvent was used as a viscosity of
sample. Number of measuring was set for 3, duration of one measuring was selected as
automatic. Multi-parameter analysis was selected for analysis of aggregation point and
automatic searching for optimal measuring position was set. One measuring was set for
measuring of zeta potential, containing in itself from 10 up to 100 repeating. Detector at
measuring of zeta potential was located at direct direction under angle 17°.
All samples of one set were tempered to the laboratory temperature before measuring of
dynamic light scattering, and they were also filtrated through a filter with size of 0.45 µm into
plastic cuvettes with the optical length of 1 cm. Samples were left to stabilize for approx 30
minutes to stabilize possible structure of analytes after filtration. After completion of
DLSmeasuring of all eight samples in one set, their zeta potential was measured. Then
measuring DLS and zeta potential was once more repeated: new samples were filtrated into
new cuvettes with the same size of pores, and the second measuring was performed under
the same conditions.
3.2.3. SEC-MALLS measuring of samples
The SEC-MALLS measuring was performed by the SEC-MALS (GPC-MALS) system.
Used detectors are: MALSmultiple-angle detector (17 angles) for the statistic light scattering,
18th
detector under angle 90° for dynamic light scattering; VISKOSTAR detector to determine
viscosity; difference refractometer using for determination of concentrations. The used
mobile phase was 0.1M solution NaNO3. Increment of fraction index was set for 0.165 for
Hya and mixtures Hya and BSA and 0.185 for pure BSA. Volume of spraying was defined for
50 µL. Flow rate was used 0.4 ml/min. Measuring of one sample run approx 85 minutes.The
used separation column was PL aquagel-OH MIXED 8 μm. It is a hydrogel on porous silica
base. Analytical standard for GPC Dextran 410 kDa and 150 kDa were used as standard for
molar mass.
Samples were always as in case of DLS measuring filtrated through the filter
0.45 µm.Sampling was performed by an automatic dozer (autosampler).
RESULTS AND DISCUSSION ABOUT DLS
4.1. DLS of low-molecular set HA in Milli-Q and 0.1 M NaNO3
Owing to relatively high number of data, only certain part of results will be included into
the final result and evaluated.
Set of low-molecule HA was selected for evaluation,prepared in water with addition of
NaNO3.
It is necessary to note that deviations occurred for individual measuring, be specific for all
sets of samples during DLS measuring. It was caused with high probability due to high poly-
dispersity of samples, it was observed on the quality of correlation curves at most. Therefore
28. 28
we cannot consider resultant measuring values and the measuring itself as accurate. The
third peak is neglectable with respect to its low representation, secondly due to absence of
any trend.
Legend:
Velikost = size
Z průměr = Z diameter
Graph1. Dependence of Z-diameter of size of particles on concentration of added BSA in
samples Hya with addition of BSA with different concentrations(NMHya in Milli-Q and 0.1 M
NaNO3)
It is evident from Legend:
Velikost = size
Z průměr = Z diameter
Graph1increase of medium size particles within the range of concentration BSA 0-1 g/L,
which can be caused due to the fact that elimination of ions of sodium nitrate through
molecule BSA from HA molecule takes place within this range. Competition to bind or as the
case may be replacement of charged BSA molecules with soda ions takes place here.
Thereby HA becomes highly charged and therefore it expands. Zeta potential data confirm
that HA charge is decreased, i.e. decreased after addition of BSA. With concentration BSA
1 g/L particles acquire their maximum size 20.4 nm,the most expanded particles of
hyaluronic acid or complexes occur in solution, be specific it is caused by certain ratio of
charges and with it associated electrostatic repelling. Particles of complexes are significantly
smaller commencing from BSA 2 g/Lconcentration and above. A certain proportion of bound
BSA molecules to free carboxy groups at Hya are with probability achieved, when due to
effect of BSA the complexes reduce. Size of BSA particles amount to 9 nm.According to data
division to 2 peaks we can claim that particles are divided into two populations.
29. 29
Graph2. Dependence of particles size and percentage of area of 1st
peak on concentration of
added BSA in Hya samples with addition of BSA with different concentrations(NMHya in Milli-
Q and 0.1 M NaNO3)
Legend:
Velikost = size
Velikost 1. pík = Size of 1st
peak
Velikost BSA – Size of BSA
1. Pík = 1st
peak
It is evident from Graph2that size of smaller particles goes down from 15.3 up to 9.5 nm.
This is caused, because they do not have much carboxyl groups on their surface, charged
and available for BSA, and thereby high ion shading, so small concentration of BSA is
sufficient firstly for elimination of existing ion force and secondly for interaction with all
available groups, thereby “shrinkage” of particles takes place immediately. Area of 1st
peak in
percentage goes down to concentration BSA 1 g/L from83.6 % to 52 %,be specific with high
probability due to transfer of some particle to the second larger population. However, despite
of drop in area of peak 1, the area is still above 50%, which confirms that small-size particles
are still the most represented. Then percentage of area starts to increase from concentration
BSA 2 g/L up to exceeding the limit ratio of concentration BSA toHA, probably due to
“shrinkage” of larger particles of second population and their transformation to the first
smaller one. With the concentration BSA 10 g/L the area percentage amounts to 75%.Size of
pure BSA is 8 nm. Area in percentage of 1st
peak BSA is 82%.So BSA in the solution of
samples predominates in form of smaller particles.
30. 30
Graph3. Dependence of size of particles and area percentage of 2nd
peak on concentration
of added BSA in samples Hya with addition of BSA with different concentrations(NMHya
in Milli-Q and 0.1 M NaNO3)
It is evident from Graph3that size of particles of second populations is increased after
adding of BSA with concentration 0.25 g/L from the value 550 nm to 968 nm, probably due to
expansion of charged particles. With increase of BSA concentration up to 2 g/L the size of
particles decreases, probably due to “shrinkage” of complexes with BSA molecules.The
value of size of particles with concentration BSA 5 g/L it is more likely distant. Area
percentage of 2nd
peak is substantially lower than in case of smaller particles of first
population. It increases to the value up to 45% with lower concentrations of BSA (1 g/L),
where they have with probability the greatest importance. However they are approximately
on the value 25% from the concentration BSA 2g/L. Size of pure BSA is 279 nm, which
proves the fact that BSA also forms two populations of particles, but obviously with higher
concentrations. Area of 2nd
peak of pure BSA is only 18 %, which proves the fact that larger
aggregates BSA are present in solution, but with significantly smaller representation.
31. 31
Graph4. Dependence of zeta potential on BSA concentration for different sets of HA samples
with addition of BSA with different concentrations in different environments (NMHyA)
It is evident from the Graph4that the set prepared in Milli-Q water has irregular and the
lowest zeta potential, due to negatively charged carboxyl groups and with probability also
due to absence of any shading ion force. Other sets almost do not differ in the zeta potential
and they are slightly decreasing with growth in BSA concentration. Almost constant value of
zeta potential is with probability caused due to effect of high ion force, which overall shadow
the HA molecule against BSA molecules.I suppose that ion force has also effect on slight
decrease of zeta potentials of complexes. The lowest zeta potential between pure BSA has
the BSA particle prepared in water (- 21 mV), the highest is at buffer with pH 4 with addition
of NaNO3 (0,22 mV).It is logical due to value of pH of solution 7 and iso-electric point BSA
around 5.
32. 32
4.2. DLS sets of high-molecule HAinMilli-Q and 0.1 M NaNO3
Graph5. Dependence of Z-diameter of size of particles on concentration of added BSA in
samples Hya with addition of BSA with different concentrations(VMHya in Milli-Q and 0.1 M
NaNO3)
Legend:
Velikost částic = Size of particles
Z průměr = Z diameter
It is evident from Graph5that the medium size is only going down as opposite to the set
with low-molecular HA. This drop is eliminated above concentration BSA approx. 2 g/L, when
the size of particles does not change significantly. The high-molecule HA also with presence
of ions has sufficient number of available carboxyl groups for BSA. Therefore displacement
of shading ions with BSA molecule from shadowed carboxyl groups HA and interactions of
free COO groups with other BSA molecules occur simultaneously. Bound BSA molecules try
to get to the most convenient position, be specific by means of interactions with other bound
BSA, so they probably “pack” HA chain into a more compact shape, a particle is then
reduced. Interactions with other BSA molecules with higher concentrations of HA much more
"shrink". Medium size of BSA is 10 nm.
33. 33
Graph6. Dependence of the size of particles and area percentage of 1st peak on
concentration of added BSA in samples Hya with addition of BSA weight different
concentrations (VMHya in Milli-Q with 0.1 M NaNO3)
It is evident from the Graph6that size of particles of the 1st
peak is descending with it that
it rapidly drops from the value 17.8 to 11.2 nm immediately with the minimum concentration
BSA 0.25 g/L. Then the drop is small from 11.2 to 9.3 nm. According to growing area of 1st
peak it can be said that together with increasing concentration of BSA that particles of 1st
population predominant with high ratios of concentrations of BSA to HA. As opposite to the
set with low-molecular HA it is evident that representation of population of smaller particles is
low at the beginning and it grows with growing concentration of BSA. Size of BSA is 8.5 nm.
Area percentage of 1st
peak of pure BSA is 78.2 %.
Graph7. Dependence of size of particles and area percentage of 2nd
peak on concentration
of added BSA in samples Hya with addition of BSA with different concentrations (VMHya in
Milli-Q and 0.1 M NaNO3)
34. 34
Legend:
Velikost = Size
Velikost 2. pík = size of 2nd
peak
It is evident from the Graph7that size of particles of the 2nd
peak is increasing up to
concentration BSA 5 g/L, be specific from 733 nm up to 2262 nm. Growth in size of particles
of the 2nd
peak is with probability caused due to flocculation of complexes as a consequence
of decrease of their total surface charge with the growing concentration of BSA. A similar
situation like was described for particles of the 2nd
peak of the low-molecular HA could
probably concern. With the concentration BSA 10 g/L the size decreases to the value
1181 nm, be specific most likely due to conformation change of the chain of Hya molecule,
which will probably pack. The HA molecule has with probability minimum of free carboxyl
groups at this point. Area percentage of the 2nd
peak show a constant drop within the range
of values from 40% with pure HA up to 11.2 % with concentration BSA 10 g/L. So,
representation of particles of the 2nd
population of larger particles as opposite to set of
NMHya only decrease. Size of pure BSA is 361 nm. Area of peak BSA is 27%.
Graph8. Dependence of zeta potential on concentration of BSA for different sets of HA
samples with addition of BSA with different concentrations in various environments (VMHya)
Legend:
Zeta potenciál = Zeta potential
It is evident from the Graph8that the lowest zeta potential is for the set prepared only in
pure water, be specific due to understandable reasons, negatively charged groups HA does
not shadow proteins. As opposite to the set with low-molecular HA it has quite regular course
and it grows together with increasing concentration of BSA, which would mean that after
binding BSA the total surface (negative) charge of particles is decreasing. This increase of
zeta potential immediately with the lowest concentrations BSA, as opposite to the set
NMHya, confirms presence of non-shadowed carboxyl groups HA, of which number is
evidently decreasing after addition of BSA.
Other sets with different ion force differ with respect to zeta potential only slightly and
together with growing concentration of BSA its value almost does not change in framework of
35. 35
individual sets. It is probably caused with it that added anti-ions stabilize HA conformation in
environments with different ion force. Relatively high values of zeta potentials compared to a
set in clean water are with probability caused due to different ion force, which will remove
shadowing of the charge of Hya molecule.
The lowest zeta potential for pure BSA has again BSA prepared in water in the value
mV9,3 . Clean BSA prepared at buffle with pH 4 and with addition of NaNO3, has the
highest zeta potential, of which value isje mV2,2 .
RESULTS AND DISCUSSION ABOUT SEC-MALLS
5.1. SEC-MALLS sets of low-molecular HA inMilli-Q and 0.1 M NaNO3
Owing to relatively high number of data, the NMHyA set, prepared in 0.1 M of solution
NaNO3was prepared for evaluation.
Furthermore an illustrative chromatography of samples (Graph9) is mentioned. The
second peak was deemed insignificant in most measuring. Only samples with higher
concentration BSA 5-10 g/L had the most significant peak, which is evident from the
chromatography. The second peak for these concentrations represents in the larger part
discharged BSA, which did not take place in interactions with Hya.
Graph9. Chromatography. Dependence Mwon the retention time with effect of concentration
BSA for Hya samples with added BSA with different concentrations (NMHya in Milli-Q and
0.1 M NaNO3)
Results of the output show presence of more peaks at some sets. Only the first most
significant peak was included into evaluation. Significance was assessed from height and
width of peak provided from LS (light scattering) of signal. It is evident from the
36. 36
chromatography given that particles get off column between 15-20 minutes. The highest
concentration signal is for clean BSA, which also has the narrowest peak. Furthermore
height of concentration signal drops together with decrease in BSA concentration in samples.
In general, the molar mass drops alongside individual peaks, as it is evident from the
Graph 10.
Graph10. Mw, [η] complexes dependent on concentration BSA for Hya samples with added
BSA with different concentrations(NMHya in Milli-Q and 0.1 M NaNO3)
It is evident from the Graph10that Mwgrowths up to concentration BSA approx1 g/Lup to
the value of 101.3 up to 113.5 kDa. With higher concentrations of BSA the molar mass drops
up to the value of 74.7 kDa. Decrease in molar mass of complexes with growing
concentration of BSA can be caused with setting of increments of fracture index, when it is
set in the apparatus´s setting as constant. In our case, the increment of fracture index was
selected both for the clean HA or clean BSA. Since interactions occur for complexes, their
entire increment of fracture index is changed. Indirect dependence of increment of fracture
index on the molar mass can be derived from the equation (21). So, the higher is increment,
the lower is molar mass. Concentration can also participate in it in the same manner, by
which constant on the left side of equation containing increment is multiplied. This is also set
as constant. So, decrease of molar mass for particles of low-molecular HA in water with
addition of NaNO3most likely means increase of increment of fracture index. It is quite logic,
since the set increment of fracture index BSA in the value of 0.185 is higher than the
increment HA 0.165.The intrinsic viscosity shows a constant drop within the range of values
from 300 to 88.8 mL/g, which confirms that interactions occur, and resultant complexes
become more compact with the growing concentration of BSA.Molar mass of pure BSA is
69.7 kDa and it corresponds with its theoretical molar mass. The molar mass BSA at the
second peak is extremely high (1300 kDa), which could be probably caused by impurities.
Intrinsic viscosityBSA is 3.117 mL/g, which is much higher value than for HA-BSA
complexes.
37. 37
Graph11. Rw and RHQ complexes dependent on concentration of BSA for Hya samples with
added BSA with different concentrations (NMHya in Milli-Q and 0.1 M NaNO3)
It is evident from the Graph11that the gyration ratio Rwalmost does not change within the
range of concentrationsBSA 0 up to 2 g/L, and it equals to the value of approx 30 nm. It
decrease from the concentration BSA 5 g/L up to the value of 16.7 nm with the concentration
BSA 10 g/L. It is evident from the equation 24 that the gyration ratio is directly proportional to
the constant on the left side of equation. So, change of increment of fraction index in the
constant should affect also the root mean square (rms) radius. Decrease of the root mean
square (rms) radius and intrinsic viscosity(see the Graph10) of particles most likely means
that interactions between molecules HA and BSA occur and thereby “shrinkage” of creating
complexes.
Hydrodynamic radius has the same course as the root mean square (rms) radius, but it is
lower than the root mean square (rms) radius, which is from the view of theory of solutions of
polymers quite logical (see the equation 36):
2
1
2
68,0 sRH (36)
Particles are not compact, but quite extensive within distribution of their weight. With
increasing concentration of BSA both two radiuses drop by reason of creation of more
compact complexes HA and BSA.
The gyration ratio of pure BSA is zero, the hydrodynamic radius has the value of 4 nm and
it is higher than the gyration ratio. This could mean that they are compact particles, more
likely in the shape of a ball with a strong solvation package
38. 38
5.2. SEC-MALLS of the set of high-molecular HA in0.1 M of solution ofNaNO3
In addition to the set NMHya the set VMHya in0.1 M of solution NaNO3was selected for
evaluation. Furthermore the indicative chromatography is given.
Graph12. Chromatography. Dependence of molecular weigh on the retention time with effect
of concentration of BSA for Hya samples with added BSA with different concentrations
(VMHyain Milli-Q and 0.1 MNaNO3)
The main difference between the high-molecular and low-molecular Hya is evident from
the chromatographyGraph12. It begins to exclude from the column a little bit sooner between
12-20 minutes. Drop in monitored values is evident (molar masss, intrinsic viscosity, gyration
and hydrodynamic radius) in framework of individual peaks. Individual peaks are more
separated one from another than in case of low-molecular HA.It is also evident that together
with growing concentration of BSA peaks are extended, which with high probability shows
creation of complexes with extensive distribution of size of particles. It can be deduced from
the chromatography that in case of VMHya more significant division of particles into two
populations occurred than in case of NMHya. Firstly free BSA was with probability released
at the second peak, which did not take place in interactions, and also the smallest
complexes.
39. 39
Graph13Dependence Mw and [η] on BSA concentration for Hya samples with added BSA
with different concentrations(VMHya in Milli-Q and 0.1 M NaNO3)
Legend:
Koncentrace BSA = BSA concentration
It is evident from the Graph13a constant drop Mwfrom 1107.6 kDa for pure HA up to
673.6 kDa with the concentration BSA 10 g/L. The drop is more significant and the total value
is naturally higher than for the low-molecular HA. Drop of the molar mass of complexes with
growing concentration BSA has probably the same reason as for the low-molecular HA
(growing increment of the fracture index). The intrinsic viscosity goes down from 1565 mL/g
with pure HA up to 642 mL/g,with concentration BSA 10 g/L it is much higher than for the set
of low-molecular HA. The intrinsic viscosity of complexes goes down by reason of creation of
more compact particles. The molar massBSA is 98 kDa, which is probably an incorrect value
owing to the theoretical value 67 kDa.The measured intrinsic viscosity of pure BSA was
3.2 mL/g.
40. 40
Graph14. DependenceRw and RH(Q) on concentration BSA for Hya samples with added BSA
with different concentrations(VMHyA in Milli-Q and 0.1 M NaNO3)
Legend:
Koncentrace = concentration
It is evident from the Graph14that the root mean square (rms) radius sheerly goes down
from 124 nm for pure HA up to 67.3 nm with the concentration BSA 2 g/L. With higher
concentrations it goes down only slightly, with the concentration BSA 10 g/L it is 53.6 nm.
The root mean square (rms) radius of complexes decreases by reason of creation of more
compact particles, as well as for the set with low-molecular HA, but it has by understandable
reasons higher values.
Compared to the low-molecular HA the gyration and hydrodynamic radius of the set
VMHya are higher. It is also evident that the root mean square (rms) radius is higher than the
hydrodynamic, which is in compliance with the theory of solutions of polymers, and it means
that they are particles with high weight distribution. The root mean square (rms) radiusBSA is
higher at the value 12.6 nm than the hydrodynamic radius 5.7 nm. This difference from the
set with NMHya (together with incorrect value of the molar mass98 kDa) was probably
caused due to error during preparation of solution or during measuring itself.
5.3. Comparison of the sets of low-molecular HA
Particles of complexes HA-BSA manifest themselves in different environments with
irregular course of the molar mass, gyration and hydrodynamic radius within the range of
concentrations BSA 0 up to approx 2 g/L. It is probably caused by different types and sizes of
ion forces and their effect on the ability of BSA to integrate with HA.
41. 41
Graph15. DependenceMw on concentration BSA for samples Hya with added BSA with
different concentrations(different environments, NMHya)
It is evident on
42. 42
Graph15
Graph15. DependenceMw on concentration BSA for samples Hya with added BSA with
different concentrations(different environments, NMHya)that molar mass drops with
increasing concentration of BSA in different environments. Sets differ the most one from
another and they also have irregular course within the range of concentrations BSA 0-2 g/L.
The highest molar mass is for the set prepared in buffer with pH 4 with addition of NaNO3.
The lowest molar mass is for the set prepared in buffer withpH 7 with addition of NaNO3with
probability due to the highest value of increment of fracture index, Which could be caused by
high density of charge. In this case, difference from sets prepared in pure water, where pH is
also neutral, is with probability caused by higher shadowing of ions of molecules HA in buffer
with pH 7.Drop of the molar mass of the set in Milli-Q water is the most regular. It can be
probably explained with absence of any ion force. Non-linearities at other sets are more or
less associated with the process of displacement of shadowing ions from HA molecule with
BSA molecules, which mostly occur within the range of concentrations BSA 0 up to approx
2 g/L. BSA shows at all sets roughly the same molar mass, which corresponds with
theoretical value.
43. 43
Graph16. Dependence of Rwon BSA concentration for Hya samples with added BSA with
different concentrations(different environments, NMHya)
It is evident on the Graph16that the root mean square (rms) radius drops in every
environment with growing concentration of BSA. The highest range of the root mean square
(rms) radius is for the set prepared in buffer with pH 7 with addition of NaNO3, which is
probably associated with high charge of carboxyl groups HA, which repel one another
electrostatically. Therefore expanded chains predominant under low concentrations of BSA,
complexes “shrink” under high concentrations of HA due to interactions and ion force. The
smallest root mean square (rms) radius is for the set prepared in pure water, be specific with
probability due to absence of any ion force. The only one non-zero Rwis for pure BSA
prepared in buffer with pH 7 with addition of NaNO3, be specific by reason of change of
conformation of BSA molecule with this pH and ion force. BSA has zero root mean square
(rms) radius at other sets, which proves the fact that BSA molecule is very compact in other
environments.
44. 44
Graph17. RHQ in dependence on concentration of BSA for Hya samples with added BSA
with different concentrations (different environments, NMHya)
It is evident from the Graph17that the hydrodynamic radius drops with growing
concentration of BSA in every environment. The hydrodynamic radius is smaller than the root
mean square (rms) radius at all sets, which proves the fact that particles of all sets have
extended shape. The highest range of the hydrodynamic radius is for the set prepared in
buffer with pH 7 with addition of NaNO3.. It could be explained by high charge of more
dissociated HA, like for the root mean square (rms) radius. The smallest one is in pure water,
which is naturally by reason of absence of any ion force. The hydrodynamic radius of pure
BSA is roughly the same at all sets. In addition to the set of buffer with pH 7, it is also higher
than root mean square (rms) radius. It proves the fact that BSA molecule is compact and
highly solvated. The pure BSA has smaller hydrodynamic radius than the root mean square
(rms) radius at the set in buffer with pH 7. This could be caused by disruption of BSA
structure with this pH and ion force.
45. 45
Graph18. Dependence of intrinsic viscosity [η] on concentration BSAfor Hya samples with
addition of BSA with different concentrations (different environments, NMHya)
It is evident from the Graph18that intrinsic viscosity drops with growing concentration of
BSA in every environment. The highest intrinsic viscosity is for a set prepared in buffer with
pH 4 with addition of NaNO3, be specific with probability due to the fact that it has the biggest
particles (even though the most significant interactions should occur with this pH).
Adequately big ion force of buffer contribute with probability to it. With higher concentrations
BSA is the smallest in buffer with pH 7 with addition of NaNO3, which with probability
confirms compactness of particles with higher concentrations of BSA in this environment.
Addition of NaNO3in water causes increase of ion force and thereby shading of HA chain and
enlargement of particles of complexes, which causes a little bit bigger viscosity of set in
water with addition of NaNO3compared to the set only in water. Molecules of pure BSA have
low intrinsic viscosity in all environments. We can claim together with the previous
derivations that BSA are very compact particles bearing a big solvating package.
A signal from the viscosity detector is missing for some samples, and thereby their data
were not included to graph 18 and to evaluation (for the set in buffer with pH 4 with 0.1 M
NaNO3 with the concentration BSA 0.25 g/L, and for the setin Milli-Q water with the
concentration BSA 2 g/L)
46. 46
5.4. Comparison of sets of high-molecular HA
The second peak was mostly important only for higher concentrations of BSA, be specific
in all environments, it was with probability in predominant part freely released BSA.
Graph19. Dependence of Mwon the concentration BSA for Hya samples with added BSA with
different concentrations(different environments, VMHya)
It is evident on the Graph19that the course of dependence of the molar mass on
concentration of BSA is more regular at sets of high-molecular HA compared to sets of low-
molecular HA. It can be due to lower shading of carboxyl groups HA with ion force, with
which displacement of ions by BSA molecules applies in smaller extent, and thereby more
significant interactions of HA with BSA should occur. Due to the presence of free carboxyl
groups HA also with presence of ion force, binding of some BSA molecules and
displacement of a part of shading ions with probability occur. These opposing processes
have with probability compensation effect. The highest molar mass is for the set prepared in
buffer with pH 7 with addition of NaNO3,this set has on the contrary the smallestMwin the low-
47. 47
molecular HA (see the
Graph15). It is with probability associated with lower increment of the fracture index and
smaller charge of complexes. Smaller charge of complexes can be initiated with more
significant interference shielding of the total charge of HA through interactions with BSA. For
the low-molecular HA, it does not occur with probability due to larger shading by ion force,
and with lower concentrations of BSA displacement of ions from HA occurs rather than
interactions with BSA. The lowest values of molar mass are for the set prepared in pure
water, be specific with probability due to the biggest increment of fracture index, which is
associated with more extensive interactions. Compared to NMHya, where the set in pure
water does not have the smallest molar mass, it is with probability caused by different ratio of
carboxyl groups per one molecule VMHya, and also by different conformation of HA and
thereby also dissociation of carboxyles, which has with probability impact on the range of
interactions between HA and BSA and thereby the values of molar masss of complexes. The
highest molar mass is for pure BSA prepared in Milli-Q water with addition of NaNO3, the
lowest molar mass is in buffer with pH 4 with addition of NaNO3.Even though they slightly
differ one from another, they run within the range of theoretical values.
48. 48
Graph20. Dependence of Rwon the concentration BSA for Hya samples with added BSA with
different concentrations(different environments, VMHya)
It is evident on the Graph20that the highest values of root mean square (rms) radius are
for the set prepared in buffer with pH 7 with addition of NaNO3, the smallest values are for
the set prepared in pure water. The root mean square (rms) radius remains within the range
of concentrations BSA 0.5 up to 10 g/L almost constant for the set in buffer with pH 7 as
opposite to the same set with low-molecular HA. Such process could be explained with the
fact that interactions with pH 7 are the weakest and manifest themselves in concentration
BSA approx 1 g/L.
The only one non-zero root mean square (rms) radius is for pure BSA prepared in Milli-Q
water with addition of NaNO3, be specific with the value 12.6 nm.
Graph21. Dependence of RHQ on concentration of BSA for Hya samples with added BSA
with different concentrations(different environments, VMHya)
49. 49
It is evident on the Graph21that up to concentration BSA approx2 g/La set prepared in
Milli-Qwater with addition of NaNO3has the highest value of hydrodynamic ratio. However
from concentration BSA approx5 g/L, it is the highest for the set in buffer with pH 7with
addition of NaNO3. The lowest values of hydrodynamic radius are for the set prepared in
Milli-Qwater, be specific again by reason of absence of ion force. Generally, the
hydrodynamic radius is lower than the root mean square (rms) radius, and the difference is
more significant compared to the set with low-molecular HA. This with probability proves the
fact that complexes are more extended with non-arranged distribution of weight. At the
course of dependence of size on concentration of BSA the hydrodynamic radiuses
completely correspond with the trend of root mean square (rms) radiuses. Both radiuses drop
with increasing concentration of BSA, which confirms the same mechanism of creation of
complexes, both VMHya and NMHya. Pure BSA has almost the same hydrodynamic radius,
approximately in the value of 4 nm in all environments.
Graph22. Dependence of intrinsic viscosity [η] on the concentration of BSA for Hya samples
with added BSA with different concentrations(different environments, VMHya)
It is evident on the Graph22that the intrinsic viscosity drops with increasing concentration
of BSA for all sets, which completely corresponds with the course of dependence of their
hydrodynamic radiuses and it also confirms creation of complexes in all environments, at
minimum in buffer with pH 7with addition of NaNO3. The highest intrinsic viscosity is for the
set prepared in buffer with pH 7 with addition of NaNO3, the smallest one is in pure Milli-
Qwater. As opposite to these data, for the low-molecular HA the set in buffer with pH 7 has
the lowest intrinsic viscosity (see the Graph18), which with probability proves the fact that
complexes VMHya are bigger in this environment compared to complexes NMHya.
Intrinsic viscosity of pure BSA is almost the same at all sets and it equals approximately
3.1 mL/g.
50. 50
CONCLUSION
With measuring of different sets of samples by the SEC-MALLS and DLS method it was
determined that electrostatic interactions between HA and BSA more or less occurred for
each of the measured sets.
Sets of low-molecular and high-molecular HA prepared in pure water with addition of
NaNO3were evaluated by the DLS method. It was determined that for low-molecular HA
growth of medium-size particles within the range of concentrations BSAapprox 0-
1 g/Loccurred, from the concentration BSA approx2 g/L the particles only became smaller,
when for high-molecular HA reduction of medium-size particles together with growing
concentration of BSA was monitored. It was probably caused due to higher ion shading of
low-molecular HA, when displacement of shading ions by molecules BSA and also
expansion of HA molecules occurred. Free carboxyl groups HA were still available for the
high-molecular HA also with presence of ion force. Therefore displacement of shading ions
took place with probability in smaller extent compared to interactions of HA and BSA.
It was determined from the zeta potential data that the smallest zeta potential was
recorded for sets prepared in pure water, be specific by reason of none shading with ion
force. More or less constant and in addition to that high zeta potential in other environments
is with a probability consequence of different value of ion force, which forms quite stable
solvating pack of the HA chain, and thereby stops interactions with BSA molecules.
Important SEC-MALLS data contained only the first peak. The second peak was
considered as insignificant or as the case may be the more significant 2nd
peak was recorded
only for samples with higher concentrations of BSA. The 2nd
peak represents with probability
in predominant part released BSA, which did not participate in interactions with HA.
The main trend of SEC-MALLS data with increasing concentration of BSAis: reduction of
the molar mass, gyration and hydrodynamic radius and intrinsic viscosity. This general trend
proves that interactions between HA and BSA cause contraction of particles.
It was determined for sets with high-molecular and low-molecular HA that reduction of the
molar mass was caused by increase of increment of fracture index with addition of higher
concentration of BSA. It was also recorded that the hydrodynamic radius was lower than the
root mean square (rms) radius. It is due to more extensive shapes of resultant complexes,
which would be in compliance with the theory of solutions of polymers.
For the set with high-molecular HA, more significant division of particles to two
populations occurred with probability, which was observed on chromatography.
It was discovered from comparison of SEC-MALLS data of different sets that sets of high-
molecular and low-molecular HA in buffer with pH 7 with addition of NaNO3performed
differently. The molar mass of the set with low-molecular HA in this buffer had the lowest
value, on the contrary the highest value was for the high-molecular HA. The lowest molar
mass of the NMHya set was with probability caused by the highest increment of fraction
index, the highest charge of HA chain and the smallest range of interactions associated with
a higher ion shading. A smaller charge (absolute value) was for VMHya as compared to
NMHya, due to lower ion shading and thereby larger range of interactions. However, the
range of interactions was with probability still minimal for both the low-molecular HA and the
high-molecular HA, which is evident on values of the root mean square (rms) radius and
intrinsic viscosity VMHya, which were highest in this environment. Intrinsic viscosity in this
environment was the highest for the set with high-molecular HA, with probability due to
significant size of molecules, when it was smallest for the low-molecular HA, which could be
due to higher concentration of complexes.
51. 51
The smallest gyration and hydrodynamic radius were recorded for the sets prepared in
pure water, both for VMHyaand also NMHya. It was caused with probability due to absence
of any shading ions, which could limit interactions.
BSA acquired almost all the time and in all environments the same values, both the molar
mass and both radiuses and intrinsic viscosity. The hydrodynamic radius was in most cases
higher than the root mean square (rms) radius. It was derived from it that these particles are
very compact with a significant solvating pack.
LIST OF USED ABBREVIATIONS
SECMALLS - size exclusion chromatography – multi angle laser light scattering
DLS - dynamic light scattering
HA - hyaluronic acid
Hya – hyaluronic acid/hyaluronan
BSA - bovin serum albumin
NMHya –low-molecular hyaluronic acid
VMHya –high-molecular hyaluronic acid
rms (radii) - root meen square (radii)
Rw – gyration (rms) radius
RhQ – hydrodynamic radius
Mw – molar mass
52. 52
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