Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces
The behavior of globular proteins (lysozyme, human serum albumin) and humic acids of coal (Powhumus) in aqueous/oil and aqueous/graphene systems was studied by means of tritium tracer. Tritium labeled biomolecules were obtained by tritium thermal activation method. Adsorption isotherms were obtained...
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| Cite this: | Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces / M.G. Chernysheva, G.A. Badun // Хімія, фізика та технологія поверхні. — 2010. — Т. 1, № 3. — С. 355-359. — Бібліогр.: 19 назв. — англ. |
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| citation_txt | Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces / M.G. Chernysheva, G.A. Badun // Хімія, фізика та технологія поверхні. — 2010. — Т. 1, № 3. — С. 355-359. — Бібліогр.: 19 назв. — англ. |
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| description | The behavior of globular proteins (lysozyme, human serum albumin) and humic acids of coal (Powhumus) in aqueous/oil and aqueous/graphene systems was studied by means of tritium tracer. Tritium labeled biomolecules were obtained by tritium thermal activation method. Adsorption isotherms were obtained by liquid scintillation spectrometry of tritium either in traditional performance or in scintillation phase technique
Метод радіоактивних індикаторів був застосований для дослідження поведінки глобулярних білків (лізоцим, сироватковий альбумін людини) та гумінових кислот вугілля (Powhumus) в системах вода/олія та вода/графен. Мічені тритієм біополімери були одержані методом термічної активації тритію. Ізотерми адсорбції біологічних макромолекул на межі поділу вода/графен і на міжфазній границі вода/олія були отримані за допомогою відповідно рідинно-сцинтиляційної спектрометрії тритію в традиційному варіанті та методом сцинтилюючої фази.
Метод радиоактивных индикаторов был применен для исследования поведения глобулярных белков (лизоцим, сывороточный альбумин человека) и гуминовых кислот угля (Powhumus) в системах вода/масло и вода/графен. Используемые меченые тритием биополимеры были получены методом термической активации трития. Изотермы адсорбции биологических макромолекул на поверхности раздела вода/графен и межфазной границе вода/масло были найдены с помощью жидкостной сцинтилляционной спектрометрии трития в традиционном варианте и в варианте метода сцинтиллирующей фазы соответственно.
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Хімія, фізика та технологія поверхні. 2010. Т. 1. № 3. С. 355–359
_____________________________________________________________________________________________
* Corresponding author masha.chernysheva@gmail.com
ХФТП 2010. Т. 1. № 3 355
UDC 546.11+544.723
RADIOCHEMICAL STUDY OF BIOPOLYMERS SORPTION
ON HYDROPHOBIC SURFACES
M.G. Chernysheva*, G.A. Badun
Lomonosov Moscow State University, Division of Radiochemistry, Department of Chemistry
1/3 Leninskiye Gory, Moscow 119991, Russia
The behavior of globular proteins (lysozyme, human serum albumin) and humic acids of coal (Powhu-
mus) in aqueous/oil and aqueous/graphene systems was studied by means of tritium tracer. Tritium labeled
biomolecules were obtained by tritium thermal activation method. Adsorption isotherms were obtained by
liquid scintillation spectrometry of tritium either in traditional performance or in scintillation phase technique.
INTRODUCTION
The behavior of biomacromolecules in hy-
drophilic/hydrophobic systems plays an important
role in different fields of technology and industry.
The interest was extremely increased with the
development of nanotechnology. Mechanisms of
the processes which occur at the interfaces are
under the high influence of both nature of hydro-
phobic surface and biomolecule itself. There are
a number of instrumental methods used for
studying the adsorption either at liquid/solid or
liquid/liquid interfaces. X-ray photoelectron
spectroscopy [1], reflectometry [2], and Raman
spectroscopy [3] are most frequently used for the
investigation of adsorption on aqueous/solids.
Liquid/liquid interfaces are more challenging
for the experimental studying. The most prevalence
experiments are conducted by pendant-drop tech-
nique [4], Fourier transform infrared spectroscopy
[5], total internal reflection fluorescence micros-
copy [6] or by radiotracer method with 14C-labeled
proteins first introduced by D.E. Graham and
M.C. Phillips in 1978 [7] and still used for study-
ing proteins adsorption at aqueous/oil interfaces
[8]. Thus the universal method for aqueous/solids
and aqueous/oil interface has not yet developed.
For this purpose radiotracer assay is rather
promising. In our previous researches we have
shown that tritium is a perspective tracer in surface
experiments [9–13]. Since tritium is a radioactive
isotope of hydrogen, it is related to all classes of
organic molecules. On the other hand, the advan-
tages of tritium as a tracer would not have become
applicable to such a wide extent if it were not rela-
tively easy to label organic molecules with tritium
at high specific activities and in versatility that is
not obtained with any other isotopes. For interfa-
cial researches tritium-labeled compounds can be
obtained by means of tritium thermal activation
method [14]. The technique is based on the bom-
bardment of solid target of organic compounds
with tritium atoms which forms on tungsten fila-
ment at 1500–2000 K. Since tritium can substitute
protium in any possible position, double purifica-
tion is usually conducted. First, treated compound
is purified from labile tritium (OH–, COOH–,
NH2–, SH-groups). Then [3H]-product is released
from the mixture of labeled compounds. This la-
beling technique is applicable to most kinds of or-
ganic materials from low molecular weight amino
acids [15] to humic substances [16].
Because of unique nuclear-physical properties
of the isotope (T1/2=12.4 years, Emax=18.6 keV) the
path length of tritium β-particles reaches few mi-
crons in the condensed media. This fact was used
when tritium labeled compounds were applied to
studying the adsorption of individual surfactants
and their mixtures with polymers at aque-
ous/organic liquid interface [10, 11].
The only way to determine tritium labeled
compound is liquid scintillation spectrometry. For
sorption experiments with solid/liquid surfaces, it is
used in the traditional performance for measuring
aqueous solution [17]. In case of the systems of two
immiscible liquids, liquid scintillation spectrometry
is applied in variant of scintillation phase method
[10–13]. It is a direct method of the determination
of both bulk concentration of tritium labeled com-
pound and its excess at aqueous/organic scintillator
interface. The technique is based on the measuring
counting rate of the system of aqueous solution of
tritium labeled compound / organic scintillator
which is immiscible with water. The counting rate
M.G. Chernysheva, G.A. Badun
_____________________________________________________________________________________________
356 ХФТП 2010. Т. 1. № 3
results in both distribution of labeled compound in
the bulk of scintillator and its concentration at the
liquid/liquid interface. The radiochemical theory of
the method was previously described in [13].
In this manuscript we have compared the ad-
sorption for biomacromolecules on hydropho-
bic/hydrophilic interfaces including aqueous/solid
and aqueous/organic liquid interfaces. Since re-
cent increased interest in nanoscale particles gra-
phene was used as hydrophobic solids. In case of
liquid/liquid experiments scintillators based either
on toluene or p-xylene were applied. Two globular
proteins lysozyme and human serum albumin and
coal humic acid were under the test. Based on our
previous experimental data the conditions of tritium
labeling were chosen according to formation of la-
beled product with high specific radioactivity on the
background of small amount of by-products.
EXPERIMENTAL
Graphene was synthesized in Institute of Ele-
mentoorganic Compounds RAS, Moscow. Specific
surface 107 m2/g was determined by BET.
Lysozyme and human serum albumin were
purchased from MP Biomedicals and used with-
out further purification. Coal humic acid (CHA-
Pow) was a commercially available preparation
Powhumus (Humintech GmbH, Germany) de-
salted using dialysis before the experiments [16].
Tritium label was introduced into the bio-
polymers by means of tritium thermal activation
method [16, 18]. Briefly, 0.8 mL of aqueous solu-
tion of compound (1.25 g/L) was distributed on the
walls of glass reactor and lyophilized. Then the
reactor was connected to the gas tritium devise
which includes W-wire in the middle of glass reac-
tor and a cooler. Air was pumped out and the reac-
tor was filled with tritium gas till 1.2 Pa. W-wire
was heated with electric current up to 1800 K dur-
ing 10 s. After the reaction the compound was dis-
solved in 2 mL of aqueous solvent: 0.4% NaOH
for CHA-Pow and phosphate saline buffer (PBS,
pH 7.2±0.1) for globular proteins. To purify la-
beled compound from the labile tritium and la-
beled by-products 30 days dialysis and size exclu-
sion chromatography were used as it described in
[16, 19]. Specific radioactivities of final products
were 1.5, 3.2 and 12.8 Ci/g for human serum al-
bumin, lysozyme, and CHA-Pow, respectively.
Adsorption experiments were conducted for
aqueous/oil and aqueous/solids interfaces. First were
carried out by means of scintillation phase method.
To 1 mL of aqueous solution of [3H]-compound
(specific radioactivity ca. 1.5 µCi/mL) 3 mL of scin-
tillation phase (non-aqueous scintillator based on p-
xylene or toluene) was added. The amount of [3H]-
compound in both oil phase and interfacial excess
were calculated as it was previously described [10].
Sorption experiments on solids were conducted
for graphene. To 4–5 mg sample of carbon nanoma-
terial 0.8 mL of aqueous solution of [3H]-compound
(specific radioactivity ca. 3 µCi/mL) was added.
Ultrasonication of the dispersion was carried out for
20 min using a bath sonicator with rated power of
50 W. Then systems were incubated at room tem-
perature during 24 hrs. 200 µL of suspension was
picked out and centrifuged. 100 µL of the solution
was filtered throw the syringe filter with 0.2 µm
PVDF membrane (Life Science). 10 µL aliquot of
filtered solution was picked out for radioactivity
measuring. Radioactivity of aqueous solution was
measured in scintillation cocktail OptiPhase Hi
Safe 3 (PerkinElmer) by means of scintillation spec-
trometer RackBeta 1215 (Finland).
Protein concentration in aqueous solution was
calculated as
1
1 V
I
с
⋅
=
ε
. (1)
Here I is counting rate, V1 is volume of aliquot, ε
is the registration efficiency of tritium β-radiation
(for OptiPhase Hi Safe 3 ε = 53±2%).
The value of protein sorption was calculated as
( )
Sm
Vсс
Г
⋅
⋅−= 010 , (2)
c0 is the initial concentration of protein solution,
V0 is the initial volume, m is mass of graphene, S
is specific surface of nanomaterial.
RESULTS AND DISCUSSIONS
Tritium labeled compounds are useful in study-
ing their behavior in different systems. Here we
have labeled biological macromolecules with trit-
ium to investigate their hydrophobic and surface
active properties. Experiments were conducted for
aqueous/oil and aqueous/graphene interfaces. Fig. 1
and Fig. 2 show the comparison of adsorptions of
lysozyme, human serum albumin and CHA-Pow at
aqueous/arene and aqueous/graphene interfaces in
semi-logarithmic coordinates. One can see that for
all tested compounds the adsorption at liquid/liquid
interface is higher than one obtained for aque-
ous/graphene. It can be explained by the strong in-
teraction between molecules of organic phase either
with hydrophobic amino acids residue of proteins or
with hydrophobic core of humic acids.
Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces
_____________________________________________________________________________________________
ХФТП 2010. Т. 1. № 3 357
Fig. 1. The comparison of the adsorption of globular
proteins at different aqueous/hydrophobic inter-
faces. 1 – human serum albumin at aqueous/p-
xylene interface; 2 – human serum albumin at
aqueous/graphene interface; 3 – lysozyme at
aqueous/p-xylene interface and 4 – lysozyme at
aqueous/graphene interface
Fig. 2. The adsorption of coal humic acids at aque-
ous/toluene (1) and aqueous / graphene (2) in-
terfaces
Such associates possess high surface activity
compared with aqueous/air interface [8] and can pe-
netrate into the bulk of organic liquid with the parti-
tion coefficients (1.0±0.2)×10-3, (3.2±0.6)×10-3 and
(3.7±0.2)×10-3 for lysozyme, albumin, and CHA-
Pow, respectively . It has to be emphasized that trit-
ium labeling procedure and purification secured the
radioactivity of organic phase provide only by la-
beled compound not by exchangeable tritium.
As to liquid/liquid interface tritium tracer have
unquestionable advantage compared with methods
based on measuring interfacial tension which lies
in the fact that it is applicable in both monolayer
region and higher concentration limited by the
formation of stable emulsion of water in the or-
ganic phase in presence of surfactant [10]. The
formation of stable associates with biomolecules
was confirmed with the experiment when organic
phase without scintillation additives saturated with
albumin that was subjected to vacuum evaporation
and then it was dissolved in water. We have ob-
served the 2.5×104 nm emulsion by means of pho-
ton correlation spectrometry that allowed suggest
that associates of proteins with molecules of or-
ganic phase do not destruct even under vacuum
while organic phase itself is volatile liquid.
Not only interaction with hydrophobic interface
but also the nature of sorbate itself is of significance
in the adsorption mechanism. Fig. 3 shows the
comparison of adsorption isotherms of globular pro-
teins on graphene surface. While albumin adsorp-
tion was subjected to Langmuir model in monolayer
region, adsorption of lysozyme linearly increased in
whole concentration range (r2 = 0.97). Henry con-
stants calculated from the liner part of the isotherms
were 4×10-2 and 1×10-2 mol/m2/mol/L for albumin
and lysozyme, respectively.
The data obtained for albumin were compared
with one obtained for carbon single-walls nanotubes
in [2]. In cited paper, adsorption of bovine serum
albumin was tested by reflectometry at different pH.
At pH 7 the authors observed a plateau with the
value of maximum adsorption 2.3±0.2 mg/m2 at
concentration 0.5 mg/mL (7.24×10-3 mmol/L) and
did not discuss future behavior. In case of graphene
we also observe first plateau at concentration range
from 1.71×10-3 to 7×10-2 mmol/L. The value of ad-
sorption corresponds to the formation of monolayer
of this protein.
One can see that the values of lysozyme adsorp-
tion in ten times higher than for albumin at the same
concentration range. The difference in adsorption
Fig. 3. Comparison of adsorption of human serum al-
bumin (top) and lysozyme (bottom) on aque-
ous/graphene interface
M.G. Chernysheva, G.A. Badun
_____________________________________________________________________________________________
358 ХФТП 2010. Т. 1. № 3
mechanism of albumin and lysozyme can also be
explained by peculiarities of the physical properties
of tested globular proteins. Since lysozyme is rather
small and structural stable molecule, it probably
preserves the possibility to interact with molecules
in the bulk of aqueous phase, when adsorbed on
graphene surface. Human serum albumin is a mole-
cule of large size and non-stable structure. When
adsorbed on hydrophobic surface, it loses the in-
teraction possibility with proteins both in aqueous
phase and adsorbed on graphene surface. One can
see that at high concentrations of albumin adsorp-
tion corresponds to formation of polylayers but
this adsorption is a reversible process. Desorption
at monolayer region for albumin and in case of
lysozyme was less than 2% even if 10 order ex-
cess of sodium dodecylsulfate was added. When
albumin polylayers were formed, desorption
started in pure buffer.
Furthermore, samples of initial graphene and
of that modified by proteins were analyzed by
photon correlation spectrometry after purification
from free protein that was performed by centrifu-
gation of the system then supernatant was care-
fully collected followed by the addition of pure
PBS and further ultrasonication. The purification
procedure was controlled by radioactivity meas-
uring and it was continued until radioactivity of
supernatant archived a background value.
It was found that graphene modified by ly-
sozyme was identical with the initial. In both cases,
average size of the particles was ca. 1.3×104 nm on a
background of high polydespersity. In case of albu-
min modification (Fig. 4), the suspension contains
particles with average size 556 nm (88% intensity).
Fig. 4. Particle size distribution of graphene-albumin associ-
ates determined by photon correlation spectrometry
CONCLUSIONS
In present research we have shown that adsorp-
tion of biomacromolecules at hydropho-
bic/hydrophilic interfaces included aqueous/solids
and aqueous/oil can be studied by radiochemical as-
say where tritium is used as a tracer. The adsorption
process is hydrophobic interaction controlled and its
mechanism is under high influence of the structural
peculiarities and stability of sorbate molecules.
ACKNOWLEDGEMENTS
This work was supported by RFBR (grant # 08-
03-00819) and by Federal Targeted Program "Scien-
tific and scientific-pedagogical personnel of innova-
tion Russia" for 2009–2013 years (project # 2351P).
We also acknowledge Dr. Kvacheva (Institute of
Elementoorganic Compounds RAS) and Dr. Cher-
vonobrodov (LLC Carbonlight) for graphene synthe-
sis; Dr. Perminova (Lomonosov Moscow State Uni-
versity) and her scientific group for their help in puri-
fication and analysis of humic compounds; Dr.
Vlasenko (Lomonosov Moscow State University) for
graphene specific surface determination.
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Received 19.05.2010, accepted 17.08.2010
Вивчення сорбції біополімерів на гідрофобних поверхнях за допомогою
радіохімічних методів
М.Г. Чернишева, Г.О. Бадун
Московський державний університет ім. М.В. Ломоносова, хімічний факультет, кафедра радіохімії
Ленінські гори 1, будівля 3, Москва 119991, Росія, masha.chernysheva@gmail.com
Метод радіоактивних індикаторів був застосований для дослідження поведінки глобулярних білків (лізоцим, си-
роватковий альбумін людини) та гумінових кислот вугілля (Powhumus) в системах вода/олія та вода/графен. Мічені
тритієм біополімери були одержані методом термічної активації тритію. Ізотерми адсорбції біологічних макро-
молекул на межі поділу вода/графен і на міжфазній границі вода/олія були отримані за допомогою відповідно рідин-
но-сцинтиляційної спектрометрії тритію в традиційному варіанті та методом сцинтилюючої фази.
Изучение сорбции биополимеров на гидрофобных поверхностях с помощью
радиохимических методов
М.Г. Чернышева, Г.А. Бадун
Московский государственный университет им. М.В. Ломоносова, химический факультет, кафедра радиохимии
Ленинские горы 1, строение 3, Москва 119991, Россия, masha.chernysheva@gmail.com
Метод радиоактивных индикаторов был применен для исследования поведения глобулярных белков (ли-
зоцим, сывороточный альбумин человека) и гуминовых кислот угля (Powhumus) в системах вода/масло и во-
да/графен. Используемые меченые тритием биополимеры были получены методом термической активации
трития. Изотермы адсорбции биологических макромолекул на поверхности раздела вода/графен и межфаз-
ной границе вода/масло были найдены с помощью жидкостной сцинтилляционной спектрометрии трития в
традиционном варианте и в варианте метода сцинтиллирующей фазы соответственно.
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| id | nasplib_isofts_kiev_ua-123456789-29007 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 2079-1704 |
| language | English |
| last_indexed | 2025-11-24T11:37:07Z |
| publishDate | 2010 |
| publisher | Інститут хімії поверхні ім. О.О. Чуйка НАН України |
| record_format | dspace |
| spelling | Chernysheva, M.G. Badun, G.A. 2011-11-27T18:12:26Z 2011-11-27T18:12:26Z 2010 Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces / M.G. Chernysheva, G.A. Badun // Хімія, фізика та технологія поверхні. — 2010. — Т. 1, № 3. — С. 355-359. — Бібліогр.: 19 назв. — англ. 2079-1704 https://nasplib.isofts.kiev.ua/handle/123456789/29007 546.11+544.723 The behavior of globular proteins (lysozyme, human serum albumin) and humic acids of coal (Powhumus) in aqueous/oil and aqueous/graphene systems was studied by means of tritium tracer. Tritium labeled biomolecules were obtained by tritium thermal activation method. Adsorption isotherms were obtained by liquid scintillation spectrometry of tritium either in traditional performance or in scintillation phase technique Метод радіоактивних індикаторів був застосований для дослідження поведінки глобулярних білків (лізоцим, сироватковий альбумін людини) та гумінових кислот вугілля (Powhumus) в системах вода/олія та вода/графен. Мічені тритієм біополімери були одержані методом термічної активації тритію. Ізотерми адсорбції біологічних макромолекул на межі поділу вода/графен і на міжфазній границі вода/олія були отримані за допомогою відповідно рідинно-сцинтиляційної спектрометрії тритію в традиційному варіанті та методом сцинтилюючої фази. Метод радиоактивных индикаторов был применен для исследования поведения глобулярных белков (лизоцим, сывороточный альбумин человека) и гуминовых кислот угля (Powhumus) в системах вода/масло и вода/графен. Используемые меченые тритием биополимеры были получены методом термической активации трития. Изотермы адсорбции биологических макромолекул на поверхности раздела вода/графен и межфазной границе вода/масло были найдены с помощью жидкостной сцинтилляционной спектрометрии трития в традиционном варианте и в варианте метода сцинтиллирующей фазы соответственно. This work was supported by RFBR (grant # 08-03-00819) and by Federal Targeted Program "Scientific and scientific-pedagogical personnel of innovation Russia" for 2009–2013 years (project # 2351P). We also acknowledge Dr. Kvacheva (Institute of Elementoorganic Compounds RAS) and Dr. Chervonobrodov (LLC Carbonlight) for graphene synthesis; Dr. Perminova (Lomonosov Moscow State University) and her scientific group for their help in purification and analysis of humic compounds; Dr. Vlasenko (Lomonosov Moscow State University) for graphene specific surface determination. en Інститут хімії поверхні ім. О.О. Чуйка НАН України Хімія, фізика та технологія поверхні Біомедичні аспекти поверхневих явищ Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces Вивчення сорбції біополімерів на гідрофобних поверхнях за допомогою радіохімічних методів Изучение сорбции биополимеров на гидрофобных поверхностях с помощью радиохимических методов Article published earlier |
| spellingShingle | Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces Chernysheva, M.G. Badun, G.A. Біомедичні аспекти поверхневих явищ |
| title | Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces |
| title_alt | Вивчення сорбції біополімерів на гідрофобних поверхнях за допомогою радіохімічних методів Изучение сорбции биополимеров на гидрофобных поверхностях с помощью радиохимических методов |
| title_full | Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces |
| title_fullStr | Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces |
| title_full_unstemmed | Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces |
| title_short | Radiochemical Study of Biopolymers Sorption on Hydrophobic Surfaces |
| title_sort | radiochemical study of biopolymers sorption on hydrophobic surfaces |
| topic | Біомедичні аспекти поверхневих явищ |
| topic_facet | Біомедичні аспекти поверхневих явищ |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/29007 |
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