ISSN 2224-5308 (Print)
ҚАЗАҚСТАН РЕСПУБЛИКАСЫ ҰЛТТЫҚ ҒЫЛЫМ АКАДЕМИЯСЫНЫҢ
Өсімдіктердің биологиясы жəне биотехнологиясы институтының
Х А Б А Р Л А Р Ы
ИЗВЕСТИЯ
НАЦИОНАЛЬНОЙ АКАДЕМИИ НАУК РЕСПУБЛИКИ КАЗАХСТАН
Института биологии и биотехнологии растений
N E W S
OF THE NATIONAL ACADEMY OF SCIENCES OF THE REPUBLIC OF KAZAKHSTAN of the Institute of Plant Biology and Biotechnology
БИОЛОГИЯ ЖƏНЕ МЕДИЦИНА СЕРИЯСЫ
СЕРИЯ
БИОЛОГИЧЕСКАЯ И МЕДИЦИНСКАЯ
SERIES
OF BIOLOGICAL AND MEDICAL
6 (330)
ҚАРАША – ЖЕЛТОҚСАН 2018 ж.
НОЯБРЬ – ДЕКАБРЬ 2018 г.
NOVEMBER – DECEMBER 2018
1963 ЖЫЛДЫҢ ҚАҢТАР АЙЫНАН ШЫҒА БАСТАҒАН ИЗДАЕТСЯ С ЯНВАРЯ 1963 ГОДА
PUBLISHED SINCE JANUARY 1963 ЖЫЛЫНА 6 РЕТ ШЫҒАДЫ
ВЫХОДИТ 6 РАЗ В ГОД PUBLISHED 6 TIMES A YEAR
АЛМАТЫ, ҚР ҰҒА АЛМАТЫ, НАН РК
ALMATY, NAS RK
Б а с
р е д а к т о р
ҚР ҰҒА академигі, м. ғ. д., проф. Ж. А. Арзықұлов
Абжанов Архат проф. (Бостон, АҚШ), Абелев С.К., проф. (Мəскеу, Ресей),
Айтқожина Н.А., проф., академик (Қазақстан) Акшулаков С.К., проф., академик (Қазақстан) Алшынбаев М.К., проф., академик (Қазақстан) Бəтпенов Н.Д., проф., корр.-мүшесі(Қазақстан) Березин В.Э., проф., корр.-мүшесі (Қазақстан) Берсімбаев Р.И., проф., академик (Қазақстан) Беркінбаев С.Ф., проф., (Қазақстан)
Бисенбаев А.К., проф., академик (Қазақстан) Бишимбаева Н.Қ., проф., академик (Қазақстан) Ботабекова Т.К., проф., корр.-мүшесі (Қазақстан) Жансүгірова Л.Б., б.ғ.к., проф. (Қазақстан) Ellenbogen Adrian prof. (Tel-Aviv, Israel),
Жамбакин Қ.Ж., проф., академик (Қазақстан), бас ред. орынбасары Заядан Б.К., проф., корр.-мүшесі (Қазақстан)
Ishchenko Alexander prof. (Villejuif, France) Исаева Р.Б., проф., (Қазақстан)
Қайдарова Д.Р., проф., академик (Қазақстан) Кохметова А.М., проф., корр.-мүшесі (Қазақстан) Күзденбаева Р.С., проф., академик (Қазақстан) Локшин В.Н., проф., корр.-мүшесі (Қазақстан) Лось Д.А., prof. (Мəскеу, Ресей)
Lunenfeld Bruno prof. (Израиль)
Макашев Е.К., проф., корр.-мүшесі (Қазақстан) Миталипов Ш.М. (Америка)
Муминов Т.А., проф., академик (Қазақстан) Огарь Н.П., проф., корр.-мүшесі (Қазақстан) Омаров Р.Т., б.ғ.к., проф., (Қазақстан) Продеус А.П. проф. (Ресей)
Purton Saul prof. (London, UK)
Рахыпбеков Т.К., проф., корр.-мүшесі (Қазақстан) Сапарбаев Мұрат проф. (Париж, Франция) Сарбасов Дос проф. (Хьюстон, АҚШ)
Тұрысбеков Е.К., б.ғ.к., асс.проф. (Қазақстан) Шарманов А.Т., проф. (АҚШ)
«ҚР ҰҒА Хабарлары. Биология жəне медициналық сериясы».
ISSN 2518-1629 (Online), ISSN 2224-5308 (Print)
Меншіктенуші: «Қазақстан Республикасының Ұлттық ғылым академиясы» РҚБ (Алматы қ.)
Қазақстан республикасының Мəдениет пен ақпарат министрлігінің Ақпарат жəне мұрағат комитетінде 01.06.2006 ж. берілген №5546-Ж мерзімдік басылым тіркеуіне қойылу туралы куəлік
Мерзімділігі: жылына 6 рет.
Тиражы: 300 дана.
Редакцияның мекенжайы: 050010, Алматы қ., Шевченко көш., 28, 219 бөл., 220, тел.: 272-13-19, 272-13-18, www:nauka-nanrk.kz / biological-medical.kz
© Қазақстан Республикасының Ұлттық ғылым академиясы, 2018 Типографияның мекенжайы: «Аруна» ЖК, Алматы қ., Муратбаева көш., 75.
Г л а в н ы й
р е д а к т о р
академик НАН РК, д.м.н., проф. Ж. А. Арзыкулов Абжанов Архат проф. (Бостон, США),
Абелев С.К. проф. (Москва, Россия),
Айтхожина Н.А. проф., академик (Казахстан) Акшулаков С.К. проф., академик (Казахстан) Алчинбаев М.К. проф., академик (Казахстан) Батпенов Н.Д. проф. член-корр.НАН РК (Казахстан) Березин В.Э., проф., чл.-корр. (Казахстан)
Берсимбаев Р.И., проф., академик (Казахстан) Беркинбаев С.Ф. проф. (Казахстан)
Бисенбаев А.К. проф., академик (Казахстан) Бишимбаева Н.К. проф., академик (Казахстан) Ботабекова Т.К. проф., чл.-корр. (Казахстан) Джансугурова Л. Б. к.б.н., проф. (Казахстан) Ellenbogen Adrian prof. (Tel-Aviv, Israel),
ЖамбакинК.Ж. проф., академик (Казахстан), зам. гл. ред.
Заядан Б.К. проф., чл.-корр. (Казахстан) Ishchenko Alexander, prof. (Villejuif, France) Исаева Р.Б. проф. (Казахстан)
Кайдарова Д.Р. проф., академик (Казахстан) Кохметова А.М. проф., чл.-корр. (Казахстан) Кузденбаева Р.С. проф., академик (Казахстан) Локшин В.Н., проф., чл.-корр. (Казахстан) Лось Д.А. prof. (Москва, Россия)
Lunenfeld Bruno prof. (Израиль)
Макашев Е.К. проф., чл.-корр. (Казахстан) Миталипов Ш.М. (Америка)
Муминов Т.А. проф., академик (Казахстан) Огарь Н.П. проф., чл.-корр. (Казахстан) Омаров Р.Т.к.б.н., проф. (Казахстан) Продеус А.П. проф. (Россия)
Purton Saul prof. (London, UK)
Рахыпбеков Т.К. проф., чл.-корр. (Казахстан) Сапарбаев Мурат проф. (Париж, Франция) Сарбасов Дос проф. (Хьюстон, США)
Турысбеков Е. К., к.б.н., асс.проф. (Казахстан) Шарманов А.Т. проф. (США)
«Известия НАН РК. Серия биологическая и медицинская».
ISSN 2518-1629 (Online), ISSN 2224-5308 (Print)
Собственник: РОО «Национальная академия наук Республики Казахстан» (г. Алматы)
Свидетельство о постановке на учет периодического печатного издания в Комитете информации и архивов Министерства культуры и информации Республики Казахстан №5546-Ж, выданное 01.06.2006 г.
Периодичность: 6 раз в год Тираж: 300 экземпляров
Адрес редакции: 050010, г. Алматы, ул. Шевченко, 28, ком. 219, 220, тел. 272-13-19, 272-13-18, www:nauka-nanrk.kz / biological-medical.kz
© Национальная академия наук Республики Казахстан, 2018 Адрес типографии: ИП «Аруна», г. Алматы, ул. Муратбаева, 75
E d i t o r
i n
c h i e f
Zh.A. Arzykulov, academician of NAS RK, Dr. med., prof.
Abzhanov Arkhat, prof. (Boston, USA), Abelev S.K., prof. (Moscow, Russia),
Aitkhozhina N.А., prof., academician (Kazakhstan) Akshulakov S.K., prof., academician (Kazakhstan) Alchinbayev М.K., prof., academician (Kazakhstan) Batpenov N.D., prof., corr. member (Kazakhstan) Berezin V.Ye., prof., corr. member. (Kazakhstan) Bersimbayev R.I., prof., academician (Kazakhstan) Berkinbaev S.F., prof. (Kazakhstan)
Bisenbayev А.K., prof., academician (Kazakhstan) Bishimbayeva N.K., prof., academician (Kazakhstan) Botabekova Т.K., prof., corr. member. (Kazakhstan) Dzhansugurova L.B., Cand. biol., prof. (Kazakhstan) Ellenbogen Adrian, prof. (Tel-Aviv, Israel),
Zhambakin K.Zh., prof., academician (Kazakhstan), deputy editor-in-chief Ishchenko Alexander, prof. (Villejuif, France)
Isayeva R.B., prof. (Kazakhstan)
Kaydarova D.R., prof., academician (Kazakhstan) Kokhmetova A., prof., corr. member (Kazakhstan) Kuzdenbayeva R.S., prof., academician (Kazakhstan) Lokshin V.N., prof., corr. member (Kazakhstan) Los D.А., prof. (Moscow, Russia)
Lunenfeld Bruno, prof. (Israel)
Makashev E.K., prof., corr. member (Kazakhstan) Mitalipov Sh.M. (America)
Muminov Т.А., prof., academician (Kazakhstan) Ogar N.P., prof., corr. member (Kazakhstan) Omarov R.T., Cand. biol., prof. (Kazakhstan) Prodeus A.P., prof. (Russia)
Purton Saul, prof. (London, UK)
Rakhypbekov Т.K., prof., corr. member. (Kazakhstan) Saparbayev Мurat, prof. (Paris, France)
Sarbassov Dos, prof. (Houston, USA)
Turysbekov E.K., cand. biol., assoc. prof. (Kazakhstan) Sharmanov A.T., prof. (USA)
News of the National Academy of Sciences of the Republic of Kazakhstan. Series of biology and medicine.
ISSN 2518-1629 (Online), ISSN 2224-5308 (Print)
Owner: RPA "National Academy of Sciences of the Republic of Kazakhstan" (Almaty)
The certificate of registration of a periodic printed publication in the Committee of information and archives of the Ministry of culture and information of the Republic of Kazakhstan N 5546-Ж, issued 01.06.2006
Periodicity: 6 times a year Circulation: 300 copies
Editorial address: 28, Shevchenko str., of. 219, 220, Almaty, 050010, tel. 272-13-19, 272-13-18, http://nauka-nanrk.kz / biological-medical.kz
© National Academy of Sciences of the Republic of Kazakhstan, 2018 Address of printing house: ST "Aruna", 75, Muratbayev str, Almaty
N E W S
OF THE NATIONAL ACADEMY OF SCIENCES OF THE REPUBLIC OF KAZAKHSTAN SERIES OF BIOLOGICAL AND MEDICAL
ISSN 2224-5308
Volume 6, Number 330 (2018), 5 – 12 https://doi.org/10.32014/2018.2518-1629.11
UDC 577.3 620.3 578.08.002.5
S. Zh. Ibadullaeva1, M. G. Fomkina2, N. O. Appazov1, L. A. Zhusupova1
1Korkyt Ata Kyzylorda State University, Kyzylorda, Kazakhstan,
2Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Russia.
E-mail: [email protected], [email protected], [email protected], [email protected]
DEVELOPMENT OF A BIOSENSOR OF UREA
WITH THE APPLICATION OF POLYMER TECHNOLOGIES FOR BLOOD AND URINE ANALYSIS
Abstract. Based on polymeric nanotechnologies, enzyme sensors and microreactors have been developedin the way, that they can determine urea in liquids. The technology of manufacturing an enzymatic biosensor does not differ significantly from the known technology of manufacturing microcapsules with an enzyme by the laer-by-laer method. This allows us, when constructing a biosensor, to use the information obtained on encapsulated enzymes by other authors. It is shown that urea biosensor is able to work for a long time (up to 2 months) without significant loss of enzyme activity. Polymer technology for manufacturing sensors is less laborious and expensive compared to other similar technologies. We propose to develop biosensor devices – urea analyzers with polymer enzyme chips for express diagnostics of biological fluids (blood, urine). One of the significant results of this work from our point of view is two factors. The first factor is the optimization of the conditions for the production of a functionally active enzyme immobilized in a polyelectrolyte coating, when the enzyme after the immobilization procedure shows an activity comparable to that of a freshly prepared free enzyme. Such a result will allow reducing the cost of enzymes when creating a sensitive layer of the developed urea analyzer. And the second factor is that the polymer coating with the enzyme is able to work not only as an enzyme electrode, but also as an enzyme microreactor, without de- creasing the rate of signal registration after passing the catalytic urease-urea reaction.
Keywords: enzyme biosensors, polymeric nanomaterial, portable analyzer, microreactor, microcapsules, urea.
Introduction. The volume of laboratory research worldwide is steadily increasing and reaches 45 billion analyzes per year, and in industrialized countries the number of analyzes per person reaches 40-60 per year. Universal biochemical analyzers analyze any biological fluids (substrates, enzymes, lipids, drugs, hormones, proteins, electrolytes, drugs). They are produced by about 60 companies, the main pro- ducers are Abbott (USA), ABC1 (Austria), Koné (Finland), Nova (USA), Corning (England), Beckmann (USA), "Radiometer" (Denmark). Ready-made sets of reagents are in great demand. Their market is about 27 billion dollars in the world market of laboratory instruments in 6 billion dollars.
Spectroscopic analyzers are used for biochemical studies (determination of organic and inorganic chemicals, such as potassium, sodium, calcium, magnesium, lithium, chlorine, substrates, metabolites, enzymes of biochemical processes in blood and other human biological fluids). Universal biochemical analyzers with the help of which an analysis of any biological fluids for the content of various compo- nents are recognized as promising. However, at the present time there are no portable devices of this class.
The development of portable devices for the analysis of biological fluids is an urgent task of modern medical diagnostics. Of particular interest among portable analyzers of various substances undoubtedly represent analyzers based on biosensors. Any biosensor consists of two functional elements: a biosensor containing a bioselective material, and a physical converter that transforms any generated signal (ion concentration, mass, color, etc.) into an electrical signal. In the role of biosecting material are all types of biological structures - enzymes, antibodies, receptors, nucleic acids and even living cells. In biosensors are used a variety of physical converters: amperometric, conductometric, optical, luminescent, fluorescent, acoustic, gravitational, etc.
The development of biosensors is an extremely time-consuming process. The most important stage in the development of enzyme sensors is proper immobilization of enzymes on solid supports (substrates).
We have developed a method for immobilizing enzymes using polymer technologies, in which the immobilized enzyme is in a functionally active state [1-3]. Immobilization of enzymes was carried out in a biosensor sensitive coating, which is a combination of nanometer polyelectrolyte layers and micro- encapsulated enzymes placed between these layers (figure 1).
А В С
Figure 1 – Enzyme electrode with sensitive biosensor coating:
A – is a glass pH electrode with a sensitive coating containing the enzyme urease;
B – image of a polyelectrolyte coating with microcells in a light microscope;
С – is a schematic representation of a sensitive coating with an enzyme
As it was shown in these works, enzymes in microcells of a polymeric material are reliably protected from aggressive influences of environment (microbes, proteases, etc.); able to detect substrates for a long time (up to 3 weeks in storage at room temperature). This work continues to improve the characteristics of the developed urea biosensor.
Materials and methods. For the production of enzymatic biosensors and enzyme micro-reactors, lyophilized urease (EC 3.5.1.5) was used from the Canavalia ensiformis beans of Sigma and Fluka, an urease solution from the Urea KT(200) kit, (Deacon-DS) with an activity of 253000 U/l., Urea extra clean (Reachim), MES buffers (Sigma), Tris-HCl (Sigma). Salts of CaCl2, Na2CO3, NaCl and KCl had a gradation of chemically pure or pure for analysis. Ethylene glycoltetraacetic (EGTA) and ethylenedia- minetetraacetic (EDTA) acid (both Sigma-Aldrich, USA). To form films and shells of microcapsules, domain enzymes, polyelectrolytes were used: polyethyleneimine (PEI) weight 600000-1000000, polystyrene sulfonate (PSS), polyallylamine hydrochloride (PAAH), (all - Aldrich) with a mass of 60000-70000. The test substances were used as solutions in 0.33 M NaCl. All salt solutions were prepared on deionized water obtained by purifying distilled water with Arium 611-UF (Sartorius). The conductivity of the water was 1 μS/cm.
The following instruments were used in the work: spectrophotometer Bekman UV/Vis DU 520 (USA), Nikon eclipse E200 microscope, 4-channel potentio-microamperometric analog-digital amplifier
"Record-4usb" with computer connection (development of IBK RAS), pH- meter Bekkman F 690 pH / Temp/mV/ISE Meter (USA), Axiovert 200 microscope, photometer (model 680 BIO-RAD, USA), Vortex (shaking and mixing device), ultrasonic bath, magnetic stirrer, table centrifuge, semi-automatic micro- pipette for 2-20 μl, 20-200 μl, 200-1000 μl, 5000 μl, chamber Goryaev.
Preparation of enzyme-containing calcium carbonate crustal particles. Composive microspherolites CaCO3– protein were used as core microparticles for the preparation of polyelectrolyte capsules.
CaCO3 microspherolites were obtained by the ion exchange reaction when mixing solutions of calcium chloride and carbonate in the presence of protein (enzyme) – by biomineralization [4-7].
Preparation of enzyme-containing polyelectrolyte microcapsules. Polyelectrolyte microcapsules with urease were produced by the method of alternate layer-by-layer adsorption with the application of polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAAH) molecules to composite calcium- carbonate spherulites containing urease as described in [4-6, 8].
Alternate layering of oppositely charged macromolecules of polyelectrolytes on colloidal particles was carried out three to five times, obtaining three/five shells with the architecture of PAAH/(PSS/PAAH)n and PSS/(PAGE/PSS)n where n=1.2. The procedure for the formation of micro- capsules was carried out at room temperature (15-25°C). Microcapsule size and sphericity of calcium carbonate particles were monitored with a Nikon eclipse E200 light microscope. The removal of calcium carbonate particles from the microcapsules was carried out while maintaining the solution with microcapsules in dialysis bags for 3 hours to 12-15 hours in 25 mM EGTA or EDTA at a temperature of 4°C or 20°C with basic alkalinization (pH 7.2-7.5). The number of capsules in the solution was counted using a cameraGoryaev.
Potentiometric method for determination of urea concentration with a standard pH electrode. Using the technique described in [1, 2], a potentiometric polymer biosensor of urea was prepared on the basis of a modified glass pH electrode (figure 1A). Measurements of the hydrogen ion concentration in the test solution were carried out using a four-channel ADC – "Record 4usb". The solution was stirred with a magnetic stirrer and maintained at 25 ± 1 °C with a U-1 thermostat (Germany). Then, the enzyme prepa- ration was added thereto in the required quantities or a modified pH electrode was introduced. The alkaline pH shift recorded (in mV) was saturated for 20-30 seconds.
Results and discussion. For the first time, the possibility of measuring the urea concentration by a modified glass pH electrode on which an ultrathin sensitive polymer coating with urease was deposited was demonstrated by us in [1, 2]. The following properties of the polymer coating provided this possi- bility: good permeability of polyelectrolyte multilayers for the substrate (urea) and its decomposition products by urease; impermeability of these layers for the enzyme; preservation of the enzyme in the cells of the coating, high activity for a sufficiently long time; as well as significant alkalization of the medium during the decomposition of urea to carbon dioxide and ammonia. Improving the characteristics and properties of the polymer sensitive coating of the urea sensor is associated with an increase in the initial activity of the immobilized enzyme, an increase in the duration of the sensor operation, and the ability to measure urea in biological fluids. As was shown in [9], we managed to achieve a sufficiently high activity of the immobilized enzyme, which amounted to 40-50% of the activity of the free freshly prepared enzyme.
In this paper, data are presented on the continuation of studies related to an increase in the initial activity of the urease sensor. Figure 2 shows the data on the dependence of the response of the glass pH electrode on urea concentration in the measuring cell for the free (line 1) and encapsulated (line 2) enzyme.
It can be seen from the figure that the activity of the encapsulated enzyme is comparable to the activity of a free freshly prepared enzyme and was about 75% of its activity. In the encapsulation process, the enzymes are partially damaged, and in the first studies on capsules with the enzyme, a high initial
Figure 2 – Dependence of the response of the glass pH electrode on the urea concentration (0.5 μg enzyme concentration was determined by the Bradford method): 1 - free enzyme (urease);
2 - encapsulated enzyme contained in microcapsules with the architecture of the PSS-PAAG-PSS envelope.
Study medium: 1 mM Tris-HCl, 1 mM MES, 100 mM NaCl, initial pH 5.3.
activity of the encapsulated enzyme was not achieved. Usually, the activity of encapsulated enzymes decreased by a factor of 6-7 [1, 6, 10-15]. Close results to our experimental data presented in this study on encapsulated urease were obtained in [16-19]. Authors, using the enzyme dextranase, obtained encap- sulated enzymes with a catalytic activity equal to 80% of the activity of the free enzyme (the capsules were formed from calcium alginate with the inclusion of silica).
Since unmodified glass pH electrodes were used for measurements in cells with a free and en- capsulated enzyme, we tried to compare pH measurements during the passage of the urease-urea catalytic reaction using a modified by our method an electrode and an unmodified electrode that were simul- taneously placed in a measuring cell. In this case, the decomposition reaction of urea passed in the biosensitive layer of the modified electrode (figure 3).
Figure 3 – Diagram of the experiment for measuring urea concentration with unmodified (upper curve) and modified pH electrodes.
A sensitive coating with urease is deposited on the ball of the lower electrode. Microcells of sensitive coating with the architecture of the shell of PAAG-PSS-PAAG.
Study medium: 1 mM MES, 100 mM NaCl, initial pH 6.0
Since unmodified glass pH electrodes were used for measurements in cells with a free and encap- sulated enzyme, we tried to compare pH measurements during the passage of the urease-urea catalytic reaction using a modified by our method an electrode and an unmodified electrode that were simul- taneously placed in a measuring cell. In this case, the decomposition reaction of urea passed in the biosensitive layer of the modified electrode (figure 3).
It can be seen from the experimental diagram that the response time after the catalytic reaction of the enzyme-substrate with the help of the modified and unmodified electrodes is practically the same. This is due to the fact that the substrate – urea and the decay products of the urease-urea catalytic reaction – carbon dioxide and ammonia easily penetrate through the nanometer polyelectrolyte shell that separates urease from the external solution. Such experimental results allowed us to create not only enzyme electrodes, but also enzyme microreactors (when the recording electrode is separated from the sensitive layer).
As a microreactor, plastic and glass cuvettes with a polyelectrolyte coating were applied, the same as for a ball of a modified pH electrode. This coating, which is a multilayer film, between layers of which was a layer of polyelectrolyte capsules with a diameter of about 2-5 microns filled with urease molecules, was applied to one of the walls of the cuvette. The presence of several, not less than five polyelectrolyte layers separating enzymes from the external environment, prevented the latter from inactivation, for example, by foreign enzymes or microbes. One of the features of the coating was that the total thickness of the polymer layers was less than 2% of the inner cell diameter in it.
Figure 4 presents data on the catalytic activity of the urease microreactor.
1 - fre Study
Thus on a glass limit of th on the pro loping a u since the n
Studi temperatu in activity other thing environme the numbe stability o microcaps It was sho and a load nate nucle trolytes is The n Investigat conducted follows th 100 times measuring suring ure measurem The s transistors As ca more than transistor used for o
Figur ee enzyme (ure in the micr medium: 1 mM
, it has been s pH electrod he measurem
operties of th urea sensor f normal urea ies of the sta ure of 4 °C, th
y of immobi gs, be due to ent in the mi er of polyele of the polye sules contain own that the d value of 25 eus dependin
of interest fo necessary com
ions of the d using urine hat if the acc approaches g urea in ser ea in blood s ments to 5%.
sensitivity of s (figure 5).
an be seen fr n an order o
coating was ne or two me
re 4 – Dependen (enzyme con ase); 2 - enzym rocells of the se M MES, 100 mM
shown that de it is possi ent depends he electrode.
for medical d content in th ability of a n he sensor is lized urease o the stability crocells of th ectrolyte laye electrolyte co ning a calciu
destruction 5 mN. Inves ng on the siz for improving
mponent of r urease poly e and blood curacy of me the error obt rum. Blood i serum, we te f the urease
om the figur of magnitude s 20 μl from
easurements
nce of the respo ncentration 3 μg me immobilized
nsitive coating M NaCl, initial
by potentiom ible to measu
on the conce We were ab diagnosis, it he human blo new type of u
capable of o is initially 4 y of the poly
he coating. P ers forming t oating.We c um-carbonate of a single m stigation of th ze of microca
g the stability research in th ymer sensor as an examp easuring by o tained during in different p ested the "d sensor can b re, the field e e. The enzy m the 3 ml se in enzyme a
onse of the glas g was determin on the lateral s with the archite pH 6.0
metric metho ure urea con entration of u ble to measur
is not neces ood is betwee
urea sensor operating for
40-50%. Th electrolyte sh Perhaps in th
the microcap carried out p e core with t
microcapsul he strength o apsules and t y of microcap
he developm for the det ple. These e our method g dilution of
people has i double additi be significan effect transist yme consump
et (see Mater analysis by th
s pH electrode ned by the Bradf
surface of the sp ecture of the PA
od using a ne ncentrations r
urease place re more than
sary to meas en 1.8 and 7.
showed that up to 2 mon e stability o hells that pro is case, the s psule shell, w preliminary the help of a e occurred w of microcaps the number psules and a ment of biosen
termination xperiments a the concentr
urine, then a its own pH a ves" method ntly increased tor has incre ption during rials and Me he usual spec
on urea concen ford method):
pectrophotomet AAG- (PSS-PA
ew type of p ranging from
d in the poly n 100 mM ur sure such hig
5 mM, depen when stored nths. At the s
f the sensor otect the enz size of the m will be impo studies of t a NanoScan- when it was sules with a of layers of new polyele nsors is testi of urea in are presented ration of ure a different pi and buffer c d, which inc d by using p ased the sens g the creatio
ethods). This ctral method
ntration tric cell and con AGE) 2 shell.
polymeric ure m 10-20 μM.
yelectrolyte c rea. In fact, w
gh urea conc nding on the d in distilled same time, th
over time c zymes from t microcapsules ortant for inc the strength -4D nanodid
compressed remote calc capsule shel ectrolyte coa ing on biolog biological f d in [9], from a in daily ur icture is obse capacity, so w creased the a pH-sensitive
sitivity of the on of a sens
s amount of d in polyclinic
ntained
ease sensor The upper coating and when deve- centrations, e age.
d water at a he decrease can, among the external s, as well as creasing the of 10 μm domer [20].
by 1.1 μm ium carbo- ll polyelec- ating.
gical fluids.
fluids were m which it rine diluted erved when when mea- accuracy of
field effect e sensor by sitive field- f enzyme is
cs.
Figure 5 – Comparison of the response of the pH-sensitive field-effect transistor and the glass pH electrode to the urea concentration.
The sensitive coating is applied to the glass electrode ball and to the surface of the recording element of the transistor.
Study medium: 2 mM Tris-HCl, 200 mM NaCl, initial pH 7.8
Conclusion. The urea biosensor manufactured with the help of polymer technologies and repre- senting a combination of polyelectrolyte layers and microcapsules with an enzyme inside and a shell of the same polyelectrolytes, as shown by the experimental data, is perfectly suitable for determining the urea concentration in blood and urine. The technology of manufacturing an enzymatic biosensor does not differ significantly from the known technology of manufacturing microcapsules with an enzyme by the laer-by-laer method [4-6]. This allows us, when constructing a biosensor, to use the information obtained on encapsulated enzymes by other authors. In this case, the urea biosensor is able to work for a long time (up to 2 months) without significant loss of enzyme activity. One of the significant results of this work from our point of view is two factors. The first factor is the optimization of the conditions for the pro- duction of a functionally active enzyme immobilized in a polyelectrolyte coating, when the enzyme after the immobilization procedure shows an activity comparable to that of a freshly prepared free enzyme.
Such a result will allow reducing the cost of enzymes when creating a sensitive layer of the developed urea analyzer. And the second factor is that the polymer coating with the enzyme is able to work not only as an enzyme electrode, but also as an enzyme microreactor, without decreasing the rate of signal regis- tration after passing the catalytic urease-urea reaction. This is due to the fact that the layers of poly- electrolytes separating the enzyme from the external analyte solution have a nanometer thickness and are easily permeable to urea and decomposition products of the urease-urea catalytic reaction. Separation of the sensitive sensor from the recording electrode provides many opportunities for designers of urea analyzers based on a polymer ultrathin coating.
Acknowledgments.Work was accomlished by grant financingof theCommittee of Scienceof the Ministry of Education and Scienceof the Republic of Kazakhstan by AP05134201 project.
REFERENCES
[1] Montrel' M.M., Ternovskij V.I., Fomkina M.G., Petrov A.I. (2008). Ul'tratonkoe polimernoe pokrytie, sposob ego izgotovlenija i fermentativnyj biosensor na ego osnove [Patent RU №2333231 ot 10.09.2008] Bull. 25 (in Rus.).
[2] Ternovskij V.I., Chernohvostov Ju.V., Fomkina M.G., Montrel' M.M. (2007). Potenciometricheskij sensor na osnove ureazy, immobilizovannoj v polijelektrolitnyh mikrokapsulah [Biofizika] 5:825-829 (in Rus.)
[3] Fomkina M.G., Minkabirova G.M., Montrel' A.M. (2015). Sposob poluchenija podlozhek s mnogoslojnym pokrytiem na osnove polijelektrolitnyh mikrokapsul, soderzhashhih biologicheski aktivnye materialy [Patent RU №2567320 ot 10.11.2015]
Bull. 31 (in Rus.).
[4] Sukhorukov G.B., Volodkin D.V., Günther A.M., Shenoy D.B., Möhwald H., Petrov A.I. (2009). Porous calcium carbo- nate microparticles as templates for encapsulation of bioactive compounds[Journal of Materials Chemistry]14:2073-2081 (in Eng.).
[5] Volodkin D.V., Prevot M., Sukhorukov G.B., Petrov A.I. (2004). Matrix polyelectrolyte microcapsules: new system for macromolecule encapsulation [Langmuir: the ACS journal of surfaces and colloids]8: 3398-3406 (in Eng.).
[6] Petrov A.I., Volodkin D.V., Sukhorukov G.B. (2005). Protein-calcium carbonate coprecipitation: a tool for protein encapsulation [Biotechnology Progress] 3:918-925 (in Eng.).
[7] Amer L., Ouhenia S., Belabbas I., Chateigner D. (2018.) The effect of ergocalciferol on the precipitation of calcium carbonate, Journal of Crystal Growth, 1: 49-59. DOI: 10.1016/j.jcrysgro.2018.08.032 (in Eng.).
[8] Gaddes D., Jung H.H., Pena-Francesch A., Dion G., Tadigadapa S., Dressick W.J., Demirel M.C. (2016). Self-Healing Textile: Enzyme Encapsulated Layer-by-Layer Structural Proteins, ACS Applied materials & Interfaces, 31: 20371-20378. DOI:
10.1021/acsami.6b05232 (in Eng.).
[9] Fomkina M.G., Grohlina T.I., Minkabirova G.M., Montrel' A.M., Maevskij E.I. (2011). Jekspress-diagnostika mocheviny v biologicheskih zhidkostjah na osnove novogo polimernogo datchika [Medline.ru. Rossijskij biomedicinskij zhurnal]
3:701-710 (in Rus.).
[10] Suhorukov B.I., Tihonenko S.A., Saburova E.A., Dubrovskij A.V., Dybovskaja Ju.N., Shabarchina L.I. (2007). Inkap- sulirovanie belkov v polijelektrolitnye nano- imikrokapsuly i problemy razrabotki fermentnogo mikrodiagnostikuma [Biofizika]
6:1041-1048 (in Rus.).
[11] Azmid N.E., Ab Rashid A.H., Abdullah J., Yusof N.A., Sidek H. (2018). Fluorescence biosensor based on encapsulated quantum dots/enzymes/solgel for non-invasive detection of uric acid, Journal of luminescence, 202: 309-315.
DOI: 10.1016/j.jlumin.2018.05.075 (in Eng.)
[12] Wilkerson J.W., Yang S.O., Funk P.J., Stanley S.K., Bundy B.C. (2018). Nanoreactors: Strategies to encapsulate en- zyme biocatalysts in virus-like particles // New biotechnology. 44: 59-63. DOI: 10.1016/j.nbt.2018.04.003 (in Eng.).
[13] Kucharzyk K.H., Benotti M., Darlington R., Lalgudi R. (2018). Enhanced biodegradation of sediment-bound heavily weathered crude oil with ligninolytic enzymes encapsulated in calcium-alginate beads // Journal of hazardous materials. 357:
498-505. DOI: 10.1016/j.jhazmat.2018.06.036 (in Eng.).
[14] Pauly J., Groger H., Patel A.V. (2018). Design, characterisation and application of alginate-based encapsulated pig liver esterase // Journal of biotechnology. 280: 42-48. DOI: 10.1016/j.jbiotec.2018.05.017 (in Eng.).
[15] Zhang Y., Ren T.T., Tian H., Jin B.B., He J.H. (2018). Hydrogel-Encapsulated Enzyme Facilitates Colorimetric Acute Toxicity Assessment of Heavy Metal Ions // ACS Applied materials & Interfaces, 31: 26705-26712.
DOI: 10.1021/acsami.8b08949 (in Eng.)
[16] Pachariyanon P., Barth E., Agar D.W. (2011). Enzyme immobilisation in permselective microcapsules [Micro and Nano Carriers Journal of Microencapsulation] 5:370-383 (in Eng.).
[17] Dogac Y.I., Teke M. (2016). Synthesis and Characterisation of Biocompatible Polymer-Conjugated Magnetic Beads for Enhancement Stability of Urease // Applied biochemistry and biotechnology. 1: 94-110. DOI: 10.1007/s12010-016-1981-3 (in Eng.).
[18] Reshetilov A.N., Plekhanova Y.V., Tikhonenko S.A., Dubrovskii A.V. (2015). Polyelectrolyte microcapsules with urease and paramagnetic particles as a basis for a potentiometric biosensor for determining urea // Journal of analytical chemistry. 11: 1368-1372. DOI: 10.1134/S1061934815110131 (in Eng.).
[19] Borase H.P., Salunkhe R.B., Patil C.D., Suryawanshi R.K., Salunke B.K., Wagh N.D., Patil S.V. (2015). Innovative approach for urease inhibition by Ficus carica extract-fabricated silver nanoparticles: An in vitro study // Biotechnology and applied biochemistry. 6: 780-784. DOI: 10.1002/bab.1341 (in Eng.).
[20] Useinov A., Kravchuk K., Maslenikov I., Reshetov V., Fomkina M. (2015). Issledovanie prochnosti mikroobektov s pomoshh'ju skanirujushhego nanotverdomera "NANOSKAN" [Nanoindustrija] 4:54-61 (in Rus.).
С. Ж. Ибадуллаева1, М. Г. Фомкина2, Н. О. Аппазов1, Л. Ə. Жусупова1
1Қорқыт Ата атындағы Қызылорда мемлекеттік университеті, Қызылорда, Қазақстан,
2 Ресей Ғылым Академиясының Теориялық жəне тəжірибелік биофизика институты, Пущино, Ресей
ҚАН ЖƏНЕ НЕСЕПТІ ТАЛДАУ ҮШІН ПОЛИМЕРЛІ ТЕХНОЛОГИЯЛАРДЫ ПАЙДАЛАНУ АРҚЫЛЫ МОЧЕВИНА БИОДАТЧИГІН ЖАСАУ
Аннотация. Полимерлі нанотехнологиялар негізінде сұйықтықтарда мочевинаны анықтай алатын ферментті тіркеуіштер мен микрореакторлар жасалды. Ферментті тіркеуішті жасау технологиясы laer-by-laer əдісімен ферментті микрокапсулалар жасаудың белгілі технологиясынан айтарлықтай ерекшеленбейді. Бұл бізге басқа авторлармен инкапсуляцияланған ферменттерден алынған мəліметтерін биотіркеуіш жасауда ақ- парат ретінде мүмкіндік береді. Мочевина биосенсоры ұзақ уақыт бойы ферменттің белсенділігін айтарлық- тай жоғалтпай ұзақ уақыт бойы (2 айға дейін) жұмыс жасай алатындығы табылды. Полимерлі технология басқа да ұқсас əдістерге қарағанда жеңіл жəне арзан болып табылады. Биологиялық сұйықтарды (қан, несеп) экспресс анықтау үшін полимерлі ферментті чипі бар мочевина анализаторы ұсынылады. Бұл жұмыста біз- дің ойымызшаайтарлықтай екі артықшылық факторы бар. Бірінші фактор – полиэлектролитті жабынға иммобилизацияланған функционалды-белсенді фермент алу жағдайын оңтайландыру, мұнда иммобилизация əрекетінен кейін фермент жаңа даярланған бос ферменттің белсенділігіне ұқсас белсенділік көрсетеді. Мұн-
дай нəтиже мочевина анализаторы қондырғысын жасауда сезімтал қабатты дайындауда ферменттерге кете- тін шығындарды арзандатады. Екінші фактор, ферменті бар полимерлі жабын ферментті электрод ретінде ғана жұмыс жасап қоймай, уреаза-мочевина каталитикалық реакциясы өткеннен кейін тіркеу жылдамдығын төмендетпей ферментті микрореактор ретінде де іс атқарады.
Түйін сөздер: ферментті биосенсорлар, полимерлі наноматериал, портативті анализатор, микрореактор, микрокапсулалар, мочевина.
С. Ж. Ибадуллаева1, М. Г. Фомкина2, Н. О. Аппазов1, Л. А. Жусупова1
1Кызылординский государственный университет им. Коркыт Ата, Кызылорда, Казахстан,
2Институт теоретической и экспериментальной биофизики Российской Академии наук, Пущино, Россия
РАЗРАБОТКА БИОДАТЧИКА МОЧЕВИНЫ С ПРИМЕНЕНИЕМ ПОЛИМЕРНЫХ ТЕХНОЛОГИЙ ДЛЯ АНАЛИЗОВ КРОВИ И МОЧИ
Аннотация. На основе полимерных нанотехнологий созданы ферментные датчики и микрореакторы, способные определять мочевину в жидкостях. Технология изготовления ферментного биодатчика сущест- венно не отличается от известной технологии изготовления микрокапсул с ферментом методом laer-by-laer.
Это позволяет нам при конструировании биодатчика пользоваться информацией, полученной на инкапсу- лированных ферментах другими авторами. Показано, что биосенсор мочевины способен работать в течение длительного времени (до 2 месяцев) без значительной потери активности фермента. Полимерная технология изготовления датчиков менее трудоемкая и дорогостоящая по сравнению с другими аналогичными техноло- гиями. Предлагаются к разработке биосенсорные приборы – анализаторы мочевины с полимерными фер- ментными чипами для экспресс-диагностики биологических жидкостей (кровь, моча). Одним из существен- ных результатов настоящей работы с нашей точки зрения являются два фактора. Первый фактор – это оптимизация условий получения функционально-активного фермента, иммобилизованного в полиэлектро- литное покрытие, когда фермент после процедуры иммобилизации показывает активность сравнимую с ак- тивностью свежеприготовленного свободного фермента. Такой результат позволит удешевить расходы на ферменты при создании чувствительного слоя разрабатываемого прибора-анализатора мочевины. И второй фактор, это то, что полимерное покрытие с ферментом способно работать не только как ферментный элект- род, но и как ферментный микрореактор, при этом не уменьшая скорость регистрации сигнала после про- хождения каталитической реакции уреаза-мочевина.
Ключевые слова: ферментные биосенсоры, полимерный наноматериал, портативный анализатор, мик- рореактор, микрокапсулы, мочевина.
Information about authors:
Ibadullaeva Saltanat Zharilkasynovna, professor of Department biology, geography and chemistry at the Korkyt Ata Kyzylorda State University, doctor of biological sciences, professor, Kyzylorda, Kazakhstan;
[email protected]; https://orcid.org/0000-0003-3270-8364
Fomkina Maria Grigorevna, leading researcher of laboratory of energy biological system at the Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, candidate of biological sciences, Pushchino, Russia; [email protected]; https://orcid.org/0000-0001-9905-8226
Appazov Nurbol Orybassaruly, head of laboratory of engineering profile at the Korkyt Ata Kyzylorda State University, candidate of chemical sciences, professor, Kyzylorda, Kazakhstan; [email protected];
https://orcid.org/0000-0001-8765-3386
Zhusupova Laila Azhibaevna, head of Department of ecology and chemical technology at the Korkyt Ata Kyzylorda State University, candidate of technical sciences, associate professor, Kyzylorda, Kazakhstan;
[email protected]; https://orcid.org/0000-0002-0561-2458
N E W S
OF THE NATIONAL ACADEMY OF SCIENCES OF THE REPUBLIC OF KAZAKHSTAN SERIES OF BIOLOGICAL AND MEDICAL
ISSN 2224-5308
Volume 6, Number 330 (2018), 13 – 19 https://doi.org/10.32014/2018.2518-1629.12
UDC 577.29 МРНТИ 34.15.29
N. D. Deryabina1,2,, D. A. Gritsenko1,2, N. N. Galiakparov1
1Institute of plant biology and biotechnology, Almaty, Kazakhstan,
2Al-Farabi Kazakh National University, Almaty, Kazakhstan.
E-mail: [email protected], [email protected], [email protected]
GENERAL CHARACTERISTICS OF INFLUENZA VIRUS A MOLECULAR STRUCTURE
Abstract. The influenza virus is one of the most abundant viruses in the world. It causes both mild seasonal infections and severe pandemics killing thousands of people and mammals. Two main extracellular receptors – neuraminidase (NA) and hemagglutinin (HA) are responsible for infection symptoms development and spread.
Error-prone RNA-polymerase incorporates mutations into both neuraminidase and hemagglutinin per replication cycle, which complicates the development of highly effective drugs against animal influenza. Incorporated mutations are also involved in the transition of influenza from animal to human species and vice versa. Transited influenza subtypes are the most dangerous, because it is unpredictable now, where the mutation might arise. However, it starts to become clear, which molecular regions are the most common for the mutation to occur.
This article revises the molecular structure of influenza extracellular receptors, including critical regions of receptors binding sites and susceptible mutation sites. The clear understanding of molecular structures and critical regions of HA and NA might facilitate the development of an effective vaccine and/or drug development.
Key words: influenza, neuraminidase, hemagglutinin, mutation, sialidase, virus.
INTRODUCTION. General description of influenza viruses. Influenza A viruses belong to a viral family of Orthomyxoviridae, what can be interpreted from Greek language as viruses which bind to mucoproteins. Influenza A viruses cause flu in many representatives of animal species, the most common hosts are: salmon, pigeon, poultry, fowl, especially chicken, swine, camel, bats and human. It should be considered that an avian virus does not cause such a wide range of symptoms in birds but as it is trans- mitted to humans, disease conditions might be very severe [1].
There are several types of influenza viruses: A, B and C types. Influenza A is the most studied and common type: has a wide range of hosts including mammals, fish and birds and is responsible for most flu epi- and pandemics. Influenza B virus infects only humans, and influenza C infects both human and swine [1, 2].
Influenza A virus causes a wide range of non-specific symptoms: high fever, sore throat, mild headache, chills, malaise, cough and muscular pain. These symptoms do not necessarily indicate that a person has flu. Otherwise, additional analyses should be certainly made to ensure a final diagnosis. Most harm done by influenza is towards infants and elders due to their suppressed or non-developed immune system. Sometimes it could be even fatal due to chronic illness or lack of special medical care and me- dicine. However, as vaccines and drugs are being improved, those fatal cases are getting much lower than in previous century [1].
There were several pandemics caused by initially swine or avian flu through the development of their transmittance to humans. It is uncommon that once in the future another pandemic virus would arise leading to enormous problems including lack of effective vaccine, inefficiency of available drugs, and may be financial losses. It is very hard to predict next pandemics and subtype of virus involved in particular, so detailed studies of viral subtypes might be a solution to overcome next potential pandemics.
Molecules mostly involved in the development of influenza infections are envelope proteins: hemag- glutinin (HA) and neuraminidase (NA). Most of the drugs against flu use HA or NA as their main targets through reducing their affinity to sialic acid, destroying covalent inter- and intramolecular bonding or by using antagonistic features. For example, zanamivir and oseltamivir are NA-inhibiting drugs [1].
So, in this review we would consider molecular structure, functions and subtypes of envelope glycoproteins HA and NA, discuss interactions between viral and host molecules and briefly mention how the transition from avian to human flu is established, which is the main cause of unexpected pandemics [1].
Hosts of influenza A virus. Influenza A virus has a variety of hosts. Well known examples are chic- ken, domesticated birds, swine, camels, horses, bats and humans.
There is a great variety of bird species susceptible to influenza A virus: ducks, wigeons, teals, mer- gansers, geese, swans, redshanks, gulls, grebes and etc. Most of the avian hosts are waterfowl and only several are terrestrial such as pigeons and chicken. The most known birds infecting influenza A subtypes are highly pathogenic H5N1 initially identified in Hong Kong, H7N3 outbreak in England and Australia in 1963 and 1992-1994 respectively, H7N7 several outbreaks in Germany, England, Australia and Netherlands and H5N2 with outbreaks in USA, Mexico, Italy and China.
Swine influenza is another major source of virus reservoir in wild environment. Outbreaks of swine influenza were firstly identified in Spanish influenza pandemic in 1918 year and had occurred several times after that. Major swine influenza subtypes: H1N1, H1N2 and H3N2 [3].
Equine influenza was described as a disease having similar symptoms as human influenza since Romanian times. It has potentially evolved together with human influenza virus due to their living in close proximity with horses, mules and donkeys. Influenza virus is still considered as the most important respiratory pathogen in horse and related species. The first isolate of equine influenza was obtained in Eastern Europe in 1956 year and identified as H7N7 subtype. Another example of horse influenza is H3N8, which was isolated in 1963 and since those times is considered as enzootic in Europe and Ame- rica [3].
Recently two novel influenza viral subtypes from bat species were identified and studied– H17N10 and H18N11. However, they are considered as influenza-like viruses, because their characteristics are rather distinct: a different binding site to sialic acid receptors and neuraminidase not being a sialidase [4].
However, human infecting influenza subtypes have emerged from animal infecting subtypes through the adaptation to the surroundings and incorporation of mutations per replication cycle. So, wild subtypes of influenza virus should be thoroughly studied to prepare and possibly prevent next pandemics.
General description of influenza A virus. Influenza virions have roughly spherical or filamentous shape. Newly synthesized ones have more filamentousvirions, and as they become mature, shape becomes roughly spherical. Gene segments are wrapped into helical nucleocapsid, which is then packaged into lipid envelope mostly derived from a host’s plasma membrane.
Influenza A viruses are encoded by six-eight strains of negative-sense RNA. RNAs are error-prone due to RNA-dependent RNA-polymerase, which drives rapid adaption and evolution of influenza [5].
Each of the genome segments encodes one or two proteins, functions of proteins encoded together on one gene segmentare rather similar. Envelope glycoproteins, which are located on the outermost layer, HA and NA are encoded by genome segments 4 and 6, respectively [1]. Three RNA polymerase proteins (PA, PB1 and PB2) are encoded by distinct genome segments from 1st to 3rd. 7thgene segment codes for an integral membrane protein (M1) with ion channel activity and an envelope protein by subsequent splicing of mRNA. Influenza A virus expresses 11 proteins in total, and 9 of them are packaged into new virions.
Two proteins, which are not packaged, facilitate assembly of viral particles.
Only in case all RNA gene segments are packaged into a viral particle, the virus is capable of survi- val and infection [5].
Influenza A virus replicates in the host nucleus, unlike most other RNA viruses such as bynuaviruses, paramyxoviruses and rhabdoviruses replicating in the cytoplasm. Viral mRNA of influenza stealscapped 5’ends of cellular mRNA in the nucleus, which facilitates rapid synthesis of new virion particles. Rapid evolution of these viruses is due to the presence of both error-prone RNA-polymerase and frequentreas- sortment (antigenic shift) of whole genome segments or some parts between related strains. This reassortment is the one most responsible of pandemics due to mutated surface antigens NA and HA [1].
Influenza A viruses are classified according to NA and HA subtypes, which together show different antigenic reactivity to poly- and monoclonal antibodies and show different nucleotide sequences [6].
The main difference between avian and human adapted viruses is their preferential binding to sialylated glycan receptors, hemagglutinin in particular. Human viral hemagglutinin exemplified by H1N1, H2N2, H3N2 subtypes preferentially binds to long α2-6 sialylated glycan receptors, which are mostly expressed in human upper respiratory epithelium. HA of avian influenza exemplified by H1N8, H2N9, H3N2, H3N8, H5N8 binds to short α2-3 sialylatedglycans. Due to this feature, there are only several avian influenza viruses capable tocause diseases in humansuch as H5N1, H7N7, H7N3, H9N2 se- rotypes. Differences in receptor specificity of human and avian influenza A viruses are also considered as features responsible for tissue tropism, host species barrier and interspecies transmission blocking. So, there is a need to constantly monitor these avian viruses to be able to detect changes in external glycan structure as they play the most important role in influenza evolution [6].
Swine influenza viruses are able to bind sialic acid in both α2-3 and α2-6 linkages, so they combine features of both human and avian viruses.
HA and NA both recognize sialylated receptors on the outside of a host cell membrane. Influenza infection is promoted by multiple HA binding to sialic acids found on the carbohydrate side chains outside regions: on surface glycoproteins and glycolipids. NA’s primary function is to release accidentally bound newly synthesized HA from sialylated glycoproteins and glycolipids, however it performs other functions as well.
Neuraminidase. NA is a viral cell surface receptor of a tetramericglycoprotein nature. It is encoded by 6thgene segment of influenza A genome. Its length is approximately 1413 base pairs in length with slight variations, mature protein size is 454 Daltons.
It consists of four identical polypeptides of approximately 470 amino acids with slight variations in sequence. Four domains of this protein are a membrane-anchoring hydrophobic domain, a thin variable stalk, a globular head domain which is a carrier of enzyme active site and a calcium-binding site [7].
The stalk domain’s length varies significantly, and its shortening is associated with adaptation of waterfowl to poultry [8]. Subunits consist of six bladed propeller-like structures, and blades are made up of four antiparallel β-strands [9]. An enzyme active site with conserved charged amino acid residues can be found in the central region of each subunit.
The function of NA is to cleave sialic acid residues from cellular and viral glycoproteins expressed outside the host cell membrane. It is crucial to prevent HA mediated aggregation of newly synthesized viral particles at the surface right after their leakage from damaged host cell, because this would prevent further dissipation. NA fulfills that function by removing newly synthesized HA, which were accidentally bound to sialylatedreceptors of dead cell [10].
Besides the release of budding virion particles through HA release, NA plays a role in promoting cellular infection by promoting glycosylation of the HA and cleaving potential inhibitory Sia-s from mucins. It was shown, that NA recognizes sialic acid residues on host glycoproteins and glycolipids in a different manner in comparison with HA [11].
Different structural features of NA and HA form influenza subtypes.
Neuraminidase subtypes. 10 structurally different NA circulate in birds, which are classified into two main groups [12]. The group 1 includes N1, N4, N5 and N8, and group 2 includes N2, N3, N6, N7 and N9. The classification is based on similar features within the following regions: in the 150-loop (residues 147-152), the 270-loop (residues 267-276), and the 430-loop (residues 429-433), which are regions adjacent to the enzyme’s active site.
The only conserved site for all influenza A and B types NA is Asn146 glycosylation region, which is located on the membrane-distal surface close to the active site [13]. Besides that highly conserved region, neuraminidase shows a great diversity in nucleotide sequence, which results in various structural con- formations. Mutations incorporated per replication cycle add changes into already present pool of neura- minidase structural diversity.
Hemagglutinin. Name of this protein comes from its ability to form aggregates of red blood cells, the carriers of hem – hem agglutinating protein. Widely spread, rapid and moderately sensitive technique of most viruses’ identification – is the hemagglutination assay, in which hemagglutinin glycoproteins
clump cells by binding to their surface receptors. There are 18 subtypes of hemagglutinin, and two recent ones H17 and H18 were found in bats [14].
HA is expressed as a trimeric surface receptor on the outside of the viral membrane. It facilitates entry through the receptor binding to the target cell and recognizes sialylated cell receptors for conse- cutive chemical binding. It is encoded by 4th gene segment of influenza A viral genome with approxi- mately 566 nucleotides in length, although some nucleotide variations are possible.
Sialylated receptor-bound viruses fuse with the cell membrane, and then are engulfed into endosomes once entering cytoplasm. HA becomes acidified by endosomal enzymes, which is followed by confor- mational changes in its molecular structure and subsequent activation. Different kinds of conformational changes constitute HA subtypes classification system.
The linkage between glycan structures and galactose is crucial in host determination. Avian viruses are characterized by binding to α2-3Sia and are so-called avian-type receptors, while mammalian viruses bind to α2-6Sia and are so-called human-type receptors. However, it should be noted, that cells in human upper epithelium mostly possess α2-6Sia receptors, whereas cells in lower epithelium possess α2-3Sia cell surface receptors. This means that influenza bearing only avian-type receptors is able to cause a moderately mild infection without any serious consequences.
The schematic representation of hemagglutinin structure [15]
However, influenza viruses typically reproduce in cells of human upper respiratory tract through the recognition of sialic acid, or N-acetylneuraminic acid, by HA. N-acetylneuraminicacid is terminated by glycan structures, which are linked to galactose in a β1-4 linkage to glucosamine, and thislinkage in particular is associated with HA recognition and binding to the target cell [10, 16].
The HA is a homotrimer consisting of a globular head with sialic acid binding domain and a fibrous stalk region. Three identical subunits have resulted from proteolytic cleavage of a single precursor, and the process occurs by cleaving single arginine residue extracellularly by serine proteases before entering the host cell. However, some members of H5 and H7 subtypes have acquired several cleaving residues (arginine and lysine), which is partly responsible for H5 and H7 high infectivity and pathogenicity [9].
The coiled-coil structure of the stalk domain stabilizes HA trimers and anchors the protein in the membrane through its transmembrane subdomain [12]. The only conserved amino acid throughout all subtypes of the stalk domain is Lys51.
As it was mentioned before, HA recognizes sialylated cell surface receptors of a target cell, so let’s consider these interactions more closely. The interaction occurs through hydrophobic and hydrogen bonding between HA residues from the 130-, 220-loops, 190-helix and sialylated receptors [17].
Sialic acid binding site contains four main structural regions made up from antiparallel β-sheets [12]:
a base with highly conserved Tyr98, Trp153 and His183, a 190-α helix (residues 184-190), a 130-loop (residues 126-135) and a 220-loop (residues 215-224) [19].
Amino acids Tyr98, Trp153, His183, Glu190 and Tyr195 directly interact through hydrogen bonding with the side chains of sialic acid, which was shown on H3 subtype in particular.
The 130-loop with crucial residues at 135-137 forms chain interactions with receptor’s sialic acid moiety. Mutations within 220-loop constitute differences in host specificity due to slight changes in loop conformation associated with glycosidic linkage type [20]. The 190-helix plays a role in species speci- ficity determination. Double mutations in the HA receptor binding domains of H1N1 at Glu190Asp and Gly225Asp and H2N2/H3N2 at Gln226Leu and Gly228Ser influenza A subtypes are associated with the adaptation of avian viruses into human pandemic viruses.
In addition, four amino acid substitutions of HA in H5N1 at Ser123Pro, Ser133Ala, Thr156Ala, and Gln192Lys are associated with increased binding of the virus to mammalian receptors [20]. Mutation of Asn158 and Thr160 were shown to increase virus affinity to human-type receptors due to the loss of the same glycosylation site on the top of the HA globular head.
In HA2, HA3 and HA9 subtypes Leu226 enables influenza A virus replication in the human airway epithelium.
HA binds to sialic acid through hydrophobic interactions and hydrogen bonding to the conserved amino acids within 130- and 220-loops, although responsible amino acid residues differ from one subtype to another [9]. For example, in HA1, glutamic acid and glycine residues at positions 190 and 225, respec- tively, are responsible for binding to avian SIA-receptors, whereas HA1 proteins that carry aspartic acid residues at these two positions interact with human SIA receptors. For HA2 and HA3, mutations of Gln226Leu and Gly228Ser correlate with a shift from avian to human receptor specificity [21]. So, slight mutations in amino acid sequenceconstitute the basis of HA subtypes classification.
Hemagglutinin subtypes. Two main groups of 18 HA circulatingin many different hosts are classi- fied according to sequence comparisons and structural characteristics. Group 1: H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16. Group 2: H3, H4, H7, H14, H15 and H10 [22]. The classification also considers conformational changes within HA molecular structure triggered by acidification due to endosomal enzymes functioning.
Ha subtypes could be also relatively classified according to their main host species. For instance, HA1, HA2 and HA3 subtypes circulate mostly in human populations.
A computer analysis by using free access applications has shown close evolutionary relationships between HA subtypes[14]. Very close relationship was shown between HA7, HA15 and HA10 constitu- ting one clade and HA4, HA14 and HA3 constituting another. The common origin was shown for HA8, HA12 and HA9 as well as for HA13, HA16 and HA11[14]. Recently described HA17 and HA18 subtypes show closest evolutionary relations towards another clade including HA1, HA2, HA5 and HA6. This evolutionary relationship formed during last century indicates rapid evolution of influenza viruses due to reassortment between different viral subtypes.
Transition between avian and human influenza types. The transition between avian and human influenza types had occurred several times and had caused several pandemics during human history.
The transition between avian and human influenza types is not so uncommon because of several reasons underlying this process. They include high degree of genetic recombination between different subtypes, error-prone RNA polymerase, which enables a multitude of mutations to occur per replication cycle, rapid generation time of virions and fast replication of virion particles inside of a host nucleus.
The major receptor binding site substitution between avian and human HA10 is Lys137Arg, although some others might also emerge due to incorporation of mutations per replication cycle. Such transitions between avian/swine/human influenza should be studied in details for the trafficking influenza evolution [22].
Conclusion. So far we have discussed some important structural features of HA and NA – envelope proteins responsible for influenza A virus infection occurrence and spread. HA plays a significant role in binding to receptors in host’s upper epithelium cells. This is the most common way for a virus to enter a cell. NA’s primary function is to release newly synthesized virions from sialylated receptors of host cell.
However, NA is also responsible for facilitating HA’s glycosylation for viral infection spread. These two
