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Research of algal polysaccharides and related natural compounds in Estonia/Vetika polusahhariidide ja nendega seonduvate looduslike uhendite uurimine Eestis.



INTRODUCTION

Although some natural algal algal

pertaining to or caused by algae.


algal infection
is very rare but systemic and udder infections are recorded. See protothecosis.

algal mastitis
the algae Prototheca trispora and P.
 polysaccharide polysaccharide: see carbohydrate.
polysaccharide

Any of a large class of long-chain sugars composed of monosaccharides. Because the chains may be unbranched or branched and the monosaccharides may be of one, two, or occasionally more kinds,
 mixtures have been used already for centuries, their fractionation fractionation /frac·tion·a·tion/ (frak?shun-a´shun)
1. in radiology, division of the total dose of radiation into small doses administered at intervals.

2.
 and structural investigations were started quite recently. Utilization of agar in bacteriology bacteriology

Study of bacteria. Modern understanding of bacterial forms dates from Ferdinand Cohn's classifications. Other researchers, such as Louis Pasteur, established the connection between bacteria and fermentation and disease.
 began more than a hundred years ago since the famous works of Robert Koch [1]. Structural research of agar took its beginning from investigations of Araki [2] and lasted about thirty years, until the end of the 1960s [3]. In the course of this troublesome study series, which involved purely chemical methods, a unique component sugar, 3,6-anhydrogalactose, was discovered [4] and an idealized extreme structure type of agarose agarose

more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments.
 (Fig. 1) was defined [5].

[FIGURE 1 OMITTED]

Full complexity of agars as a continuous spectrum of polysaccharides, depending on their natural source, was established by Duckworth & Yaphe in the early 1970s [6, 7]. Also, some generalizations regarding the structure of gelling galactans were made. Among them was the hypothesis of "masked repeating structure" [8] inherent in all the galactans, and extensive elucidation of gelling properties, principles, and mechanisms [9] in hydrated galactan systems.

Briefly, such was the general scientific background in the field of galactan chemistry of the middle of the 1970s when the research project of red algal galactans was started at the Institute of Chemistry, Estonian Academy of Sciences Founded in 1938, the Estonian Academy of Sciences (Estonian: Eesti Teaduste Akadeemia) is Estonia's national academy of science. As with other national academies, it is an independent group of well-known scientists whose stated aim is to promote research and development, . This research into galactans, especially agars, was the beginning of investigations of polysaccharides in Estonia. Later, some other related subjects such as alginate alginate /al·gi·nate/ (al´ji-nat) a salt of alginic acid; water-soluble alginates are useful as materials for dental impressions.  and lectin lectin /lec·tin/ (lek´tin) any of a group of hemagglutinating proteins found primarily in plant seeds, which bind specifically to the branching sugar molecules of glycoproteins and glycolipids on the surface of cells.  chemistry, applications in affinity chromatography Affinity chromatography is a chromatographic method of separating biochemical mixtures, based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. , immunochemical im·mu·no·chem·is·try  
n.
The chemistry of immunologic phenomena, as of antigen-antibody reactions.



im
 investigations, etc. were added to the main galactan topic. Also, the scope of galactan research was broadened to include carrageenans of the Baltic Sea Baltic Sea, arm of the Atlantic Ocean, c.163,000 sq mi (422,170 sq km), including the Kattegat strait, its northwestern extension. The Øresund, Store Bælt, and Lille Bælt connect the Baltic Sea with the Kattegat and Skagerrak straits, which lead to the , especially the furcellaran from the red alga red alga
n.
Any of various predominantly marine algae of the division Rhodophyta, characteristically red or reddish in color.



red alga  
 Furcellaria lumbricalis.

It should be mentioned that the scope of all the activities described below was exceptionally wide. The whole research process might be viewed from the following aspects.

1. Structural types of the compounds investigated: galactans (agars, agaroses, carrageenans) and products of their modification, polyuronates (alginates) and lectins.

2. Types of methods used: purifying, fractionating, and beading techniques; methods for the determination of the structure and composition of polysaccharides; structure modification; activation and cross-linking methods; technological elaborations on the pilot production level; immunological methods involved; etc.

3. Major research topics (connected with usability and/or application parameters of elaborated polysaccharide systems and carriers): kinetics (of algae algae (ăl`jē) [plural of Lat. alga=seaweed], a large and diverse group of primarily aquatic plantlike organisms. These organisms were previously classified as a primitive subkingdom of the plant kingdom, the thallophytes (plants that  extraction, gel forming, and activation); rheology (viscosity, gel strength); separation techniques tested (gel filtration, affinity chromatography, fractionating precipitation, etc); immunology (especially separation and specific activity preservation of antibodies); and some others.

4. Biological background: investigated algae species and habitats; dependence of the polysaccharide content of algae on the collection time; expeditionary activities of the groups involved; etc.

5. Organizational activities and institutions involved; cooperation projects; participation in (or organizing of) symposiums and other meetings; etc.

Of these five aspects, the two last are discussed together in the next section. The remainding three aspects are treated later.

The aim of this article is to analyse the development of the research into polysaccharides and related compounds in Estonia and to summarize the main innovative and scientific trends and results of 25 years of this complex activity.

EXPEDITIONARY AND ORGANIZATIONAL PRELIMINARIES

The whole cycle of investigations concerning algal polysaccharides algal polysaccharides,
n.pl noncellulose polysaccharides that are found in food additives and provide dietary fiber. These include algin, agar, and carrageenan.
 was introduced by elucidation of promising seaweed species containing a nearly neutral galactan fraction close to agarose (Fig. 1). For collecting samples of various algae several series of expeditions were organized. Because of the political situation of Estonia in the 1970s, the area for possible expeditions was strictly limited to the territory of the then Soviet Union. However, this large region had a very extensive coastline and many bodies of water.

Expeditions were organized yearly from 1976 to 1990, mainly in summer or autumn, in some years simultaneously by different groups (later less often). As a final result, all the large bodies of water were observed on the whole territory of the Soviet Union. At the end of the investigations attention was focused on the Far East. The major destinations of expeditions by years were as follows:

--The region near Vladivostok (Primorye and/or islets in Amur Bay, the Sea of Japan: Popov Island, Reineke Island): 1976-78, 1981-84, 1988.

--South Kamchatka: 1977, 1978, 1982, 1987, 1988.

--Sakhalin and/or the South Kuril Islands Kuril Islands (kyr`ēl, krēl`) or Kuriles (ky  (Kunashir and Iturup): 1978, 1983-86.

The extreme North-East Asian region was the target of investigations in 1977 (Bering Island Bering Island (bēr`ĭng, bâr`–), Rus. Beringa, largest of the Komandorski Islands, c.55 mi (90 km) long and up to c.15 mi (20 km) wide, off Kamchatka peninsula, E Russian Far East, in the Bering Sea. , Provideniya), in 1979 (Anadyr, Uelen, Ratmanov Island), and 1981 (Cape Schmidt, Nutepelmen). The region of Lake Baykal in Siberia was investigated in 1982.

In Europe, extensive shorelines of the Black Sea and Caspian Sea were researched mainly in 1980 and White Sea regions (Solovets Islands, Arkhangel'sk) in 1985. The Baltic Sea areas (Kassari Bay, Saaremaa Island, etc.) were studied mainly in 1993-2000. Also some other trips were made, for instance to the Aral Sea, Lake Il'men (close to Novgorod), the Barents Sea (near Murmansk), and Lake Sevan (Armenia).

As a result of the expeditionary activities, an exceptionally wide area of Eurasia was fully sampled, especially from the standpoint of resources for potential industrial use of red macroalgae containing gelling galactans of various structure and rheological properties. In case of need, algal strata were observed and procured by using aqualungs (including in such extreme northern regions as the Chukotsk Peninsula). Local practices of storing, preparation, and processing of seaweeds were studied on the spot (all the major agar/agaroid factories of this tremendous territory were visited). Seaweeds were gathered by researchers themselves, which guaranteed the necessary quality and sure geographic origin of the samples. Later, the samples served as a suitable raw material for pilot production of agarose and affinity chromatography carriers. A variety of large algae samples for voluminous extracting investigations were collected. Some preliminary experiments on artificial cultivation of red macroalgae (Ahnfeltia tobuchiensis and Gracilaria verrucosa) were also undertaken (in Tallinn). However, artificial cultivation appeared to be too expensive for extensive use.

Persons and groups participated very dynamically in the course of the development of the polysaccharide programme. The whole project was initiated by Andres Kollist in 1975; some key persons in Tallinn (Aavo Aaviksaar, Jaan Paris, Merike Vaher) and Tartu (Tonu Pussa) were involved from the very beginning. From time to time, numerous persons and groups from several biochemical and medical research institutions in (or outside) Estonia joined in. For example, all in all 55 persons took part in the complex programme "Agarose and spheron carriers for chromatography" supervised by Kollist [10], and accomplished in 1986-90. They were mainly from the Institute of Chemistry of the Estonian Academy of Sciences and the University of Tartu At different times during its history the University of Tartu was known as Academia Gustaviana, University of Dorpat, '' Universität (zu) Dorpat, University of Yuryev, and Tartu State University (Tartu Riiklik Ülikool)''.  (in addition, some people from the Institute of Chemical and Biological Physics, Tallinn, and from N. F. Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia, were involved).

Nevertheless, the main group of algal polysaccharide researchers at the Institute of Chemistry was not numerous. The group was formed and supervised by Andres Kollist in 1975-90 (since 1991 by Kalle Truus) and consisted of 11 persons as a maximum. Two smaller groups were very important as well, especially in the 1980s: one at the University of Tartu (Tonu Pussa, Peeter Toomik, Riina Mahlapuu and others) and the other at the Institute of Chemical and Biological Physics (Mati Haga and others).

The members of the groups from the Institute of Chemistry and from the University of Tartu took part in organizing expeditions and conferences. The 11th International Lectin Meeting (Tallinn-Tartu 1989) with participants from 35 countries [11] deserves special mentioning.

ALGAL EXTRACTING AND TESTING OF POLYSACCHARIDES

One of the initial aims of the red algal galactan investigations was to elucidate some important matrix properties of polysaccharides in the seaweeds growing on the territory studied. The characteristics of main interest were charge density of the matrices and average molecular mass of the polysaccharides. These characteristics determine many important properties in practical work, such as rheological behaviour (viscosity, gel strength, etc.), interactions with proteins, and so on. On the other hand, the molecular as well as rheological properties of the galactans from algae are closely connected to the extracting dynamics of algae, because seaweeds do not contain any certain compound but a wide spectrum of similar polysaccharides. The group had also an aim to elaborate isolation and purification methods for the fractionation of possibly homogeneous galactans.

The first results of the work were presented at meetings of young scientists, at conferences of the Baltic States and of the USSR USSR: see Union of Soviet Socialist Republics.  (13 abstracts in 1977-80). A series of six articles [12-17] was published in 1980.

Seven most promising algae species from this extensive Eurasian region were selected and treated in publications. Gelling substances were isolated from the following seaweeds [12]: (1) Furcellaria lumbricalis I (Hiiumaa Island in the Baltic Sea), (2) Ahnfeltia plicata and (3) F. lumbricalis II (Solovets Islands in the White Sea), (4) Phyllophora nervosa (Odessa Region in the Black Sea), (5) Ahnfeltia tobuchiensis I and (6) Gracilaria verrucosa (Gulf of Peter the Great in the Sea of Japan), and (7) A. tobuchiensis II (Kunashir Island, the Kurile Archipelago in the Pacific). According to the extraction kinetics of gelling galactans, the studied seaweeds were divided into two groups (algae species 1-4 and 5-7). Further, the changes of galactan parameters in the course of algae processing and in the fractionation of the gelling substances were investigated. Special attention was paid to the dependence of gel strength on the homogeneity and sulphur content of the galactans [13].

It was shown that the characteristics of laboratory agars and their quality strongly depend on the extraction time. A large number of algae species were tested and some fractions of very high gelling properties were selected. Agars with the highest gel strength and satisfactory electroendoosmosis (EEO EEO Equal Employment Opportunity
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EEO Extremely Elliptical Orbit
EEO Exotic Electro-Optics, Inc.
) properties were obtained from certain fractions of the red algae A. tobuchiensis (I and II) and G. verrucosa. The qualities of these agars surpass the corresponding qualities of all commercial agars [14].

At that time the relationships between various gelling properties of algal galactans were not exactly known yet, especially in the case of the seaweed species mentioned. For this reason, some correlations of rheological parameters were elucidated in the following papers of this series [15-17]. Also, some agarose fractions were already isolated from agars of A. tobuchiensis [15] and characterized by their EEO properties and zone electrophoresis zone electrophoresis Any electrophoretic technique in which components are separated into zones or bands in a buffer, and stabilized in solid, porous, or any other support medium–eg, filter paper, agar gel, or polyacrylamide gel  of human serum proteins [16]. Very promising results in this field led to some practical solutions as well: synthesis of affinity carriers on an original agarose matrix basis never used for this purpose before. These agarose sorbents found wide use in biochemistry and medicine from the early 1980s onwards (will be observed later). Further, some more extensive generalizations were presented [18] about correlations between the gel strength, average viscometric vis·com·e·ter  
n.
An instrument used to measure viscosity. Also called viscosimeter.



[Short for viscosimeter.
 molecular mass of galactans, and their EEO characteristics (-[m.sub.r]).

It was shown that the gel mechanical strength (lg G) for chemically similar agaroses increases linearly with increasing average viscometric molecular mass of the polysaccharide. When correlating the deviations ([DELTA]lg G) of the points for agars and agaroses with the EEO characteristics it was observed that the points fall into two groups. In the interval where -[m.sub.r] > 0.2 an increase in -[m.sub.r] is accompanied by a decrease in gel strength and at the values of -[m.sub.r] < 0.2 the opposite occurs (Fig. 2). The voluminous experimental results revealed also that the algae investigated initially do not contain gelling galactans of average molecular mass in the interval of 60-140 thousand [18]. Later, the conclusions of this work were analysed by Norwegian scientists [19]. Mostly on the basis of the six articles cited, Kollist defended his Cand.Sci. degree in 1981 [20].

[FIGURE 2 OMITTED]

INVESTIGATIONS OF THE COMPOSITION AND STRUCTURE OF ALGAL POLYSACCHARIDES

Some technical analyses of the composition (ash and sulphur content mainly) of galactans have been performed almost from the beginning of the project. The group started investigations on the integral structure of agarose in the second half of the 1980s. The findings were first presented at some all-USSR symposiums and at Eurocarb VI and published in the abstracts of these meetings [21-23]. The results of the study of the structure of galactans, mainly agaroses, with some additional aspects were published also elsewhere [24, 25].

These structure investigations in Estonia were based already on [sup.13]C-NMR spectroscopic spec·tro·scope  
n.
An instrument for producing and observing spectra.



spectro·scop
 methods, which have been applied to gelling polysaccharides from the end of the 1970s. This approach, developed by two independent groups [26, 27], was found to be exceptionally fruitful and is now an obligatory component for the elucidation of galactan structures instead of very troublesome chemical procedures. In our first studies, [sup.13]C-NMR spectroscopy was used to investigate structural differences between commercial preparations of agarose of various firms. By detecting the basic signal assignments of the disaccharide disaccharide /di·sac·cha·ride/ (di-sak´ah-rid) any of a class of sugars yielding two monosaccharides on hydrolysis.

di·sac·cha·ride
n.
 unit with high accuracy it was shown that the quite low methylation methylation,
n a phase-II detoxification pathway in the liver; methyl groups combine with toxins to rid the body of various substances.

methylation
(meth´
 degree actually present in agaroses does not cause, contrary to earlier results, differences in chemical shift values in the cases of real matrices [24].

In spectroscopic investigations various methods of methoxyl and sulphur content determination were comparatively applied and a large number of agars and agaroses were tested [K. Truus & M. Limberg, unpubl.] since the content and location of methyl and sulphate groups in disaccharide units are the major causes of structural and rheological differences between various galactans. Later, chemical analysis combined with NMR NMR: see magnetic resonance.  spectroscopic methods was applied also to other polysaccharides, particularly to furcellaran [28-30]. At present, alginates from the Baltic brown alga Fucus vesiculosus Fucus vesiculosus,
n See kelpware.
 (Fig. 3) are being studied by these methods.

Reliability and advantages of various methods of functional group analysis in algal galactans have been investigated during a long period. The data were summarized and analysed with structure determination methods including NMR spectroscopy and X-ray diffraction analysis [31]. Receiving high-quality agarose films and their orientation (stretching out) prior to X-ray analysis caused some problems. This orientation is important for obtaining regular and informative microzones in isotropic Refers to properties that do not differ no matter which direction is measured. For example, an isotropic antenna radiates almost the same power in all directions. In practice, antennas cannot be 100% isotropic.  agarose films. As a matter of fact, there was only one research paper concerning agarose orientation [32] at that time. Nevertheless, stretching methods were elaborated and satisfactory rontgenograms were achieved [33]. Also, a computer-controlled stretching device was constructed, and a package of programs was compiled for calculating cylindrical Patterson functions with their graphical presentation and finding the atom coordinates of agarose by the method of linked atoms [34].

[FIGURE 3 OMITTED]

Structural investigations of carrageenans (Fig. 4) from Baltic red macroalgae were carried out in the second half of the 1990s. The results were presented at some European symposiums [28-30, 35] and summarized in papers [36-38]. Also some aspects of the structure-functionality relationship were treated in these papers.

It was shown that the galactan mixture in F. lumbricalis (furcellaran) from Kassari Bay contains 6-O-methylgalactose and 2-O-methyl-3,6-anhydrogalactose in addition to the unsubstituted residues in their composition (Fig. 5). The main structural features of this complex polysaccharide are presence of kappa carrageenan car·ra·geen·an or car·ra·geen·in
n.
Any of a group of closely related colloids derived from several red algae, widely used as a thickening, stabilizing, emulsifying, or suspending agent in pharmaceuticals.
 [37] and absence of iota fragments in furcellaran [38].

The low gel strength of the native (water-extracted) furcellaran may be considerably (about five times) increased by alkaline extraction of the seaweeds. From this standpoint, the most suitable medium for extraction with simultaneous modification of these algal galactans is potassium hydroxide potassium hydroxide, chemical compound with formula KOH. Pure potassium hydroxide forms white, deliquescent crystals. For commercial and laboratory use it is usually in the form of white pellets.  solution at a concentration of 0.16 M. Another red alga occurring in Kassari Bay, Coccotylus truncatus, may also be used for the production of a gelling galactan mixture by alkaline extraction without any structural modification of the carrageenans from this species [37]. It was shown that C. truncatus contains viscous galactans of iota carrageenan type, their structure is close to those from Phyllophora brodiaei (the Black Sea) [39].

This comparative structural approach was summarized as a Master's thesis in 1999 [40]. The thesis elucidated the chemical composition (including microelements, sulphur, and monosaccharide monosaccharide: see carbohydrate.
monosaccharide

Any of the simple sugars that serve as building blocks for carbohydrates. They are classified based on their backbone of carbon (C) atoms: Trioses have three carbon atoms, tetroses four, pentoses
 residues), the localization Customizing software and documentation for a particular country. It includes the translation of menus and messages into the native spoken language as well as changes in the user interface to accommodate different alphabets and culture. See internationalization and l10n.  of sulphate and methyl groups in the repeating disaccharide units, and the structure of the latter. In this context, the criteria for the gelling ability of the carrageenans were specified.

During a long period the content of micro- and trace elements Trace elements
A group of elements that are present in the human body in very small amounts but are nonetheless important to good health. They include chromium, copper, cobalt, iodine, iron, selenium, and zinc. Trace elements are also called micronutrients.
 in algal polysaccharides and in seaweeds has been investigated mainly by methods of neutron activation analysis Neutron Activation Analysis (NAA) is a nuclear process used for determining certain concentrations of elements in a vast amount of materials. NAA allows discrete sampling of elements as it disregards the chemical form of a sample, and focuses solely on its nucleus. .

As to agarose, the main aim was to give a possibly complete characterization of the real natural matrix and develop its purification process. The level of biologically active microelements in agarose carriers is also of interest for finding them applications in biochemistry and biotechnology [41]. Depending on objects, interests, and possibilities, the content of more than 30 elements has been analysed. To follow the changes in the content of micro- and trace elements in agarose processing, the concentration of 22 elements and the dynamics (purification multiplicity) of elemental composition were studied [42].

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

It was shown that various elements get detached very differently in the course of agarose isolation and purification process. As a rule, no strong dependence upon an element's position in the periodic table was observed. Some of the elements (Na, Mg, Zn, etc.) have a considerable affinity to the agarose matrices and they cannot be removed below a certain level during the purification process. However, halogens (especially iodine) are easy to separate from agarose [42] as well as from carrageenans [38] and they should not have a noticeable affinity to these galactan matrices. Consequently, the unusually high concentrations of iodine and bromine bromine (brō`mēn, –mĭn) [Gr.,=stench], volatile, liquid chemical element; symbol Br; at. no. 35; at. wt. 79.904; m.p. –7.2°C;; b.p. 58.78°C;; sp. gr. of liquid 3.12 at 20°C;; density of vapor 7.  in algae (typically, 10-1% as an order of content for both halogens) should be related to other chemical structures of the cells.

As to brown algae of the Baltic Sea, it was elucidated that the mineral composition of the biomass from F. vesiculosus is rich in light elements (Mg, K, Ca, Na) and halogens (Br, I). Its arsenic content is also high (over 300 ppm); however, the content of other toxic elements (Hg, Sb, Se, etc.) is extremely low. The mineralization of the algal biomass practically does not depend on seasonal varieties. The content of [alpha]-L-guluronic acid residues, and therefore the viscosity of alginates from the Baltic F. vesiculosus, is relatively low [43].

AGAROSE CARRIERS FOR GEL FILTRATION AND AFFINITY CHROMATOGRAPHY

When our project was started agarose carriers were introduced to gel filtration and affinity chromatography [44]. The main stages of the preparation of novel agarose carriers are as follows: (1) finding a natural agarose matrix suitable for biochemical applications and (2) elaboration of beading (granulation granulation /gran·u·la·tion/ (-shun)
1. the division of a hard substance into small particles.

2. the formation in wounds of small, rounded masses of tissue during healing; also the mass so formed.
) procedures to give good flowing characteristics for the eluent eluent

the solution used in elution.
 to the gel filtration media. These two kinds of procedures may be followed by (3) cross-linking of the matrix for raising the mechanical strength of agarose beads and (4) activation of the granulated agarose gel to obtain carriers for affinity chromatography.

For conclusive elucidation of a suitable matrix, some technical problems faced in beading and cross-linking of the novel agarose were resolved and a starting material with good flowing characteristics (for eluent) was obtained. Single agarose gel beads of various concentration (mainly 2, 4, and 6% of the agarose content) were tested for use in gel filtration techniques. Because of the high gel strength of the original agarose from the alga A. tobuchiensis (the Gulf of Peter the Great, the Sea of Japan), the granulated medium guarantees a high flowing velocity of the eluent under a liquid column pressure. This galactan matrix has also other advantageous characteristics (particularly, a low charge density) for use in liquid chromatography, being comparable to other best agarose carriers [17].

The novel agarose was activated by methods known before: using cyanogen bromide cyanogen bromide

reagent used to fragment peptide chains, since it cleaves only those peptide bonds in which methionine contributes the carbonyl group. Also used as coupling agent in affinity chromatography where it reacts with the Sepharose matrix forming a reactive product to
 [45, 46], divinyl sulphone Sul´phone

n. 1. (Chem.) Any one of a series of compounds analogous to the ketones, and consisting of the sulphuryl group united with two hydrocarbon radicals; as, dimethyl sulphone, (CH ) s>.SO .
 [47], 1,1'-carbonyldiimidazole [48], active dyes [49], and some others (Table 1). Also, some original activating agents were introduced and new affinity carriers were developed.

The activated matrices were used directly or additional spacers via specific groups were coupled. The spacers may permit the use of milder binding conditions or a more suitable attachment of a ligand (due to the specific steric steric /ste·ric/ (ster´ik) pertaining to the arrangement of atoms in space; pertaining to stereochemistry.

ster·ic or ster·i·cal
n.
 conditions).

A new structural type of affinity carriers was worked out in collaboration with N. F. Gamaleya Scientific Research Institute of Epidemiology and Microbiology. The carriers called Odigose and Odifil (ethylsulphoactivated agarose, Fig. 6) are preparations with variable degree of active groups, suitable for synthesis of antigenic immunosorbents [50]. It was established that some antibodies and enzymes such as muscle aldolase aldolase /al·do·lase/ (al´do-las)
1. aldehyde-lyase.

2. an enzyme that acts as a catalyst in the production of dihydroxyacetone phosphate and glyceraldehyde phosphate from fructose 1,6-bisphosphate.
 retain their specific activity after immobilization Immobilization Definition

Immobilization refers to the process of holding a joint or bone in place with a splint, cast, or brace. This is done to prevent an injured area from moving while it heals.
 on these sorbents [51]. Technological regulations for the production of ethylsulphoactivated agarose and antigenic immunosorbents on its basis were worked out at the Institute of Chemistry in 1986-87 [K. Truus, unpubl.]. Also a method for the isolation of medicinal [gamma]-globulin preparations against Staphylococcus staphylococcus (stăf'ələkŏk`əs), any of the pathogenic bacteria, parasitic to humans, that belong to the genus Staphylococcus. The spherical bacterial cells (cocci) typically occur in irregular clusters [Gr.  from human plasma by these immunosorbents was elaborated with N. F. Gamaleya Institute and the Institute of Sera and Vaccines of Ufa (Bashkiriya) [52].

Another type of sorbents for medical use was elaborated with the researchers of the Oncological Scientific Centre (Moscow, Russia) for the isolation of a cancer-embryonic antigen from tumour tissue [53].

In many cases traditional carriers were synthesized and used, the reaction dynamics, optimum conditions, and/or possible mechanisms were studied. The activation kinetics of the reaction with some agents such as cyanogen bromide and 4-([beta]-oxy-ethylsulphonyl)-2-aminoanisole was thoroughly investigated [J. Paris & K. Truus, unpubl.] but insufficiently published [31, 54, 55]. From the extensive investigations only some brief overviews, mainly about the synthesis of agarose-based ion exchangers [56-58], were published. The activation dynamics of the agarose matrix with 1,1'-carbonyldiimidazole (CDI CDI compact disc interactive: a system for storing a mix of software, data, audio, and compressed video for interactive use under processor control ) and the yield of the carrier (CDI bound) in various conditions were investigated [59]. An extensive overview on affinity carriers [60] was published by researchers from the University of Tartu at the same time.

[FIGURE 6 OMITTED]

APPLICATIONS AND ELABORATION OF TECHNOLOGICAL SCHEMATA

Technological investigations and applications have been of great importance in the group's activities from the very beginning of the project. The first programme of national level in which the 'agarose group' was already involved was carried out in 1976-80. By that time the methods of isolation, fractionation, and pilot production of a novel agar and agarose had been elaborated [61]. The next step was aimed at obtaining a CNBr-activated matrix for the production of a variety of affinity sorbents for practical use in biochemistry. For this purpose, a safe method of cyanogen bromide preparation was introduced [54]. Other activation methods, described in Table 1, were also used to produce numerous affinity carriers. The main types of agarose preparations produced in Estonia were the following:

1. Agarose-based gel filtration media: beaded 4% or 6% agarose gel, and the same, cross-linked; fractionation range of molecular mass 6 x [10.sup.4]-2 x [10.sup.7] (for 4% gel) or 1 x [10.sup.4] - 4 x [10.sup.6] (for 6% gel).

2. Ion exchangers on agarose: cross-linked carriers diethylaminoethyl agarose (anion anion (ăn`ī'ən), atom or group of atoms carrying a negative charge. The charge results because there are more electrons than protons in the anion.  exchanger) and carboxymethyl agarose (cation cation (kăt'ī`ən), atom or group of atoms carrying a positive charge. The charge results because there are more protons than electrons in the cation.  exchanger).

3. Affinity chromatography carriers: a variety of agarose matrices of direct activation (Table 1) and those with additional groups (for example, aminohexyl agarose, containing hexamethylene diamine di·am·ine  
n.
Any of various chemical compounds containing two amino groups, especially hydrazine.

Noun 1. diamine - any organic compound containing two amino groups
 bound to a CNBr-activated matrix); and also dye ligand carriers (containing triazine tri·a·zine  
n.
1. Any of three isomeric compounds, C3H3N3, each having three carbon and three nitrogen atoms in a six-membered ring.

2. A compound derived from one of these isomers.
 active dyes or arylvinylsulphone groups, see Fig. 6).

As a rule, detailed methods of preparing, analysis, etc. of these carriers are described in reports [10, 62] and in numerous classified regulations. All the agarose preparations (over 30 different products) have full documentation for manufacturing on a pilot plant scale. The carriers have been produced by one to three Estonian institutions (Pilot-Production Plant of the Institute of Chemistry in Tallinn, firms Kemotex in Tallinn and Aire in Tartu County), particularly in 1983-91. Some of them are still produced though only by Kemotex at present.

The groups at the Institute of Chemistry and the University of Tartu have elaborated some technological schemata for algae processing. The first of them was a scheme of agar and agarose production from the red alga A. tobuchiensis of the Far East discussed above.

In the first half of the 1990s, particularly in 1993-94, some technological investigations concerning the Baltic red alga F. lumbricalis and a commercial carrageenan product from this seaweed were carried out at the Institute of Chemistry. As a result, the basic principles for industrial production of high-quality carrageenan from this alga species were formulated [36, 37]. In 1995-2000 the group completed an Estonian State Programme (in cooperation with researchers of the Estonian Marine Institute) of industrial utilization of algae resources of the Baltic Sea. The part of the programme concerning red algae of Kassari Bay was fulfilled in 1995-97 [63]. This investigation included also a full technological scheme for processing the algae F. lumbricalis and C. truncatus.

Some strictly technological investigations, particularly those concerning the effects of drying on the gelling properties of furcellaran, were made later by another group of Tallinn Technical University [64].

SOME OTHER RESEARCH TOPICS AND PERSPECTIVES

As it was briefly mentioned, some topics of interest concerned immunochemistry Immunochemistry

A discipline concerned both with the structure of antibody (immunoglobulin) molecules and with their ability to bind an apparently limitless number of diverse chemical structures (antigens); with the structure, organization, and rearrangement
 and blood fractionation [50-53]. The Tartu group was also interested in human plasma treatment [65].

Other objects of research were lectins and their complexes with carbohydrates, which play an important role in sugar research. Lectin from the edible snail Helix pomatia and the carbohydrate complexes were investigated in greater detail [66-68]. Also potato lectin [69] and some other lectins were investigated. Expeditions to obtain natural raw material were organized to the other Baltic states (Lithuania, Latvia) at the end of the 1980s and in 1990.

Gelling kinetics and effects of electrolytes, solvents, etc. were investigated mainly during the second half of the 1980s. The findings were presented at conferences in Estonia and elsewhere [25, 70]. Also rate constants of gel formation and energy of hydrogen bonds in agarose gels were calculated. This kind of research will be continued in the future and some visualization techniques for polysaccharide aggregates will be developed.

At the present time, the main investigation themes are connected with the polysaccharic structures in brown algae of the Baltic Sea. Although the Baltic brown algae, particularly F. vesiculosus, have been of interest for over 70 years in Estonia, their more extensive research started only in 1998 [71]. Some conclusions from these investigations [43] were mentioned before in this paper. Probably, the minor components of F. vesiculosus (fucans, fucoidan, etc) are of greater interest than the alginates of this species. Fucoidans have a very interesting structure and promising biological activity. The fraction of low molecular mass fucan of F. vesiculosus has in vitro anticoagulant activity [72]. Some fractions of this seaweed show also a high potential as antioxidants [73].

FINAL REMARKS AND ACKNOWLEDGEMENTS

Summarizing the activities and results of the groups, it should be mentioned that most of the time the conditions for creative work were relatively good. The wide area of scientific interests and investigations had found recognition and financial support from various institutions and funds. The authors of the article are thankful to the Estonian Government for the support to the State Programme of algae research. The grants of the Estonian Science Foundation, including the last, No. 4179, are gratefully acknowledged. Our thanks are due to all coworkers, colleagues, and partners from other laboratories and countries.

Received 9 July 2002

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An instrument that produces a chromatogram.

tr.v. chro·mat·o·graphed, chro·mat·o·graph·ing, chro·mat·o·graphs
To separate and analyze by chromatography.
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[73.] Ruperez, P., Ahrazem, O. & Leal LEAL. Loyal; that which belongs to the law. , A. Potential antioxidant antioxidant, substance that prevents or slows the breakdown of another substance by oxygen. Synthetic and natural antioxidants are used to slow the deterioration of gasoline and rubber, and such antioxidants as vitamin C (ascorbic acid), butylated hydroxytoluene  capacity of sulfated polysaccharides from the edible marine brown seaweed Fucus vesiculosus. J. Agric. Food Chem., 2002, 50, 840-845.

Kalle Truusa, (b) * and Merike Vaher (a)

(a) Institute of Chemistry, Tallinn Technical University, Akadeemia tee 15, 12618 Tallinn, Estonia

(b) Tallinn Pedagogical ped·a·gog·ic   also ped·a·gog·i·cal
adj.
1. Of, relating to, or characteristic of pedagogy.

2. Characterized by pedantic formality: a haughty, pedagogic manner.
 University, Narva mnt. 25, 10120 Tallinn, Estonia

* Corresponding author, truus@tpu.ee
Table 1. The main activated agarose matrices used

   Activation       Linkage       Available          Specificity
                   to agarose   reactive group         of group

Cyanogen bromide   Isourea      Cyanate ester    Amine
                   (finally)

Carbonyl-          Carbamate    Imidazolyl       Amine
diimidazole                     carbamate

Epichloro-hydrin   Ether        Epoxy            SH > [NH.sub.2] > OH
Epoxy(bis)

Vinyl sulphone     Sulphonyl    Vinyl sulphone   SH > [NH.sub.2] > OH
                   thioether

   Activation              Bond type;
                   stability of attachment

Cyanogen bromide   Isourea; moderately
                   stable

Carbonyl-          Carbamate; good
diimidazole        stability below pH 10

Epichloro-hydrin   Thioether, sec amine
Epoxy(bis)         ether; all very stable

Vinyl sulphone     Thioether, sec amine
                   ether; good stability
                   below pH 9
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