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COMPARATIVE STUDY ON HEMAGGLUTININS FROM THE RED ALGAE Bryothamnion seaforthii AND Bryothamnion 1 triquetrum
IRACEMA LIMA AINOUZ2, ALEXANDRE HOLANDA SAMPAIO3, ANA LUCIA PONTE FREITAS4, NORMA MARIA BARROS BENEVIDES4 and SHIRLEY MAPURUNGA5. Depto.de Bioqumica e Biologia Molecular, Universidade Federal do Cear. CP 6020, Fortaleza, CE. 60451-970, Brasil.
ABSTRACT- The hemagglutinins from Bryothamnion seaforthii (Turner) Kutzing and Bryothamnion triquetrum (Gmelin) Howe were purified by extraction under liquid nitrogen with 0.02M phosphate buffer pH 7.0 containing 0.15M NaCl, precipitation with 60% saturated ammonion sulfate, and DEAE-cellulose chromatography. The hemagglutinating activity of both lectins was not dependent on divalent cations and was not inhibited by simple sugars. These lectins gave single bands with similar mobility in SDS-PAGE. The average molecular masses for B.seaforthii and B.triquetrum lectins estimated by gel filtration were 4500 Da and 3500 Da respectively. Additional index terms: Lectins, red marine algae. Termos adicionais para marinhas vermelhas. indexao: Lectinas, algas

INTRODUCTION
The increasing number of biological applications of lectins (Sharon & Lis,1989) has stimulated the research on a large number of organisms in which proteins with hemagglutinating activity have been detected, including marine algae. Although several studies on hemagglutinins from marine algae have been reported, since the first work of Boyd et al. (1966), the number of lectins purified and characterized is considered small. These studies have been reviewed by Rogers & Fish (1991). Some peculiar properties of marine algal hemagglutinins such as preferential agglutination for trypsin-treatred rabbit erythrocytes, no affinity for monosaccharides, relatively low molecular masses, no requirement for metal ions, and occurence of monomeric forms (Hori et al., 1990) are demanding further studies in order to explain the actual physiological functions of these lectins. Lectins from taxonomically close species of marine algae have not been extensively investigated (Rogers et al., 1990) in an attempt to use them as a tool in chemotaxonomy, as has been suggested for land plants. It has been found extensive homologies among the amino acid sequences of lectins from leguminous plants within the same tribes. These lectins have shown most of their characteristics within related species during evolution (Etzler, 1985). A remarkable similarity between the lectins of the red algae Ptilota plumosa and P. serrata was observed by Rogers et al. (1990). They also reported the lectins from two sub-species of the green alga Codium fragile are biochemically very similar (Rogers et al., 1986).

ESTUDO COMPARATIVO SOBRE HEMATOAGLUTININAS DAS ALGAS VERMELHAS Bryothamnion seaforthii E Bryothamnion triquetrum
RESUMO- As hemaglutininas de Bryothamnion seaforthii (Turner) Kutzing e Bryothamnion triquetrum (Gmelin) Howe foram purificadas por extrao sob nitrognio lquido com tampo fosfato 0,02M pH 7,0 encerrando NaCl 0,15M, precipitao com sulfato de amnio a 60% de saturao e cromatografia em DEAE-celulose. A atividade hemaglutinante de ambas as lectinas no dependente de cations divalentes nem inibida por acares simples. Estas lectinas apresentam uma simples banda com mobilidade semelhante por SDS-PAGE. As massas moleculares das lectinas de B.seaforthii e B.triquetrum, estimadas por filtrao em gel, foram de 4500 Da e 3500 Da, respectivamente.

1Received in 08/25/94 and accepted in 04/15/1995. 2Pesquisador do CNPq 3Mestre em Bioqumica 4Professor Adjunto, Doutor em Biologia 5Bolsista PET/CAPES

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Screening of Brazilian algae for hemagglutinins showed that extract of Bryothamnion seaforthii agglutinates trypsin-treated erythrocytes from rabbit, chicken and cow (Ainouz & Sampaio, 1991) while that of Bryothamnion triquetrum agglutinated enzyme treated erythrocytes from rabbit, chicken, goat, pig and human ABO (Ainouz et al., 1992). The present paper deals with the purification of hemagglutinins from B. seaforthii and B. triquetrum in order to establish possible biochemical similarities.

MATERIAL

AND METHODS

Standard hemagglutination assay. The algal preparations (200 L) were used for serial twofold dilutions made with 0.15M NaCl. Equal volumes of 2% erythrocyte suspension were added to each tube, gently shaken, and incubated at 37oC for 30 min. The tubes were allowed to stand for 30 min at room temperature, centrifuged (2000g, 5s) and examined for agglutination (Ainouz et al., 1992). The activity was given as titer (the reciprocal of the highest twofold dilution) or expressed as the minimum protein concentration (mg L-1) which produced macroscopically visible agglutination. Thermostability. Aliquots of crude extract were incubated at the temperatures of 50, 70 and 100 oC. After 30, 60 and 90 min 200 L of samples were taken, cooled and assayed for hemagglutinating activity. Effect of pH on the hemagglutinating activity. Samples of crude extract were adjusted to different pH values (from 2 to 10) by addition of HCl or NaOH and kept for 16 h at 4 oC. Any precipitate formed was removed by centrifugation and the supernatant, brought to the inicial pH, was used for hemagglutination assay. Effect of metal ions. To evaluate the effect of metal ions aliquots of the crude extract were dialysed against 5mM EDTA for 16 h at 8 oC. The material was used for hemagglutination assay in absence of Mn2+ and Ca2+. Hemagglutination inhibition test. Equal volumes (200 L) of sugar and agglutinin solutions were mixed and incubated at room temperature for 2 h, before the addition of the erythrocyte suspension (400 L). The activity was determined as described under standard assay. Molecular mass. The molecular masses of the hemagglutinins were estimated by gel filtration on Sephadex G-50 column (870 x 15 mm) in phosphate buffer pH 7.6. Cyanocobalamin (1345 Da), ribonuclease (13400 Da) and -chymotrypsin (22500 Da) were used as reference proteins. The void volume was estimated with Blue Dextran 2000. SDS-PAGE. Discontinuos system electrophoresis was carried out in a vertical apparatus following the Laemmli method as described by Hames & Rickwood (1983). The running conditions were: plates 60 x 55mm; stacking gel 4% (T); separating gel 12.5% (T); 12 mA for 2 h. Samples were treated with SDS, SDS-beta-mercaptoetanol or 8 M urea. The proteins were fixed by 12.5% TCA for 1 h, before staining by Coomassie blue R-250.

Specimens of the red algae B. seaforthii and B. triquetrum were collected at the Atlantic coast of Brazil (Pacheco beach, Cear) and kept in plastic bags at -20oC until used. Hemagglutinin preparations. The algae were thawed, rinsed with distilled water, cleaned of epiphytes, ground to a fine powder under liquid nitrogen, stirred for 4 h with three volumes of 0.02 M phosphate buffer, pH 7.0, containing 0.15 M NaCl, filtered through nylon tissue and centrifuged. The supernatant (crude extract), after centrifugation (7000g, 30 min, 4oC), was acidified and left for 4 h under refrigeration. The precipitated pigments were removed by centrifugation and to the supernatant, adjusted to pH 7.0 (fraction 7), solid ammonium sulfate was added to 60% saturation. Precipitated proteins were recovered by centrifugation (fraction 0/60), ressuspended in a small volume of buffer, dialysed and applied to a DEAE cellulose-column. The column was equilibrated and eluted with 0.02 M phosphate buffer, pH 7.6, followed by elution with 1M NaCl in the same buffer. Fractions of 3 mL were collected at a flow rate of 30 mLh-1. The unadsorbed fractions with hemagglutinating activity were pooled and rechromatographed on the same column. Active fractions were combined, dialysed (fraction DEAE) and lyophilized. Analytical methods. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Total neutral sugar content was measured by the phenol-sulfuric acid method of Dubois et al. (1956) with glycose as reference. Erythrocyte samples. Rabbit blood samples obtained by venous puncture were collected into a preheparinized tube and washed three times with ten volumes of 0.15M NaCl. Trypsin (EC3.4.21.4) (0.1 mg per 10 mL of NaCl 0.15M) was added to packed cells to give a 2% erythrocyte suspension, incubated for 60 min at 15 oC and washed six times with cold NaCl. The treated cells removed by centrifugation were ressuspended in 0.15M NaCl to give a 2% erythrocyte concentration.

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TABLE 1- Purification steps of B. triquetrum and B. seaforthii hemagglutinins. Fraction Volume (mL) Total Protein (mg) Total Activity (HU) (g)* Total Carbohydrate (mg)

B. triquetrum
Extract pH 7 0/60 DEAE 500.0 484.0 12.2 21.5 220.0 77.5 16.7 9.5 16,000 15,492 6,230 5,507 14.0 5.0 2.7 1.7 60.0 48.4 1.9 0.3

B. seaforthii
Extract pH 7 0/60 DEAE 500.0 473.3 21.0 40.6 600.0 397.6 80.2 31.6 64,020 60,594 21,439 20,773 9.4 6.6 3.7 1.5 179.0 146.7 19.8 1.2

*Minimum Protein concentration (mg L-1) which produced agglutination of 2% erythrocyte suspension.

RESULTS
Table 1 summarizes the data concerning the purification of B. seaforthii (BS) and B. triquetrum (BT) hemagglutinins. Over 94% of the hemagglutinating activity present in the crude extract of both species was recovered after acidification used to eliminate the pigments. The fraction precipitated by ammonium sulfate to 60% saturation contained about 34 and 39% of the initial activity respectively for BS and BT. Further purification was achieved by DEAE-cel-

lulose chromatography (pH 7.6) when the bulk of the activity was obtained in one peak not retained on the column (Figs 1 and 2). The average protein concentration of the purified fractions required for definite hemagglutination of 2% trypsin-treated rabbit erythrocyte suspension was found to be 1.6 mg.L-1 for BS and BT. The average carbohydrate content were 3.2 and 3.8% for BS and BT, respectively as determined by phenol-sulfuric method. The hemagglutinating activity was not inhibited by the following sugars: arabinose, xylose, ribose, rham-

FIGURE 1- DEAE-Chromatography of 0/60 fraction B. seaforthii. The column was from equilibrated and eluted with phosphate buffer (pH 7.6) followed by elution with NaCl 1M in the same buffer. Absorbance at 280 nm (---) and hemagglutinating activity (...).

FIGURE 2- DEAE-Chromatography of 0/60 fraction B.triquetrum. The column was from equilibrated and eluted with phosphate buffer (pH 7.6) followed by elution with NaCl 1M in the same buffer. Absorbance at 280 nm (---) and hemagglutinating activity (...).

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h or addition of either CaCl2, or MgCl2. Their activities were unaffected by heating for 30 min at 90 oC. The purified hemagglutinins were eluted from Sephadex G-50 column (pH 7.6) as a single peak and apparent molecular masses were estimated to be 4500 Da and 3500 Da for BS and BT, respectively ( Fig 3). The purified fractions from BS and BT migrate as single bands on SDS-PAGE and the eletrophoretic patterns were not modified by addition of 8M urea or beta-mercaptoetanol indicating the hemagglutinins are monomeric peptides.

DISCUSSION
FIGURE 3- Molecular masses by gel filtration on Sephadex G-50 (pH 7.6). (1) Cyanocobalamin, (2) B. triquetrum agglutinin, (3) B. seaforthii agglutinin, (4) Ribonuclease and (5) a-chymotrypsin. Kav (distribution coefficient) = (Ve - Vo ) / ( Vt - Vo) Ve : Elution volume; Vo : Void volume; Vt : Total volume of the packet bed. The agglutinins from the red algae B. seaforthii and B. triquetrum , purified by saline extraction, precipitation by ammonium sulfate followed by DEAE-chromatography, show to be very similar in their properties. Their homogeneity was judged by polyacrylamide gel electrophoresis and by the presence of only one Nterminal amino acid (unpublished data). The hemagglutinating activities for both species against trypsin-treated rabbit erythrocytes are not inhibited by simple sugars but by glycoproteins, are resistent to heat at 90 oC for 30 min, and are not dependent on divalent cations. The inhibition by glycoproteins such as fetuin, avidin and mucin have been observed for the majority of algal agglutinins and has been suggested that their complex sugar moities are responsible for the inhibition. All these properties are shared with other hemagglutinins from red marine algae (Hori et al ., 1990). They show the lowest molecular masses ( 3500 and 4500 Da) of all algal hemagglutinins so far studied. Usually the lectins from land plants show higher molecular weights, even though the value of 3500 - 4000 Da have been found for the anti-A lectin from seeds of Crotalaria striata (Skidar et al. 1990) . They contain ca 3.5% of neutral sugars as determined by phenol-sulfuric method. They appear to be monomeric glycopeptides, a property also reported for algal hemagglutinins (Shiomi et al ., 1979; Okamoto et al ., 1990; Hori et al ., 1990), although it remains to be elucidated how a monomeric form causes the agglutination of cells. The biochemical similarities between the hemagglutinins from the two species of Bryothamnion suggest the lectins may be useful in taxonomy. Their properties such as amino acid composition, the primary structure and the mitogenic activity are being carried out in order to verify the possible applications of the new algal lectins.

FIGURE 4- Electrophoresis of B. and seaforthii B. triquetrum hemagglutinins.

nose, glucose, galactose, frutose, sucrose, trehalose, raffinose, cellobiose, melibiose, glucosamine, mannosamine, salicin, methyl-D mannoside, and methylglycopiranoside at the final concentrations of 25 mM. The inhibition by fetuin, mucin and avidin was observed at the concentration of 3 g L-1. BS and BT hemagglutinins were not dependent on divalent cations for their activities as shown after dialysis of the crude extract against 5 mM EDTA for 16

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REFERENCES
AINOUZ, I.L. & SAMPAIO, A.H. Screening of Brazilian marine algae for hemagglutinins. Botanica Marina, 34:211-214,1991. AINOUZ, I.L.; SAMPAIO, A.H.; BENEVIDES, M.M.B.; FREITAS, A.L.P.; COSTA, F.H.F.; CARVALHO, M.C. & PINHEIRO-JOVENTINO, F. Agglutination of enzyme treated erythrocytes by Brazilian marine algae. Botanica Marina, 35:475-479, 1992. BOYD,W.C.; ALMODOVAR, L.R. & BOYD, L.G. Agglutinins in marine algae for human erythrocytes. Transfusion, 6:82-83, 1966. DUBOIS,M.; GILES, K.A.; HAMILTON, J.K.; ROGERS, P.A. & SMITH, F. Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28:350-356, 1956. ETZLER, M.E. Plant lectins: molecular and biological aspects. Annual Review of Plant Physiology, 36:209-234, 1985. HAMES, B.D. & RICKWOOD, D. Gel Electrophoresis of Proteins. A Practical Approach. Washington, IRL Press. 1983. 27-33p. HORI, K.; MIYASAWA, K. & ITO, K. Some properties of lectins from marine algae. Hidrobiologia, 204 /205 : 561-566, 1990. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDAL, R.J. Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193:265-275, 1951.

OKAMOTO, R.; HORI, K.; MIYASAWA, K. & ITO, K. Isolation and characterization of a new hemagglutinin from the red alga Gracilaria bursa-pastoris. Experientia, 46:975-977, 1990. ROGERS, D.J. & FISH, B.C. Marine algal lectins. In: Kilpatrick, D.C., Van Driessche, E. & Bog-Hansen,T.C. Eds. Lectins Reviews. v.1 St. Louis, Sigma Chemical, 1991. p.129-142. ROGERS, D.J.; FISH, B. & BARWELL, C.J. Isolation and properties of lectins from the red marine algae: Plumaria elegans and Ptilota serrata. In: Kocourek, J. & Freed, D.L.J. Eds. Lectins: Biology, Biochemistry, Clinical Biochemistry. v.7 St. Louis, Sigma Chemical, 1990. p. 49-52. ROGERS, D.J., LOVELESS, R.W. & BALDING, P. Isolation and characterization of the lectins from sub-species of Codium fragile . In: Lectins, v.5 p.155-160. Walter de Gruyer, Berlin. NY.1986. SHARON, N. & LIS, H. Lectins. Chapman Hall. Ltd. London NY. 1989. SHIOMI, K.; KAMIYA, H. & SHIMIZU, Y. Purification and characterization of an agglutinin in the red alga Agardiella tenera. Biochimica Biophysica Acta, 576:118-127, 1970. SKIDAR, S.; AHMED, H. & CHATERJEE, B.P. A pH dependent low molecular weight blood group A specific lectin from Crotalaria striata seeds: purification and specificity. Biochemistry Archives, 6:207-215, 1990.

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