lectins are defined as proteins / glycoproteins 
 possessing at least one non - catalytic domain which binds reversibly to a 
 specific mono- or oligosaccharide . over the last few decades , lectins have 
 become a topic of interest to a large number of researchers owing to their 
 potentially exploitable biological properties including antitumor [ 2 , 3 ] , 
 immunomodulatory and anti - insect , antifungal , antibacterial , anti - hiv 
 [ 2 , 5 , 7 ] , and mitogenic   activities . because of their sugar binding 
 properties , lectins have been extensively studied and used as molecular tools for 
 the study of carbohydrate architecture and dynamics on the cell surface , and 
 have been exploited for such practical applications as distinguishing between 
 normal and malignant cells [ 9 , 10 ] , purification of glycoconjugates , and 
 coating of drugs to enhance their gastrointestinal tract absorption [ 12 , 13 ] . further , specific amino acid residues are essential for maintaining the carbohydrate 
 binding and hemagglutinating activities of lectins [ 1416 ] . identification of these amino acid residues is a prerequisite for investigating 
 the structure - function relationships of lectins . chemical modification with 
 group - specific modifying agents provides a general approach for identification 
 of the amino acid residues present in the functional or active site of proteins , 
 including lectins [ 14 , 16 ] . hence , elucidation of biological activities of 
 lectins and amino acid residues essential to these activities is a meaningful undertaking . although lectins are found ubiquitously 
 in plant species , they have variable structures and specific activities according 
 to the plants they originate from [ 9 , 10 ] . thus , purification and 
 characterization of lectins from a variety of plant species interests 
 researchers in the field of glycobiology . the more is known about the lectins , 
 the wider the applications of this type of proteins that can be achieved . this 
 study reports the purification and some properties of a new lectin isolated 
 from seeds of the anasazi cultivar of phaseolus vulgaris . to date , the isolation of a lectin 
 from the anasazi bean and examining it for various potentially exploitable 
 biological activities such as mitogenic , antitumor , immunomodulatory , and hiv-1 
 reverse transcriptase inhibitory activities have not been attempted . in this 
 study , it was assayed for the various 
 aforementioned activities . in order to further characterize the lectin , a 
 chemical modification study was undertaken to determine the involvement of 
 different amino acid residues in its hemagglutinating activity . two hundred grams of anasazi beans were soaked in 700 ml distilled water at 
 room temperature for 4 hours to soften the beans . then they were blended , after 
 addition of 700 ml of distilled water in a waring blender into a slurry . subsequently , the mixture was centrifuged at 14000 rpm for 30 minutes at 4c . tris - hcl buffer ( 1 m ) 
 at ph 7.3 was added to the supernatant collected after the centrifugation until 
 the final tris concentration reached 10 mm . the supernatant was loaded on an 
 affi - gel blue gel ( bio - rad , calif , usa ) column ( 5  18 cm ) which had been 
 equilibrated with the same buffer . after the unbound proteins had been eluted , 
 bound proteins with hemagglutinating activity were eluted with 1 m nacl in 10 mm tris - hcl buffer ( ph 
 7.3 ) , dialyzed , lyophilized , and then subjected to ion exchange chromatography 
 by fast protein liquid chromatography ( fplc ) using an akta purifier ( ge healthcare , 
 hong kong , china ) on a 1 ml mono s column ( ge healthcare , hong kong ) in 20 mm 
 tris - hcl buffer ( ph 7.3 ) . the adsorbed fraction was further purified by 
 fplc - gel filtration on a superdex 200 column ( ge healthcare ) in 20 mm tris - hcl 
 buffer ( ph 7.3 ) . sodium dodecyl 
 sulfate - polyacrylamide gel electrophoresis ( sdspage ) was carried out in 
 accordance with the procedure of laemmli and favre , using a 12% resolving 
 gel and a 5% stacking gel . at the end of electrophoresis , after destaining , the electrophoretic mobilities 
 of the marker proteins and the purified lectin were determined . the molecular 
 mass of the lectin was estimated from the standard curve plotting 
 electrophoretic mobility against molecular mass . fplc - gel filtration was 
 carried out using a superdex 200 hr 10/30 column ( ge healthcare ) that had been 
 calibrated with molecular - mass standards ( ge healthcare ) to estimate the 
 molecular mass of the purified lectin . amino acid sequence analysis was carried out using a hewlett packard 
 ( hp ) g1000a edman degradation unit and an hp 1000 hplc system . in the assay for lectin ( hemagglutinating ) activity , a serial twofold dilution of 
 the lectin solution in microtiter u - plates ( 50 l ) was mixed with 50 l of a 2% 
 suspension of rabbit red blood cells in phosphate - buffered saline ( ph 7.2 ) at 
 20c . the results were read after about 1 hour , when the blank had fully 
 sedimented . the hemagglutination titer , defined as the reciprocal of the 
 highest dilution exhibiting hemagglutination , is reckoned as one 
 hemagglutination unit . the hemagglutinating inhibition tests 
 to investigate inhibition of lectin - induced hemagglutination by various 
 carbohydrates were performed in a manner analogous to the hemagglutination 
 test . a serial twofold dilution of each sugar samples was prepared in 
 phosphate - buffered saline . all of the dilutions were mixed with an equal volume 
 ( 25 l ) of a solution of the lectin with 16 hemagglutination units . the mixture 
 was allowed to stand for 30 minutes at room temperature and then mixed with 50 l 
 of a 2% rabbit erythrocyte suspension . the minimum concentration of the sugar 
 in the final reaction mixture which completely inhibited 16 hemagglutination 
 units of the lectin preparation was calculated . the effect of temperature on hemagglutinating 
 activity of the lectin was examined as previously described . a solution of 
 the lectin with 16 hemagglutination units was incubated at various temperatures 
 for 30 minutes : 0c , 10c , 20c , 30c , 40c , 50c , 60c , 70c , 80c , 
 90c , and 100c . the tubes were then put on ice , and assay of 
 hemagglutinating activity was then carried out . the ph stability of the lectin was determined by incubation 
 of the lectin ( 1 mg / ml ) in buffers of different ph values ranging from ph 1.014.0 . for 60 minutes . the ph of the lectin solution was adjusted to 7.0 by the addition of 0.1 n hcl 
 or 0.1 n naoh before hemagglutination activity was determined . breast cancer mcf-7 cells or hepatoma hepg2 
 cells were suspended in rpmi medium and adjusted to a cell density of 5  10 cells / ml . a 100 l aliquot of this cell suspension was seeded to a well of a 
 96-well plate , followed by incubation for 24 hours . different concentrations of 
 the lectin in 100 l complete rpmi medium were then added to the wells followed 
 by an incubation for 24 hours . after 24 hours , 50 l of a 5 mg / ml solution of 
 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) in 
 phosphate buffered saline was spiked into each well , and the plates were 
 incubated for 2 hours . the supernatant was carefully removed , and 150 l of dimethyl sulfoxide was 
 added in each well to dissolve the mtt ( formazan ) at the bottom of the wells . the absorbance at 590 nm was then measured with a microplate reader within 10 
 minuets . the assay of anasazi bean lectin for ability to inhibit hiv-1 reverse 
 transcriptase was carried out by using an enzyme - linked immunosorbent assay kit 
 from boehringer mannheim ( germany ) as described by collins et al . . the 
 assay takes advantage of the ability of reverse transcriptase to synthesize 
 dna , starting from the template / primer hybrid poly(a)oligo(dt ) 15 . instead of radio - labeled 
 nucleotides , digoxigenin and biotin - labeled nucleotides in an optimized ratio are incorporated into one and the same dna molecule , which is freshly 
 synthesized by the reverse transcriptase ( rt ) . the detection and quantification 
 of synthesized dna as a parameter for rt activity follows a sandwich enzyme - linked 
 immunosorbent assay protocol . biotin - labeled dna binds to the surface of 
 microtiter plate modules that have been precoated with streptavidin . in the 
 next step , an antibody to digoxigenin , conjugated to peroxidase ( anti - dig - pod ) , 
 binds to the digoxigenin - labeled dna . in the final step the peroxidase enzyme catalyzes the cleavage of the 
 substrate , producing a colored reaction product . the absorbance of the samples 
 at 405 nm can be read using a microtiter plate ( elisa ) reader and is directly 
 proportional to the level of rt activity . a fixed amount ( 46 ng ) of 
 recombinant hiv-1 the inhibitory activity of 
 the purified protein was calculated as percent inhibition as compared to a 
 control without the lectin . macrophages were collected from the peritoneal 
 cavity of mice after an intraperitoneal injection of a 3% thioglycolate 
 solution . the cells were washed and resuspended in rpmi medium containing 10% 
 heat - inactivated fetal bovine serum , 100 iu / ml penicillin , and 100 mg / ml 
 streptomycin . cells ( 2  10 cells / well ) were seeded in a 96-well culture 
 plate for 1 hour , before incubation with the anasazi bean lectin for 24 hours . the amount of nitric oxide in the culture medium was 
 determined by a colorimetric method . in the assay , a 100 l aliquot of 
 cell - free culture medium from each culture well was allowed to react with 50 l 
 of griess reagent ( 1% sulfanilamide in 5%   h3po4 - 0.1% 
 naphthalene - ethylenediamine dihydrochloride ) for 10 minutes before the 
 absorbance was read at 540 nm using a microplate reader . dexamethasone ( 5 m ) was used as 
 inducible nitric oxide synthase ( inos ) inhibitor in this assay . the antifungal activity of anasazi bean lectin toward botrytis cinerea , 
 mycosphaerella arachidicola , and fusarium oxysporum was examined 
 using 90  15 mm petri plates that contained 10 ml of potato dextrose agar , in 
 view of the report that some lectins displayed antifungal activity . after 
 the mycelial colony had developed , sterile blank paper disks ( 0.625 cm in 
 diameter ) were placed at a distance of 0.5 cm away from the rim of the mycelial 
 colony . an aliquot of a solution of anasazi bean lectin was added to a disk . the plates were kept at 25c for 72 hours until mycelial growth had enveloped 
 disks containing the control and crescents of inhibition had formed around 
 disks containing samples with antifungal activity . four c57bl/6 mice ( 2025 g ) were killed 
 by cervical dislocation , and the spleens were aseptically removed . spleen cells 
 were isolated by pressing the tissue through a sterilized 100-mesh stainless steel 
 sieve and resuspended to 5  10 cells / ml in rpmi 1640 culture medium 
 supplemented with 10% fetal bovine serum , 100 units penicillin / ml , and 100 g 
 streptomycin / ml . the cells ( 7  10 cells/100 l / well ) were seeded 
 into a 96-well culture plate , and serial dilutions of a solution of anasazi bean 
 lectin in 100 l medium were added . after incubation of the cells at 37c in a 
 humidified atmosphere of 5% co2 for 24 hours , 10 l methyl [ h]thymidine 
 ( 0.25 ci , ge healthcare ) was added , and the cells were incubated for further 6 
 hours under the same conditions . the cells were then harvested with an 
 automated cell harvester onto a glass fiber filter and the radioactivity was 
 measured with a beckman model ls 6000sc scintillation counter . all the lectin 
 ( 100 g ) in 0.1 ml of 50 mm tris - hcl buffer ( ph 7.4 ) was incubated with 5 mm 
 phenylmethylsulfonyl fluoride ( pmsf ) at 27c for 1 hour . excess reagent was removed by ultrafiltration , 
 followed by determination of residual hemagglutinating activity . reduction of the thiol groups of anasazi bean 
 lectin was carried out by incubating the lectin ( 100 g ) in 0.1 ml of 50 mm phosphate 
 buffer ( ph 8.0 ) with 0.1 mm 5 , 5-dithiobis-(2-nitrobenzoic acid ( dtnb ) at 27c for 1 hour . excess reagent 
 was removed by ultrafiltration , followed by determination of residual 
 hemagglutinating activity . for lysine modification , 0.5 mg of nabh4 was added to the lectin ( 5 mg ) in 2 ml of 0.2 m sodium borate buffer ( ph 9.0 ) 
 at 4c , followed by six aliquots ( 5 l each ) of 3.5% formaldehyde at 10 minutes 
 intervals . excess reagent was removed by ultrafiltration , followed by 
 determination of residual hemagglutinating activity . lectin incubated in the 
 absence of sodium borohydride ( nabh4 ) served as a control . modification of tryptophan residues was carried out 
 according to the method of spande and witkop . the lectin was dissolved in 
 naoac buffer ( 0.1 m , ph 5.0 ) to 1 mg / ml . the modification was carried out at 20c . n - bromosuccinimide ( nbs ) ( 10 l , 10 mm ) was added every 5 minutes . the 
 number of tryptophan residues was calculated as described by spande and witkop 
 . the samples were then desalted by ultrafiltration to remove excess 
 reagent , and then assay of hemagglutinating activity was carried out . acetylation of the tyrosine side chain phenoxy 
 groups of the lectin was done at room temperature by incubating 300 g lectin 
 in 1.0 ml of 25 mm phosphate buffer ( ph 7.5 ) containing 60-fold molar excess 
 ( 0.110 mm ) of 
 n - acetylimidazole . excess reagent was removed by ultrafiltration , 
 followed by determination of residual hemagglutinating activity . purification of the anasazi bean seed lectin 
 involved initial extraction in 10 mm tris - hcl buffer ( ph 7.3 ) and three - step 
 chromatography including affinity chromatography on affi - gel blue gel , ion - exchange 
 chromatography on mono s , and gel filteration on superdex 200 . fractionation of 
 the crude seed extract using affi - gel blue gel revealed the presence of a 
 slightly larger unadsorbed fraction designated as b1 , and a slightly smaller 
 adsorbed fraction designated as b2 ( figure 1(a ) ) . the latter fraction , in which 
 hemagglutinating activity was concentrated , was subsequently fractionated on 
 mono s into a large unadsorbed fraction and a small adsorbed fraction ( data not 
 shown ) . the adsorbed fraction with hemagglutinating activity was resolved into a 
 large peak ( su1 ) and a small peak ( su2 ) by fplc - gel filtration on superdex 200 . the purified lectin , 
 represented by su1 , appeared as a single band with a molecular mass of 30 kda 
 in sds - page ( figure 2 ) 
 and a 60 kda absorbance peak in gel filtration on superdex 200 ( figure 1(b ) ) . the 
 protein yield and specific hemagglutinating activity of the product at each 
 stage of the purification scheme are shown in table 1 . anasazi bean lectin 
 demonstrated remarkable n - terminal sequence similarity to those of phaseolus lectins ( table 2 ) . the hemagglutinating activity of purified anasazi bean 
 lectin could not be inhibited by any of the simple sugars tested at 1.56100 mm , -l - fucose , d(+)-galactose , d(+)-glucose , d(+)-glucosamine , 
 d(+)-galactosamine , ( + ) -lactose , d(+)-melibiose , l(+)-mannose , d(+)-mannose , 
 d - mannosamine , d(+)-raffinose , l - rhamnose , ( + ) -xylose , galacturonic acid , and n - acetyllactosamine , 
 and also not by the glycoproteins heparin , thyroglobulin , lactoferrin , human 
 chorionic genadotropin , and ovalbumin . the 
 lectin exhibited remarkable stability over the entire range of ph 114 ( data not 
 shown ) . the anasazi bean lectin inhibited hiv-1 reverse transcriptase with an 
 ic50 of 7.6 m 
 ( figure 4 ) . the antiproliferative activity of anasazi 
 bean lectin toward hep g2 and mcf-7 cells is shown in figure 5 . the lectin 
 displayed only slight inhibition of proliferation toward hepg2 cells with an ic50 beyond 8 m while its ic50 toward mcf-7 cells was 1.3 m . the lectin 
 stimulated the mitogenic response of mouse splenocytes with an optimal response 
 at 1.04 m 
 ( figure 6 ) . however , the hemagglutinin did not affect nitric oxide 
 production by mouse macrophages ( figure 7 ) . anasazi bean lectin exhibited no 
 effect on mycelial growth in the fungal species examined , that is , botrytis 
 cinerea , mycosphaerella arachidicola , and fusarium oxysporum ( data 
 not shown ) . the effects of various types of chemical modifications on 
 hemagglutinating activity of the purified lectin are summarized in table 3 . dtnb , reductive methylation and n - acetylimidazole treatments did not produce 
 any alterations in the hemagglutinating activity of anasazi bean lectin , suggesting 
 that cysteine , lysine and tyrosine residues , respectively , did not play any 
 important role in its hemagglutinating activity . however , 67% loss of 
 hemagglutinating activity after nbs treatment was noted , whereas no change in 
 the control was detected . these results strongly suggest a considerable 
 involvement of tryptophan residues in hemagglutinating activity , and stability 
 of the lectin . pmsf treatment resulted in 43% loss in hemagglutination activity 
 suggesting partial 
 involvement of serine in the lectin activity . spectrophotometric monitoring 
 revealed that the controlled addition of 10 l of 10 mm nbs under 
 mildly acidic conditions ( ph 5.0 ) led to a progressive decrease in absorption 
 at 280 nm , indicative of oxidation of tryptophan residues . a concomitant drop in lectin activity in this study , a plant lectin has been purified by 
 three - step chromatography from seeds of the anasazi bean . similar to pinto bean 
 lectin , anasazi bean lectin is adsorbed on affi - gel blue gel . however , 
 unlike the former lectin , the latter is adsorbed on mono s as well . the homogeneity 
 of the anasazi bean lectin preparation was evidenced by the presence of a 
 single band in sds - page . the results of sds - page and gel filtration 
 chromatography together revealed that the lectin exists as a dimer of two 
 subunits . the molecular mass and dimeric nature of anasazi bean lectin are 
 similar to those of pinto bean lectin and most of the other phaseolus lectins 
 [ 2 , 28 ] . on the other hand , it differs from a tetrameric 115120 kda lectin 
 from tepary bean ( phaseolus acutifolius )   , phaseolus acutifolius var . latifolius 
 lectin from which consists of four subunits of 21 kda molecular mass , and 
 a tetrameric 94 kda immunosuppressive lectin isolated from seeds of phaseolus 
 vulgaris cv cacahuate . lectins from some cultivars of phaseolus vulgaris are oligomeric , whereas 
 anasazi bean lectin is dimeric . the subunit molecular weight of the oligomeric 
 lectins is similar to that of anasazi bean lectin . isolectins are absent in anasazi beans but 
 present in some cultivars of p. vulgaris such as red kidney bean [ 3234 ] . the isolectins differ from one another by the 
 number of erythrocyte - reactive ( e ) subunits and lymphotcyte - reactive ( l ) 
 subunits that they possess . there are 
 five such isolectins : l4 , l3e1 , l2e2 , l1e3 , and e4 . although there is striking homology between anasazi 
 bean lectin and other phaseolus lectins in n - terminal sequence , anasazi bean 
 lectin exhibits absence of simple sugar specificity . simple sugars , the 
 n - acetyl sugar n - acetyllactosamine and the glycoproteins heparin thyroglobulin , 
 lactoferrin , human chorionic gonadotropin , and ovalbumin are not able to 
 inhibit the hemagglutinating activity of anasazi bean . in this aspect , anasazi bean lectin is 
 dissimilar from arcelin-1 , a lectin - like protein from wild varieties of kidney 
 bean which interacts with complex glycans , and also from lectins from many 
 other p. vulgaris cultivars such as the galactose specific pinto bean 
 lectin . similar to pinto bean lectin , anasazi bean lectin is fairly 
 thermostable because its hemagglutinating activity is stable at temperatures up 
 to 80c , and is reduced 
 only at 90c . haricot bean and ground bean 
 lectins , however , have been shown to lose activity beyond 40c in a temperature - dependent manner [ 19 , 36 ] . the 
 lectin shows remarkable ph stability , its activity being unaffected throughout 
 the entire range of ph from 1 to 14 . this is in contrast to lectin from parkia 
 javanica beans which is stable in ph 710 . like 
 many lectins such as mushroom lectins , haricot bean lectin , and others [ 2 , 3 , 31 ] , anasazi bean lectin exhibits an antiproliferative activity toward tumor 
 cell lines . interestingly , the anasazi bean lectin exhibits only slight 
 antiproliferative activity toward hep g2 cells while it has been shown to have 
 a significant antiproliferative effect against mcf-7 cells . only a very small 
 number of lectins have been shown to have antifungal activity . anasazi bean 
 lectin inhibits hiv-1 reverse transcriptase with an ic50 below those 
 of some anti - hiv natural products . however , it is almost one - tenth of that of ground 
 bean lectin . it is known that hiv-1 protease inhibits hiv-1 reverse transcriptase with a 
 similar mechanism . antifungal proteins [ 4042 ] , ribosome 
 inactivating proteins [ 4345 ] , and lectins [ 2 , 5 , 7 , 21 , 46 ] of plant origin 
 have been shown to inhibit hiv-1 reverse transcriptase . the ability of anasazi bean 
 lectin to inhibit hiv-1 reverse transcriptase is interesting in view of the 
 inhibition of dna polymerase alpha activity by lectins including concanavalin a and 
 ricin [ 47 , 48 ] and dna polymerase beta activity by red kidney bean agglutinin . induction of mitogenicity is a common feature elicited by most lectins of phaseolus genus , such as pinto bean lectin , haricot bean agglutinin , and pha - l4 , 
 a lectin from kidney beans . however , gnl-2 , a lectin from great northern 
 beans , is devoid of this activity . the magnitude of the maximal response is about one - fourth of 
 that of con a ; the lectin concentration needed to bring about the maximal 
 response is 64 times less than that of con a. nitric oxide ( no ) is known to 
 play an important role in immunomodulation . proinflammatory cytokines , for 
 example , tumor necrosis factor- ( tnf- ) and bacterial products , for example , 
 lipopolysaccharide ( lps ) are well established no stimulators . these agents up- or 
 downregulate the activity of the inducible form of nitric oxide synthase . no is also involved in cytotoxity against tumor cells , and it is possible 
 that no is implicated in tumor lysis induced by lectins with an antitumoral 
 effect [ 5355 ] . previously , 
 emperor banana lectin   has been reported to induce nitric oxide production 
 by mouse macrophages . anasazi bean lectin , in the present report , is not 
 capable of augmenting nitric oxide production by mouse macrophages . chemical 
 modification studies were carried out to investigate the role of specific amino 
 acids in the hemagglutinating activity of anasazi bean lectin . the results 
 disclose that tryptophan and serine are important to the hemagglutinating activity , 
 the contribution of tryptophan being more important . previous studies have 
 reported that lysine , tyrosine , and tryptophan ( e.g. , in dolichos lab - lab bean 
 ) or tryptophan alone are indespensable for the hemagglutinating activity 
 of some legume lectins . specific amino acids may be involved in either 
 direct interaction with the sugar or may have a role in maintaining 
 conformation of the sugar binding pocket , and hence contribute to the 
 hemagglutinating activity of lectins . the application of three successive 
 chromatographic steps , namely , affinity chromatography on affi - gel blue gel , 
 ion exchange chromatography on mono s , and gel filtration on superdex 200 , is 
 effective in isolating anasazi bean lectin , with an approximately 15-fold 
 purification . it is known that the sugar snap produces an antifungal protein , 
 miraculin , whereas the garden pea produces a ribosome inactivating 
 protein , pisavin , instead , although they are two cultivars of the same 
 species , pisum sativum . anasazi bean lectin differs from pinto bean lectin , 
 its counterpart from another cultivar of the same species , in the 
 presence / absence of some features , such as sugar specificity , antiproliferative 
 activity , hiv-1 reverse transcriptase inhibitory activity , although the two 
 beans are two different cultivars of the same species ( phaseolus vulgaris ) . the present study on anasazi bean lectin has revealed that tryptophan and 
 serine residues are involved in the activity of the lectin . the involvement of 
 other amino acid residues and groups in maintaining the conformation of the 
 lectin could be further investigated by circular dichroism analysis . the 
 interesting features of anasazi bean lectin comprise the following : absence of 
 simple sugar specificity which is dissimilar to phytohemagglutinins from many 
 other p. vulgaris cultivars , antiproliferative activity against tumor 
 cells , potent hiv-1 reverse transcriptase inhibitory activity , mitogenic activity 
 in mouse splenocytes , and remarkable ph stability and thermostability .
a lectin has been isolated from seeds of the phaseolus vulgaris cv . 
 anasazi beans  using a procedure that involved affinity chromatography on affi - gel blue gel , fast protein liquid chromatography ( fplc)-ion exchange chromatography on mono s , and fplc - gel filtration on superdex 200 . 
 the lectin was comprised of two 30-kda subunits with substantial n - terminal sequence similarity to other phaseolus lectins . 
 the hemagglutinating activity of the lectin was stable within the ph range of 114 and the temperature range of 080c . 
 the lectin potently suppressed proliferation of mcf-7 ( breast cancer ) cells with an ic50 of 1.3 m , and inhibited the activity of hiv-1 reverse transcriptase with an ic50 of 7.6 m . 
 the lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [ 3h - methyl]-thymidine incorporation . 
 the lectin had no antifungal activity . 
 it did not stimulate nitric oxide production by murine peritoneal macrophages . 
 chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin .