techniques involving solid supports play crucial roles in the development of genomics , proteomics , and in molecular biology .
however , solid - phase tools have been employed to a much lesser extent in glycobiology and glycomics .
there are a number of  classical  methods for immobilization of mono- and oligosaccharides to commercially available matrices and supports .
these methods have , for example , been used for the preparation of affinity columns with specific ligands .
increased attention has been given to the development and application of magnetic separation techniques , which employ small magnetic particles .
antibodies , dna / rna / oligonucleotide / aptamer binding proteins , albumin , hemoglobin , and enzymes have been purified by magnetic techniques .   in our laboratory fe3o4 magnetite particles prepared by coprecipitating fe2 and fe3 with either dacron or a network of polysiloxane - polyvinyl alcohol
, magnetic particles containing levan , a homopolysaccharide of fructose residues in 2 , 6-glycosidic linkage , are proposed to purify lectins .
the latter glycoproteins and/or oligomeric proteins are found in a diverse assortment of organisms and have the extraordinary property of binding specifically , reversibly , and noncovalently to carbohydrates .
lectin - carbohydrate interactions are extensively studied in different scientific disciplines , from basic to applied natural and clinical sciences .
such inter- and multidisciplinarity corroborates the importance to develop new methodologies for the study of lectin - saccharide interactions and the potential applications in clinical diagnostics . in the present work a composite of the carbohydrate levan and magnetite
lectins complexed specifically to the composite were separated from other contaminant proteins by washing with a high ionic strength solution and obtained from composite with specific monosaccharide .
the washing procedures were facilitated by the magnetic field and all process can be automated .
the seed lectins from cratylia mollis ( camaratu bean ) , cramoll [ 9 , 10 ] and canavalia ensiformis ( con a ) were used as models .
con a and potato lectin from solanum tuberosum have already been purified by magnetic techniques using dextran   and chitosan   as ligands , respectively .
the aim of this work was to evaluate the use of zimomonas mobilis levans insolubilized and ferromagnetized ( fmzag-12l ) , to purify fructose / specific lectins using lectin preparations of c. mollis seeds .
cramoll 1 , 4 and cramoll 1 were obtained through a previously established protocol    from a c. mollis seed extract ( 10% , w / v ) that was ammonium sulfate fractionated ; fraction ( f ) 40 to 60% saturated ( f4060 ) was affinity chromatographed in sephadex g-75 ( cramoll 1 , 4 ) followed by ion exchange chromatography in cm - cellulose ( cramoll 1 and cramoll 4 ) .
cramoll 3 was also obtained from the above mentioned seed extract through a previously described protocol ; the 0 to 40%   ammonium sulfate fraction ( f040 ) was molecular exclusion chromatographed in sephadex g-100 .
levan ( l ) was produced by zimomonas mobilis strain zag-12 ( departamento de antibiticos , universidade federal de pernambuco , brazil ) and abbreviated from now on as zag-12l .
a solution containing fe and fe ions in a molar ratio of 1.1 m : 0.6 m was prepared from fecl36h2o and fecl24h2o in distilled water ; 50 ml of 2% zag-12l in distilled water was then added and the ph was raised to 11.0 by adding drop wise 1 m nh4oh .
mixture was then heated up to 85  3c and incubated for 30 minutes with vigorous stirring .
the ferromagnetic levan obtained ( fmzag-12l ) was centrifuged 5 times to remove solid material .  
the product was dried for 24 hours at 50c , ground and kept at room temperature .
aqueous suspensions of magnetic particles were prepared by coprecipitation of fe ( iii ) and fe ( ii ) in the presence of nh4oh and polymer .  
the protein content was carried out by lowry et al .   using bovine serum albumin as standard , at a range of 0500 g / ml and absorbance reading at 720 nm .
lectin sample solutions ( 50 l ) were serially 2-fold diluted in 0.15 m nacl , in microtiter u - plates and incubated with of a 2.5% ( v / v ) suspension of glutaraldehyde treated erythrocytes from new zealand white rabbit ( 50 l ) .
the titer , defined as the lowest sample dilution which showed hemagglutination , was established after 45 minutes incubation according to correia and coelho .   hemagglutinating activity ( ha ) corresponded to the reciprocal titer . the ha inhibition ( hai )
was assayed by 2-fold serial dilution of lectin sample solutions ( 50 l ) in 50 l of 200 mm levan or fructose solutions , followed by 15 minutes incubation and addition of erythrocyte suspension .
h unidimensional spectra were recorded in a bruker drx 400 mhz ( bruker , germany ) with a triple resonance 5-mm probe .
the lectins ( 1 ml ) con a , cramoll 1 , 4 , cramoll 3 , and f4060 were each incubated with fmzag-12l particles ( 1 ml containing 10 mg ) for 2 hours at 4c , under constant agitation .   afterwards , the magnetic particles were recovered by a magnetic field ( 6 000 oe ) and supernatant was collected .
the remaining unspecific bound proteins were eluted with 0.15 m nacl ( 1 ml ) by recovering the magnetic particles under the magnetic field and collecting the supernatant . finally , adsorbed lectin was eluted with either 0.3 m d - glucose ( con a and cramoll 1 , 4 ) or d - galactose ( cramoll 3 incubation ) in 0.15 m nacl ( 1 ml ) recovering the magnetic particles .
the collected supernatants had their absorbancies at 280 nm and ha analyzed ; page for native and basic proteins were performed according to reisfeld et al . .

 c. mollis ( camaratu bean ) is a native forage from the semi - arid region of pernambuco state , northeastern of brazil , and belongs to fabaceae family , taxonomically related with c. ensiformis species from which seeds are obtained con a. c. mollis seeds have been considered an important lectin source , giving multiple cramoll molecular forms with different carbohydrate specificities :   cramoll 1 , cramoll 2 , and cramoll 4 are specific for glucose / mannose whereas cramoll 3 is galactose specific .
a preparation containing cramoll 1 and cramoll 4 together ( cramoll 1 , 4 ) showed a higher hemagglutinating activity ( ha ) when compared with the isolated cramoll 1 and cramoll 4 as well as con a. these lectin preparations ( cramoll 1 , cramoll 4 , and cramoll 1 , 4 ) are inhibited by different carbohydrates ( d - glucose , d - mannose , -d - methyl - mannoside , and d - fructose , among others ) in distinct concentrations .
cramoll 1 , 4 and cramoll 1 were successfully used in different biological assays as well as in structural and electrochemical studies [ 1521 ] .
the nmr analysis of zag-12l used in this work revealed a spectrum profile corresponding to the fructose residues protons .
h nmr spectrum shows seven protons between 3.4 and 4.2 ppm indicating that the polysaccharide produced by z. mobilis was levan type with the linkage of ( 2  6 ) fructofuranoside ( figure 1 ) .   no signals in the anomeric region ( 5.3 to 4.3 ppm )
levans from erwinia herbicola , acetobacter xylinum , and bacillus subtilis ( natto )   showed the same structural characteristics analyzed by nmr .  
hai of cramoll 1 , cramoll 1 , 4 , cramoll 3 , and con a by fructose and levan revealed that all lectins presented ha inhibited by fructose and its polymeric derivative ( table 1 ) . however , inhibition of cramoll 1 , 4 activity by the commercially acquired levan was less intensive .
previous studies of this preparation specificity using different monosaccharides showed that d - fructose inhibited cramoll 1 , 4   ha at the same d - mannose proportion .
mo et al .   reported that con a did bind to d - fructofuranosyl groups present in plant and microorganism levans whereas banana ( musa acuminate ) lectin reacted only with microorganism levans .  
it is important to observe that banana lectin and con a ( structurally similar to cramoll 1 ) are both glucose / mannose specific .  
the inhibition of lectins by levans suggested that magnetized levan could be potentially used as an affinity matrix to investigate or purify lectins that recognize fructose .  
figure 2 shows the con a elution profile by using particles of ferromagnetic levan composite ( fmzag-12l ) .
proteins unspecifically bound to the particles were completely washed out with 0.15 m nacl from the 1st to the 8th washes ( 8 ml ) and a second peak emerged after 0.3 m glucose addition at the 10 - 11th fractions .
it is worthwhile to draw attention to the fact that equal profile was attained four times indicating its reproducibility and the reuse of the particles .  
this preparation containing both lectin isoforms was previously purified from c. mollis seed extract by ammonium sulfate fractionation and affinity chromatography in sephadex g-75 .
the particles washed with 0.15 m nacl again showed that from the 1st to 8th fraction ( 8 ml of washes ) all unspecifically bound proteins were removed and a second peak emerged after 0.3 m glucose introduction .
similar to con a purification this procedure was four times reproduced using the same fmzag-12l .
the protein peak eluted at the 14th fraction showed two bands ( cramoll 1 and cramoll 4 ) by polyacrylamide electrophoresis to basic and native proteins ( figure 3(b ) ) similar to previously reported pattern .
none specific binding to fmzag-12l was detected when cramoll 3 was used ( data not shown ) .
recently , the importance of protein - protein interaction has been pointed out in certain oligomeric lectins since differences among their quaternary organizations appear to be directly related to those among their functions .
lectin binding sites have been deeply characterized by many workers to understand carbohydrate interaction of these versatile proteins [ 2729 ] .   
the evaluation of fmzag-12l to purify cramoll lectins from f4060 preparation is presented in figure 4(a ) .
three different concentrations of the ammonium sulfate preparation per 10 mg of fmzag-12l were used : 1.8 mg / mg , 1 mg / mg , and 0.5 mg / mg of protein / mg magnetic particles . all concentrations showed a second protein peak ( 9th-10th fractions ) eluted by 0.3 m glucose addition at the 8th washing with 0.15 m nacl .
the polyacrylamide gel electrophoresis to basic and native proteins of the fraction ( 10th ) collected from the 10 mg f4060 purification showed only cramoll 1 protein band ( figure 4(b ) ) .
furthermore , the pure lectin cramoll 1 showed ha of 256 and was inhibited by all levans ( table 1 ) .
the purification of this lectin is relevant due to its several applications such as neoplastic tissue marker .
cramoll 1 showed a higher intensity of staining to transformed tissues than normal ones : also , encapsulation of cramoll 1 into liposomes produced an improvement in its in vivo antitumor activity against sarcoma 180 compared with free cramoll 1 solution .
the lectin purification using only the ferromagnetic particles , namely , absent of levan , did not show any protein peak after 0.3 m glucose addition ( control ) .
it is interesting to notice that cramoll 4 was collected when a purified preparation was incubated with the fmzag-12l ( figure 3(b ) ) but it was not obtained when a lesser purified preparation ( f4060 ) was used ( figure 4(b ) ) . probably , higher amounts of cramoll 1 than cramoll 4 in preparation f4060 and/or different binding constant values for the magnetic levan - lectin can justify these discrepancies .   
furthermore , the ferromagnetic composite of levan is synthesized by a simple and inexpensive method .
cramoll 1 was purified by this fast two - step procedure ( ammonium sulfate fractionation and fmzag-12l affinity binding ) instead of the laborious and time consuming three - step protocol previously described .
finally , it is important to investigate if other polysaccharides could replace the levan in the composite synthesis and be used to lectin purification .
 a simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field . 
 lectins from canavalia ensiformis ( con a ) and cratylia mollis ( cramoll 1 and cramoll 1 , 4 ) did bind specifically to composite . 
 the magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution . 
 cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three - step protocol with ammonium sulfate fractionation , affinity chromatography on sephadex g-75 , and ion exchange chromatography through a cm - cellulose column . 