Lectin Chromatography

Immobilized lectins are true complements to boron-ate chromatographic materials. Lectins do not have the stability that boronates possess, but they possess a considerably higher and broader range of specificity. While there are several immobilized commercial boronate materials available for boronate affinity chromatography, there are a much greater number of different types of immobilized lectins. For example, one commercial firm offers 19 different lectins immobilized by several different methods, chiefly to agarose. The same firm offers approximately 60 purified lectins in a nonim-mobilized state, any one of which could be immobilized by a researcher for his or her own specific purposes. Since the specificity of lectins can vary from relatively narrow specificity to those which have very broad specificities, and since the specificities between lectins are so varied, they offer a very powerful tool for separating many glycoproteins. An idealized purification scheme utilizes boronate affinity chromatography in an initial purification step, which allows one to obtain first the glycated protein fraction as a whole, followed by lectin affinity chromatog-raphy to separate the glycated proteins.

Lectins are obtained from either plant or animal materials and have various biological functions. Originally, lectins were also termed phytohaemag-glutinins because they were isolated from plant sources (phyto) and were used to classify blood cells by their agglutinizing property (haemagglutinins). The agglutinization occurs because lectins are multimeric proteins with multiple sugar binding sites which can crosslink red blood cells, thus aggregating them.

There are two basic approaches to lectin affinity chromatography. At the present time, the easiest approach, and the one most commonly used, is to survey the commercially available lectins, preferably those which are already immobilized, for their ability to bind the target protein. It is helpful if the terminal sugar of the glycated protein is already known, since that makes the choice of lectin much easier. On the other hand, it is also relatively simple to carry out a trial and error procedure to determine which immobilized lectin is best in the purification of a particular protein. It is also possible to immobilize an appropriate lectin from among the many commercial lectins that are available if the terminal sugars of the glycoconjugate to be purified are known.

The second approach is to determine the terminal sugar of the glycoconjugate to be purified and then to purify, de novo, a new lectin, by immobilization of the desired terminal sugar to a matrix, such as agarose, by means of a spacer arm. This allows the screening of numerous potential lectin sources to find as many lectins as possible that will bind to the target sugar. This methodology was used for many years prior to the present significant commercial availability of purified lectins.

After the lectin has been chosen and obtained in an immobilized form, or is immobilized by one of many simple procedures, for example agarose or cyanogen-bromide-activated agarose, or Affi-gel 10, adsorption/desorption, method conditions need to be defined. The binding of glycoproteins to lectins is generally easier than their elution. The factors in binding are generally temperature and salt concentration, as well as the density of coverage of the lectin immobilized on the matrix. High densities of lectins are not desirable in most instances. Although high ligand density may yield a marginal increase in capacity, it significantly increases the difficulty of elut-ing the target protein. The use of a moderate salt concentration is often helpful during binding of the glycoprotein target to the immobilized ligand. In many instances, it is necessary to be certain that the required metal ions are included in the sample wash and elution buffers in order to prevent deforming and/or denaturation of the lectin. Once the protein binding conditions have been determined, elution conditions need to be investigated. If one wants to have the highest purity product and to be certain that binding the protein to the immobilized lectin is through the biologically significant, sugar-protein interaction, then elution should be done by a high concentration of the free sugar. This will bind competitively to the lectin, displacing the glycoconjugate and thus eluting it. The sugar concentration during elution must be high enough to compete effectively with the lectin for the glycoconjugate, particularly when the density of the immobilized lectin is high.

Nonspecific elution of the glycoprotein from the lectin is frequently used. Changes in pH, temperature and salt can affect elution by decreasing the affinity of the lectin for the glycoconjugate. If elution were either with free sugar or by changing binding conditions, for example pH, a good yield is not obtained. It is also possible to apply an eluent to the column and stop the flow, thus allowing equilib rium to be reached in the free solution. This may or may not be necessary, however, and frequently a simple clean elution can be obtained by a proper choice of conditions.

One of the more interesting applications of immobilized lectins is the determination of the structure of sugar residues bound to the glycoconjugate. For example, a protein will be applied to a specific immobilized lectin and found to bind to that lectin. If the lectin has a rather broad specificity, the investigator cannot be certain which sugar is the terminal sugar on the oligosaccharide moiety of the glycoprotein. However, by using various glycases to remove the terminal sugar, the investigator can determine which glycase yields a derivative that will no longer bind to the immobilized lectin used. The glycase specificity indicates the identity of the terminal sugar. This procedure can be done repetitively and from the results a resonable understanding of the structure of the oligosaccharide of the glycated protein can be determined.

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