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IONTOSORB - Bead Cellulose Derivatives
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Chromatography of Proteins on Selective Sorbents Chromatographic characterization of proteins is fast and simple and simple and enables evaluating proteins also in such cases, where their molecular properties are not very known or they are unknown at all as in certain more complicated cases, where more that one enzyme participate in the investigated process. In the referred paper there were characterized chosen proteins (serumalbumine, ovalbumine, catalase and others) by chromatography on immobilized dyes (DLAC) and chromatography on immobilized ions of metals (IMAC). Retention volumes and immobilization yields were compared for two immobilization manners (binding by reaction with glutaraldehyde on IONTOSORB DETA and azo-coupling on IONTOSORB AV). It is known that during binding by reaction with glutaraldehyde above all free aminogroups and reactive heterocyclic aminoacids (especially histidine) take place. This situation is the same as during IMAC. Sulkowski [2] proved that IMAC can be used as a method for the determination of protein topography because of the reactive groups position on the protein surface. For this reason it is clear that the relation between protein sorption on IMAC sorbents specific for aminogroups and histidine can be in connection with the reactivity of these proteins during immobilization by glutaraldehyde. Histidine and primary aminogroups, if they are not available, are not sorbed during IMAC and therefore cannot react during binding with glutaraldehyde. Besides that the flexibility and size of glutaraldehyde are very similar to the size and flexibility of chelating groups, res. chelates. Only the size of a metal atom in a chelate, which is much greater than the size of carbon atoms in glutaraldehyde, causes certain difficulties. The relation between the available aminogroups content and the sorption on IMAC sorbents or binding with glutaraldehyde on the other hand is undoubted and unambiguous [3,4]. As a second model binding proteins by azo-coupling on IONTOSORB AV was chosen. Detailed description of individual processes and their character is given in the work [1]. Author was dealing especially with properties of both chromatographic procedures in connection with individual immobilization methods. During model proteins IMAC always the same procedure was kept, which is
the basic condition of correlation. Immobilization of the same proteins was performed by glutaraldehyde in the usual manner [5]. Yields of bound proteins and adequate elution volumes are given in the table 1. From the table it can be seen that the agreement is very good. In statistical evaluation of the results agreement authors calculated the correlation coefficient 0.86, which was lower than authors presupposed, but nevertheless it shows a very good agreement and applicability of this method. Table 1 The Comparison of Relative Retention Volumes in Chromatography of
Model Proteins
By the same procedure authors [1] worked also during comparing retention volumes at DLAC of these proteins on IONTOSORB RED and yields of immobilized proteins at azocoupling. In this case the program of the chromatographic method was as follows: rinsing: 5 ml initiate buffer (acetate c = 50
mmol/l, pH 5.5) elution: finishing buffer (the same as the initial buffer with NaCl (c = 1 mol/l).
As it can be seen from the table 2 showing results of measurements, the agreement is very low. The correlation coefficient is only a bit higher than 0.6 (exactly 0.62), which shows only rather free dependence. Nevertheless authors [1] mean that the method can serve at least to only orientation evaluations of binding method applicability and it could be improved by more detailed investigation. Table 2 Comparing Relative Retention Volumes at Chromatography of Model
Proteins
Results of both methods showed that optimization of enzymes binding on supports by retention volumes during chromatography on selective sorbents is applicable, which is a rather encouraging. It can be supposed that this procedure could be improved by the choice of a proper chromatographic method and ligand. It involves not only measuring a greater assortment of proteins but also more detailed studying individual chromatographic methods. In the next period authors intend dealing with both these questions in the following way: They would isolate individual ingredients from complicated natural mixtures by affinity chromatography on sorbents with wide selectivity and immobilize proteins obtained in this way with exactly known retention times by several methods. References:
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