Dextran enzyme conjugates

Our present approach in the preparation of new dextran-enzyme conjugates is initially to test the standard conditions. If such conditions do not give satisfactory results, either in terms of retention of activity or extent of conjugation, the effect of the three important parameters, pH, temperature and duration of reaction are then investigated to establish appropriate conditions for coupling. In some cases it has also been found useful to examine the effect of these variables on the stability characteristics and other properties of the resulting dextran-enzyme conjugates. 

Jin R, Hiemstra C, Zhong Z et al (2007) Enzyme-mediated fast in situ formation of hydrogels from dextran-tyramine conjugates. Biomaterials 28 2791-2800... 

In this article the preparation of one class of carbohydrate-enzyme conjugates, prepared by attachment of dextran to enzymes, is described in some detail and the properties of enzymes modified in this way are discussed. The molecular basis of enzyme stabilization by coupling with dextran is also considered. 

A number of enzymes to which soluble dextran had been attached after activation by cyanogen bromide have been characterized by Marshall and coworkers.7 All of the conjugated enzymes were found to be more stable than the native enzymes to heat, proteases, denatur-ants, and the absence of calcium. The attachment of dextran appeared to have stabilized the conformation of the enzyme. It was suggested that the attachment of the enzyme to the dextran polymer at several points fixed its conformation in much the same way as do intramolecular, disulfide bridges.7... 

The most versatile method for the introduction of bioactive molecules with terminal NH2 functions such as proteins and enzymes is the coupling via formation of a Schiff base (Fig. 47). It is necessary to oxidise the dextran backbone in the first step to obtain reactive aldehyde moieties. The Schiff base formed between the amine group of the bioactive molecule and the oxidised dextran is usually reduced in a separate step to obtain a stable conjugate. 

Because of these limitations, polymer-based immunohistochemical methods that do not rely on biotin have been introduced and are gaining popularity (5). These methods utilize a unique technology based on a polymer backbone to which multiple antibodies and enzyme molecules are conjugated. In the EPOS (Enhanced Polymer One Step) system, as many as 70 enzyme molecules and about 10 primary antibodies were conjugated to a dextran backbone. This allowed the entire immunohistochemical staining procedure, from primary antibody to enzyme, to be accomplished in a single step (6). On the other hand, one limitation of this method was its restriction to a select group of primary antibodies provided by the manufacturer, and not suitable for user-supplied primary antibodies. 

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