Cross-Linking



        Immobilization of enzymes has been achieved by intermolecular cross-linking of the protein, either to other protein molecules or to functional groups on an insoluble support matrix.. Cross-linking an enzyme to itself is both expensive and insufficient, as some of the protein material will inevitably be acting mainly as a support. This will result in relatively low enzymatic activity. Generally, cross-linking is best used in conjunction with one of the other methods. It is used mostly as a means of stabilizing adsorbed enzymes and also for preventing leakage from polyacrylamide gels.
 
        Since the enzyme is covalently linked to the support matrix, very little desorption is likely using this method. Marshall (1973), for example, reported that carbamy phosphokinase cross-linked to alkyl amine glass with glutaraldehyde lost only 16% of its activity after continuous use in a column at room temperature for fourteen days.
 
        The most common reagent used for cross-linking is glutaraldehyde. Cross-linking reactions are carried out under relatively severe conditions. These harsh conditions can change the conformation of active center of the enzyme; and so may lead to significant loss of activity.


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