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RESEARCH PRODUCT

Methods for Separating Native Enzymes

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In the course of electrophoresis the stability of an enzyme depends on such conditions as (a) pH-value, (b) ion strength and ion species, (c) effector molecules, (d) temperature and (e) properties of the separation matrix. These parameters were empirically optimized for starch gel electrophoresis [1–3] and cellulose acetate electrophoresis [4, 5] when analyzing predominantly animal and human specimen. A major advantage of these types of separation media is that practically every buffer system can be used to separate enzymes whereas in disc-gel electrophoresis [6–8] the number of applicable buffer systems is limited. When using isoelectric focusing to separate native enzymes no buffer choice at all is possible [9–10]. On the other hand, starch gel electrophoresis is more time consuming than cellulose acetate electrophoresis. Cellulose acetate membranes are commercially available and easy to handle, and separation and enzyme visualization together do not take longer than 1–1.5 h. In clinical diagnosis therefore, both methods are favoured. Disc-gel electrophoresis, gradient gel electrophoresis and isoelectric focusing are used if the separation capacities of cellulose acetate membranes are insufficient or when the isoelectric point or the molecular mass of an enzyme is to be estimated. The data compiled in Tables 3.1 and 3.2 can be used to decide between methods for a particular case.