Determination of the Molecular Weight of α-Amylase, as the Enzyme-Inhibitor Complex, using Thin Layer Gel Filtration on Sephadex

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Abstract
Thin layer gel filtration on Sephadex was performed as a simple method for recognition of the small MW differences of human .alpha.-amylases from different sources. Activity was located with dry chromogenic substrate, using a replica technique. Undesirable interaction between the gel matrix and substrate binding sites on the enzyme, which causes an anomalous decrease in the migration rate of the enzyme protein, was suppressed by preincubation of the enzyme with appropriate inhibitor. Gradual masking of substrate binding sites of the enzyme by increasing concentrations of amylase inhibitor resulted in 2 distinct migration rates for the enzyme in thin layer gel filtration. This suggests the existence of 2 substrate binding sites in the enzyme molecule. Together with thin layer gel affinity chromatography on a mixture of Sephadex and ConA[concanavalin A]-Sepharose, the method yielded useful data on the MW of amylase and its glycosylated forms and served as a valuable tool for the differential diagnosis of macroamylasemia.

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