Abstract
In order to determine the amount of amylase/cell in hamster parotid epithelial cells following their purification in a previously described isokinetic gradient (1), we required a very sensitive assay which could be used to examine samples obtained from very small numbers of cells. The kinetic method reported herein requires only 50 μl of enzyme solution. The initial velocity of the reaction can be determined in 3 min by monitoring the decrease in the starch-iodine color at 610 nm in a solution containing starch, iodine, and potassium iodide. Using this method, the amylase activity of a sonicated suspension of unpurified hamster parotid cells was 0.23 mg starch degraded/min/million epithelial cells. This method eliminates the problem of decolorization of the starch-iodine complex by proteins such as albumin since relatively small amounts of enzyme solution are required, the method measures the relative decrease in absorbance with time, and excess iodine is present to combine with the protein. The method offers the additional advantage that it is not necessary to remove samples of the reacting mixture, and the problems of stopping the reaction and contamination of the reaction mixture are eliminated. The method is compared with the methods of Somogyi (3) and Jacobsen and Hensten-Pettersen (13).
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