The Estimation of the Proteolytic Activity of Trypsin and Pronase E toward Gelatin and Casein by Using Acid-base Indicators

MASAKI Takeharu, NAKAMURA Keigi, SOEJIMA Masami

doi:10.1271/nogeikagaku1924.51.3_147

A simple spectrophotometric method to estimate proteolytic activities of trypsin and pronase E toward gelatin and casein were described. The principle of the method was based on the measurement of colour changes of acid-base indicators, i. e., phenol red (PR) or neutral red (NR) in the enzyme reaction mixture.<br> The colour developments of PR and NR were decreased or increased with the changes of the concentrations of hydrogen ion liberated during the hydrolysis of gelatin and casein.<br> The absorptions at 560 nm (PR) or at 530 nm (NR) of the reaction mixture in the cuvette were liniearly changed in the correlation with the concentration of the enzymes and with the reaction time elapsed. The absorbance changes (_??_E) of acid-base indicator were well correlated with the concentration of free carboxyl groups liberated, measured by formol titration method. The reaction mixture consisted of 8.3 μg/ml of PR and 0.67_??_6.70 μg/ml of the enzyme in 5 to 10mM Tris-HCl buffer, pH 8.0, together with 1.0 to 2.5% of substrates (PR-T method).<br> The estimation could be also done in the reaction mixture not containing buffers since gelatin and casein have cosiderably potent buffer actions (PR method, NR method). The proteolytic activity of trypsin was little inhibited by PR and NR.

The Estimation of the Proteolytic Activity of Trypsin and Pronase E toward Gelatin and Casein by Using Acid-base Indicators

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