Immobilized Leucine Aminopeptidase Applications in Protein Chemistry

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Abstract
Leucine aminopeptidase (EC 3.4.1.1) has been covalentiy bound to porous glass through an azo linkage. For the hydrolysis of leucine p-nitroanilide at pH 7.3 and 25°, Km(app) for the immobilized enzyme is higher than that of the soluble enzyme: 1.30 ± 0.2 and 0.53 ± 0.03 mM, respectively. However, at saturating levels of substrate the immobilized derivative and free enzyme have similar activities; k. values for the bound and free enzymes are 46 ± 5 and 46 ± 2 sec−1, respectively. In addition, the pH and temperature dependences of the two enzyme forms are quite similar. These data suggest that the environment and conformation of the enzyme are not significantly changed after coupling. The apparent decrease in the substrate binding ability could be explained by a decrease in the effective diffusion coefficient of the substrate. The insoluble enzyme is also active against peptide substrates. After treatment to remove contaminating proteases, immobilized leucine aminopeptidase was used successfully in sequencing experiments. The bound enzyme should be useful in total hydrolysis of peptides and proteins. The aminoethylated derivatives of the A- and B-chains of insulin were hydrolyzed essentially to completion. βLactoglobulin was hydrolyzed to the extent of 93% with immobilized leucine aminopeptidase and immobilized pronase.

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