Immobilized derivatives of leucine aminopeptidase and aminopeptidase M. Applications in protein chemistry.

Leucine aminopeptidase and aminopeptidase M have been covalently bound to an arylamine derivative of porous glass. The bound forms of both enzymes retain 100% of their activities at saturating levels of substrate (leucine p-nitroanilide). For the hydrolysis at pH 7.3 and 25” kcat values for bound and free leucine aminopeptidase are 46 & 5 s-l and 46 + 2 s-l, respectively; for aminopeptidase M (pH 7.5 and 25”) k,,, values are 23 f 2 0 and 21 i 0.4 s-l, respectively. Although the Michaelis constants for both enzymes increase on binding, the pH and temperature dependencies of the bound enzymes remain unchanged. These data suggest that the environments and conformations of the enzymes are not significantly changed after coupling to the solid support. The apparent decrease in the binding of substrate could be explained by a decrease in the effective diffusion coefficient of the substrate. Both insoluble enzymes are active against polypeptide substrates. After treatment for removal of contaminating endopeptidases, the immobilized derivatives of leucine aminopeptidase and aminopeptidase M were used successfully in NH*-terminal sequence determination. The bound aminopeptidase M appears to be the better of the two for this purpose. Both bound enzymes will catalyze the hydrolysis of the aminoethylated A and B chains of insulin nearly to completion ( 287% recovery of free amino acids in all cases). These digests are carried out at pH values near neutrality in a volatile buffer with no activating metal. Immobilized pronase (ROYER, G. P., AND GREEN, G. M. (1971) Biochem. Biophys. Res. Commun. 44, 426) was used in concert with bound leucine aminopeptidase and bound arninopeptidase M for the hydrolysis of /3-lactoglobulin. In both cases the recovery of free amino acids was 93 %. These bound enzymes should be quite useful in amino acid composition determinations when acid-labile residues such as tryptophan, glutamine,