Electrophoresis of elastase like esterases from human neutrophils

Multiple esterases from individual leukocyte types of normal adult human beings were demonstrated after separation by cationic disc gel electrophoresis. Two substrates, 1 naphthyl N acetyl DL alanine (NAcAla) and 1 naphthyl butyrate, were used routinely in a simultaneous coupling azo dye method to demonstrate the esterases. Cytochemically, the neutrophil granules stain very poorly with α unsubstituted carboxylic acid esters such as 1 naphthyl butyrate or 1 naphythl acetate but stain selectively and intensely at pH 7.0 with 2 classes of naphthol esters: α halo substituted esters such as 1 naphthyl 2 bromobutyrate or naphthol AS D chloroacetate or α amino substituted carboxylic acid esters such as 1 naphthyl N acetyl DL alanine, or 1 naphtyl N acetyl alanyl L alanyl L alanine. Cationic zymograms showed 3 major neutrophil esterases which hydrolyze NAcAla and 1 naphthyl N acetyl L alanyl L alanyl L alanine vigorously and 1 naphthyl butyrate very poorly, which provided us with the first evidence that these enzymes might be proteolytic. Cationic zymograms of a mixture of purified enzymes (reference enzymes), pancreatic elastase, chymotrypsin and trypsin, were compared to zymograms of neutrophil extracts after assaying with NAcAla. The neutrophil esterase and elastase hydrolyzed NAcAla vigorously; chymotrypsin and trypsin hydrolyzed it moderately. More precise characterization of the neutrophil esterases was obtained with chloromethyl ketone inhibitors which have the advantage of being irreversible and also can be synthesized with a substrate like moiety which confers specificity. Neutrophil extracts and the mixture of reference enzymes were preincubated before electrophoresis with either N acetyl L alanyl L alanyl L alanine chloromethyl ketone (NAcAla₃CK), N acetyl L alanyl L phenylalanine chloromethyl ketone (NAcAlaPheCK) or N tosyl L lysine chloromethyl ketone (TLysCK). Elastase was selectively inhibited by NAcAla₃CK, chymotrypsin by NAcAlaPhe CK and trypsin by TLysCK. Neutrophil 'lysosomal' and neutrophil enriched cell extracts which were treated before electrophoresis with the chloromethyl ketone inhibitors at the same concentrations as used for the reference enzymes were almost completely inhibited by the NAcAla₃CK except for one enzyme fraction. This fraction was inhibited by NAcAla₃CK when neutrophil preparations were electrophoresed and then exposed to the inhibitor before assaying for activity. The neutrophil enzymes were much less sensitive to the TLysCK and NAcAlaPheCK. The evidence from these studies strongly supports the conclusion that the major esterases in human neutrophil granules are elastase like esterases.