Synthesis of N alpha-[3H]acetyl-L-lysine chloromethyl ketone and its use in the fluorographic detection of proteases.
Tritiated N alpha-acetyl-L-lysine chloromethyl ketone (ALCK) was synthesized on a laboratory scale for use as an active-site-directed affinity label in the fluorographic detection of proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The synthesis involved acetylation of N epsilon-benzyloxycarbonyl-L-lysine chloromethyl ketone with [3H]acetic anhydride just before the removal of the benzyloxycarbonyl group. By this method, [3H]ALCK with a specific activity of 250 mCi/mmol was obtained as a crystal. Trypsin, thrombin, plasmin, papain, and clostripain were inactivated by ALCK according to first-order kinetics. For fluorographic detection of proteases, enzyme samples were allowed to react with [3H]ALCK and then resolved by SDS-PAGE. Proteases that reacted with [3H]ALCK could be detected with a sensitivity equivalent to or higher than that of Coomassie brilliant blue R-250 staining. A trypsin-like protease in Pronase, clostripain as a contaminant in a commercial preparation of Clostridium histolyticum collagenase, and cysteine proteases in Porphyromonas gingivalis could be detected.
Nishikata
M
M
Central Research Division, School of Dentistry, Hokkaido University, Sapporo, Japan.
D000590:Amino Acid Chloromethyl Ketones / Q000138:chemical synthesis* / Q000494:pharmacology; D001345:Autoradiography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010450:Endopeptidases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D014316:Tritium
D000590:Amino Acid Chloromethyl Ketones; C084570:Nalpha-acetyllysine chloromethyl ketone; D011480:Protease Inhibitors; D014316:Tritium; D010450:Endopeptidases