HIGHLY SENSITIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY FLUOROMETRIC ASSAY-METHOD FOR CARBOXYPEPTIDASE-H ACTIVITY

A rapid and sensitive high-performance liquid chromatographic (HPLC)-f luorimetric assay method has been developed for the determination of c arboxypeptidase H activity based on the measurement of N-(5-dimethyl-a minonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically f rom dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyr-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N'-2-ethanesulf onic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slo wer with an acidic buffer at pH 4.6. The new HPLC method separates the product and substrate in less than 5 min using an elution buffer at p H 7.0 containing 60% methanol. Using this method carboxypeptidase H ac tivity has been detected in rat sciatic nerves. This HPLC method facil itates the assay of carboxypeptidase H activity in the enzyme samples from various tissues.