Highly sensitive high-performance liquid chromatography-fluorimetric assay method for carboxypeptidase H activity.
A rapid and sensitive high-performance liquid chromatographic (HPLC)-fluorimetric assay method has been developed for the determination of carboxypeptidase H activity based on the measurement of N-(5-dimethyl-aminonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically from dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyl-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slower 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 pH 7.0 containing 60% methanol. Using this method carboxypeptidase H activity has been detected in rat sciatic nerves. This HPLC method facilitates the assay of carboxypeptidase H activity in the enzyme samples from various tissues.
Yajima
R
R
Research and Development Center, Dainabot Co., Chiba, Japan.
Chikuma
T
T
Kato
T
T
D000595:Amino Acid Sequence; D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D012584:Sciatic Nerve / Q000201:enzymology; D012680:Sensitivity and Specificity
C038595:dansylglycine; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D005998:Glycine; D000432:Methanol