Highly sensitive high-performance liquid chromatography-fluorimetric assay method for carboxypeptidase H activity |
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| Authors: | Ryuichi Yajima Toshiyuki Chikuma Takeshi Kato |
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| Affiliation: | 1. Department of Medicine, Division of Rheumatology, University of Miami Miller School of Medicine, Miami, FL 33136, United States;2. Medical Genetics Branch, NHGR, NIH, Bethesda, MD 20892, United States;3. Department of Medicine, Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Boston, MA 02115, United States;4. Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, United States;5. Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, United States |
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| Abstract: | 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. |
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