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A fluorometric assay for peptidyl alpha-amidation activity using high-performance liquid chromatography
B N Jones1, P P Tamburini, A P Consalvo
1Department of Protein Chemistry, Unigene Laboratories, Inc., Fairfield, New Jersey 07006.
Analytical Biochemistry
|February 1, 1988
Summary
A new assay quantifies peptidyl alpha-amidation activity using HPLC and fluorometric detection. This rapid and sensitive method is ideal for enzyme screening and characterization.
Area of Science:
- Biochemistry
- Analytical Chemistry
Background:
- Peptidyl alpha-amidation is a crucial post-translational modification.
- Accurate quantification of this activity is essential for biochemical research.
Purpose of the Study:
- To develop a rapid and sensitive assay for peptidyl alpha-amidation activity.
- To enable high-throughput screening and detailed enzyme characterization.
Main Methods:
- Reverse-phase high-performance liquid chromatography (RP-HPLC) with fluorometric detection.
- Utilized a dansylated tripeptide substrate (N-dansyl-Tyr-Val-Gly-OH) for assay development.
- Quantified product formation (N-dansyl-Tyr-Val-NH2) via isocratic elution on C-18 columns.
Main Results:
- Achieved sensitive detection of both substrate and product down to 5 fmol.
- Demonstrated high reproducibility and a rapid assay time of less than 3 minutes per sample.
- Validated applicability across various pH conditions and for diverse research applications.
Conclusions:
- The developed RP-HPLC assay offers a robust, sensitive, and efficient method for determining peptidyl alpha-amidation activity.
- The assay is suitable for screening enzyme activity in tissues, monitoring purification, determining kinetic parameters, and identifying inhibitors.