Edman degradation and protein sequencing: Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP)
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a unique exo-type aminopeptidase that liberates blocking groups (formyl, acetyl, and myristyl) from proteins and peptides, releasing amino acids until it reaches the first X-Pro bond. This enzyme has a wide range of substrate specificity and is used to determine short stretches of amino acid sequence in blocked proteins and peptides via mass spectrometry. An additional advantage of using Pfu Ac-DAP is that, since its amino terminus is acetylated, the enzyme itself is not subject to Edman degradation.
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a unique exo-type aminopeptidase that liberates blocking groups (formyl, acetyl, and myristyl) from proteins and peptides, releasing amino acids until it reaches the first X-Pro bond. This enzyme has a wide range of substrate specificity and is used to determine short stretches of amino acid sequence in blocked proteins and peptides via mass spectrometry. An additional advantage of using Pfu Ac-DAP is that, since its amino terminus is acetylated, the enzyme itself is not subject to Edman degradation. Therefore, even if a high enzyme to substrate (E/S) ratio is used (e.g., E/S = 0.5–1), protein sequencing can follow Edman degradation without the need for an extra purification step.
NOTE: Pfu Ac-DAP cannot act on non-denatured proteins; therefore, the protein sample must be completely denatured by carboxymethylation for Pfu Ac-DAP digestion.
More Information
Used for the liberation of N-terminal formyl, acetyl, and myristyl blocking groups from proteins
Components
- Buffer (5X)
Once thawed, the buffer should be stored at 4°C. Avoid additional freeze-thaw cycles.
Volume: 1 ml Components: 250 mM N-Ethylmorpholine-AcOH buffer (pH 8.0, 0.5 mM CoCl2) Once thawed, the buffer should be stored at 4°C. Avoid additional freeze-thaw cycles.
Source
Recombinant yeast containing the gene encoding Pyrococcus furiosus Ac-DAP.
Storage
−20°C. Once thawed, the enzyme solution should be stored at 4°C. The thawed enzyme and buffer are stable for at least 3 months at 4°C.
Notes
This enzyme cannot react with proteins containing higher order structures; protein samples should be denatured by chemical procedures such as carboxymethylation. This enzyme cannot react with proteins blotted onto PVDF membranes.
Properties
- Specific activity: >8 units/mg
- Protein concentration: 1 mg/ml
- Molecular weight: 38,639 Da (mass spectrometry), ~451 kDa (sedimentation equilibrium method)
- Activator: CoCl2
- Inhibitor: Amastatin, EDTA
- Optimum pH: 6.5–9.0
- Optimum Temperature: 85–95°C
- Thermal stability: Enzyme retains 100% activity after 48 hr at 50°C in the supplied buffer (pH 8.0, 0.1 mM Co2+)
- N-terminal amino acid sequence: Ac-MDDYELLKKVVEADGV...
Form
In a solution of 50 mM N-Ethylmorpholine-AcOH buffer (pH 8.0)
Definition of activity
One unit of activity corresponds to the amount of enzyme required to hydrolyze 1 µmol of leucine-p-nitroanilide at 75°C and pH 8.0 in one minute.
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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