Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP)
Applications:
- Liberates the N-terminal Acyl Blocking Group from Proteins
Description:
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a unique exo-type aminopeptidase that liberates blocking groups (formyl, acetyl, and myristyl), and then releases the subsequent amino acids from proteins and peptides until it reaches the first X-Pro bond. This enzyme has a wide range of substrate specificities and is used to determine short stretches of amino acid sequence of blocked proteins and peptides whose sequence can be determined by mass difference in Mass Spectrometry. In addition, since the amino terminus of Ac-DAP is acetylated, its amino acid sequence cannot be determined by Edman Degradation. Therefore, even if a high E/S ratio is used (for example, E/S = 0.5-1), the amino acid sequence of the target protein or peptide can still be determined by Edman degradation without separation from the enzyme.
Note: Ac-DAP cannot act upon a non-denatured protein; therefore, the protein sample must be completely denatured by carboxymethylation for Ac-DAP digestion.
Purity:
Homogeneous on PAGE
Form:
Solution in 50 mM N-Ethylmorpholine-AcOH buffer (pH 8.0)
Definition of Activity:
One unit of the enzyme activity corresponds to the amount required to hydrolyze 1 μmol of Leucine-p-nitroanilide at 75°C, pH 8.0, in one minute.
Source:
Yeast carrying the plasmid which contains the gene encoding Pyrococcus furiosus DAP.
Properties:
| Molecular weight: | 38,639 kDa (mass spectrometry) |
| 451,000 kDa (sedimentation equilibrium method) | |
| Activator: | CoCl2 |
| Inhibitor: | Amastatin, EDTA |
| Optimum pH: | 6.5–9.0 |
| Optimum Temperature: | 85–90°C |
| Thermal Stability: | It was confirmed that the enzyme retains 100% activity after 48 hrs. at 50°C in the supplied buffer (pH 8.0, with 0.1 mM Co2+). |
Supplied Buffer (5X)
| Volume: | 1 mL |
| Component: | 250 mM N-Ethylmorpholine-AcOH buffer |
| (pH 8.0) containing 0.5 mM CoCl2 | |
| Once thawed, the buffer should be stored at 4°C. Avoid freeze-thaw cycles. | |
Specific Activity: 8–10 units/mg
Protein Concentration: 1 mg/mL
Storage:
–20°C. Once thawed, the enzyme solution should be stored at 4°C. The thawed enzyme and buffer are stable at least 3 months at 4°C.
Notes:
Since this enzyme cannot react with proteins containing the higher order structures, protein samples should be denatured by chemical pocedures such as carboxymethylation. This enzyme cannot act on proteins blotted on PVDF membrane.
