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 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 requiring separation from the enzyme.

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.

Purity

Homogeneous on PAGE

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)