Asp-N peptide cleavage assay

Figure 1: Schematic of Asp-N peptide cleavage

The imaging protease assay utilises the metallo-endoproteinase, Asp-N and its cleavage of an 8-mer peptide on the N-terminal side of aspartic acid (D)(1). Asp-N is often used for peptide mapping and protein sequencing. The assay format could, in principle, be adapted for use with other proteases. The assay has been configured in a quench format in which the N-terminus has been labeled with Cy™3B and the C-terminus with Cy5Q. Therefore, prior to protease cleavage the intact peptide cannot generate a fluorescent signal at Cy3B emission wavelengths (following excitation with light at Cy3B wavelengths) due to quenching by Cy5Q.

Proteolytic digestion of the peptide releases the quench effect, so excitation at Cy3B wavelengths results in the concomitant emission at Cy3B wavelengths (Figure 1).

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Methods

Assays were carried out in black opaque 384-well (maximum working volume 80 ml) or 1536-well (maximum working volume 10 ml) microtitre plates (Greiner Labortechnik GmbH, Frickenhausen, Germany) by adding substrate peptide, Cy3B-YVADAPVK-Cy5Q to a final assay concentration of 100 nM in a volume of 50 ml (384-well assay) or 5 ml (1536-well assay) of 50 mM Tris buffer, pH 8 containing 0.005% Tween™ 20. Subsequently an addition of Asp-N (Sigma Chemical Co., Poole, Dorset, U.K.) enzyme, added as 5 ml of a 1 mg/ml solution (5 ng, 384-well assay) or 0.5 ml of a 1 mg/ml solution (0.25 ng, 1536-well assay) or assay buffer only (no enzyme control) was made. The total assay volumes being 55 ml (384-well assay) and 5.5 ml (1536-well assay. Following incubation at room temperature for between 0 and 60 minutes, imaging was performed on LEADseeker™ Homogeneous Imaging System at 540 nm excitation and 570 nm emission wavelengths and exposure times of 60 seconds (384-well assay) and 90 seconds (1536-well assay) were used. A well-by-well correction method was employed with four standard concentrations of between 0 and 100 nM. The standard used for the corrections was Cy3B.

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Results

Cleavage of the peptide substrate over time was followed both in the presence (—) or absence (—) of enzyme in both (a) 384-well and (b) 1536-well assays (Figure 2). Values are mean ± SEM (n=3 for 384-well assay, error bars not seen and n=4 for 1536-well assay) and are expressed as nominal signal units obtained after the time shown. For reference maximum signal:background levels achieved were in the order 12:1 (384-well assay) and 14:1 (1536-well assay) and are expressed as the total Cy3B signal obtained after extensive cleavage (approximately 3 hours) above that of the uncleaved (no enzyme) peptide control.


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References

• DRAPEAU, G.R. (1979), Substrate specificity of a proteolytic enzyme isolated from a mutant of Pseudomonas fragi.
  J. Biol. Chem., 255, pp. 839 - 840.