Use of Novel Long Wavelength FRET Substrate for Renin Assay

By Vera Rakhmanova, Ph.D. and Rich Meyer, Ph.D. AnaSpec, Inc. San Jose, CA 95131

Introduction
The renin–angiotensin system (RAS) plays a central role in the regulation of blood pressure and electrolyte homoeostasis. At the first and rate-limiting step of the RAS cascade, renin, a highly specific aspartyl protease, cleaves angiotensinogen to angiotensin I, which is further converted into angiotensin II. Angiotensin II constricts blood vessels leading to increased blood pressure. An over-active renin-angiotensin system results in vasoconstriction and retention of sodium and water leading to hypertension. Therefore, renin is an attractive target for the treatment of hypertension.

The SensoLyte^™ 520 Renin Assay Kit provides a convenient assay for high throughput screening of renin inhibitors and for continuous testing of renin activity using FRET peptide labeled with the quencher QXL^™ 520 and the fluorophore 5-FAM. The sequence of this FRET peptide is derived from a cleavage site of renin.¹

In the FRET peptide the fluorescence of 5-FAM is quenched by QXL^™ 520. Upon cleavage into two separate fragments by renin, the fluorescence of 5-FAM is recovered, and can be monitored at excitation/emission = 490/520 nm. Compared to a previously reported commercial substrate, Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg, the QXL^™ 520/5-FAM FRET substrate is about 40 fold more sensitive and can detect 0.8 ng/ml of renin.

Advantages of QXL^™ 520 - 5-FAM FRET pair:
• The absorption spectrum of QXL^™ 520 overlaps with the emission spectrum of 5-FAM.
• Hydrophilicity of QXL^™ 520 results in better solubility of the peptide substrate.
• Higher brightness and stability of 5-FAM compared to EDANS.
• Long wavelength fluorescence of 5-FAM is less interfered by autofluorescence of drug candidates or cellular components.

Materials
• The SensoLyte^™ 520 Renin Assay Kit, Cat# 72040.
• Renin peptide inhibitor, RRPFH-Sta-IHK-NH₂ Cat#72065
• 96-well or 384-well microplate: Black microplates can provide better signal to noise ratio
• Fluorescence microplate reader: Capable of detecting emission at 520 nm with excitation at 490 nm.

Assay Performance
The SensoLyte^™ 520 Renin Assay was compared with an EDANS/DABCYL based assay. QXL^™ 520/5-FAM substrate (7 M) was incubated with titrated renin in the total volume of 150 L (96-well plate format). Signal increased up to 60 fold after 30 minutes of renin incubation with QXL^™ 520/5-FAM FRET substrate in comparison to only 2.5 fold signal increase observed for an EDANS/DABCYL based assay (Fig.1).

Fig. 1. Assay comparison at different concentration of enzyme. End-point fluorescence signal was recorded at Ex/Em=490 nm/520 nm (FlexStation 384II, Molecular Devices).

The sensitivity of QXL^™ 520/5-FAM substrate was evaluated in a continuous assay. FRET substrate (7 M) was incubated with the indicated amount of enzyme at 37oC and fluorescence signal was measured over time (Fig.2). Sensitivity of the SensoLyteTM520 Renin Assay Kit was 0.8 ng/ml renin with 30 minutes incubation time and 0.2 ng/ml with 2 hours incubation time.

Fig. 2. Sensitivity of the SensoLyte^™ 520 Renin Assay Kit measured over time with serial dilutions of enzyme. Renin concentrations tested were 0.2, 0.4, 0.8, 1.7 and 3.5 ng/mL.

To validate the SensoLyte^™ 520 Renin Assay for screening of renin inhibitors, we used previously described renin peptide inhibitors.^2,3 The peptide inhibitor Ac-His-Pro-Phe-Val-Sta-Leu-Phe-NH₂ is included in the kit. Another peptide inhibitor (Cat# 72065) is derived from sequence Z-Arg-Arg-Pro-Phe-His-Sta-Ile-His-Lys(Boc)-OMe. QXL^™ 520/5-FAM substrate was incubated with 3.5 nM renin in the presence of inhibitors (10^-3 to 10⁵ nM). Kinetic readings were taken every 3 minutes for 15 minutes at 37oC. Calculated IC₅₀ correlated well with the literature values (Fig.3).

Figure 3. Validation of the assay with renin peptide inhibitors.

Conclusion
Properties of the SensoLyte^™ 520 Renin Assay Kit are summarized in the table (Table 1). The kit provides higher sensitivity than the assay using EDANS/DABCYL substrate. The longer excitation and emission wavelengths of QXL^™ 520/5-FAM substrate minimize the interference from autofluorescence of test compounds, which ideally suited for screening of renin inhibitors.

Table 1. Summary of properties for the SensoLyte^™ 520 Renin Assay Kit

*with 0.13 ng/ml enzyme, with 30 min. incubation
**with 30 min. incubation

References
1. Paschalidou, K. et al., Biochem. J. 382, 1031 (2004).
2. Hui, KY. et al., J. Med. Chem., 31, 1679 (1988).
3. Wood, JM. et al., Hypertension, 7, 797 (1985).

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