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Gly-Pro-AMC *CAS 115035-46-6*

Gly-Pro-AMC is a sensitive fluorogenic substrate for detecting dipeptidyl peptidase IV (DPPIV), a serine protease that cleaves N-terminal dipeptides from polypeptides with L-proline or L-alanine at the penultimate position. DPPIV is a multifunctional protein expressed on the surface of several cell types including epithelial, endothelial and lymphoid cells. It is identical to the T cell activation antigen CD26 and the adenosine deaminase binding protein, and it is also released as a soluble form in plasma. The substrates of CD26/DPPIV include a wide variety of proline-containing peptides such as growth factors, chemokines, neuropeptides, and vasoactive peptides. DPPIV is involved in immune regulation, signal transduction, and apoptosis, and appears to play an important role in tumor progression. Importantly, DPPIV is a therapeutic target for type II diabetes due to its role as a serum protease that cleaves incretin hormones of the glucagon family of peptides and thus regulates glucose homeostasis. Studies indicate that a DPPIV inhibitor improves impaired glucose tolerance. The assay of DPPIV with Gly-Pro-AMC is a simple, fast and flexible assay that is ideal for high-through put screening. The assay demonstrates improved sensitivity compared to colorimetric DPPIV assays, allowing the researcher to use less enzyme while still achieving appropriate Z' values. The homogeneous coupled-enzyme format is convenient, requiring only a single reagent addition to the test samples. Maximal sensitivity is achieved in 20-30 minutes, and the signal generated is very stable.

Example protocol

AT A GLANCE

Important notes
The following protocol is recommended procedure, the optimum conditions must be determined experimentally for each test.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Gly-Pro-AMC stock solution:
Make a 10 to 25 mM Gly-Pro-AMC stock solution in DMSO. Keep from light. Note: The stock solution should be used promptly; any unused solution should be aliquoted and frozen at -20 o C. Note: Avoid repeated freeze-thaw cycles and keep from light.

PREPARATION OF WORKING SOLUTION

Gly-Pro-AMC working solution:
Take one vial out, and make 50 to 100 µM assay solution by diluting the stock solution with an assay buffer of your choice (such as 50 mM Tris-HCl, pH 7.5, 1.0 mM DTT).

SAMPLE EXPERIMENTAL PROTOCOL

  1. Mix equal volume of the DPPIV standards or samples with the assay solution, and incubate at room temperature for at least 1 hour.

  2. Monitor the fluorescence increase at Ex/Em = 380/500 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Gly-Pro-AMC *CAS 115035-46-6* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM225.545 µL1.128 mL2.255 mL11.277 mL22.555 mL
5 mM45.109 µL225.545 µL451.091 µL2.255 mL4.511 mL
10 mM22.555 µL112.773 µL225.545 µL1.128 mL2.255 mL

Molarity calculator

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Spectrum

Citations

View all 8 citations: Citation Explorer
Activation of the NLRP1 Inflammasome and Its Role in Transmissible Gastroenteritis Coronavirus Infection
Authors: He, Haojie and Wang, Wenzhe and Li, Liang and Zhang, Xin and Shi, Hongyan and Chen, Jianfei and Shi, Da and Xue, Mei and Feng, Li
Journal: Journal of Virology (2023): e00589--23
Manipulation of saliva-derived microcosm biofilms to resemble dysbiotic subgingival microbiota
Authors: Jiang, Yaling and Brandt, Bernd W and Buijs, Mark J and Cheng, Lei and Exterkate, Rob AM and Crielaard, Wim and Deng, Dong Mei
Journal: Applied and Environmental Microbiology (2021): e02371--20
Collagen peptides with DPP-IV inhibitory activity from sheep skin and their stability to in vitro gastrointestinal digestion
Authors: Wang, Beibei and Yu, Zhe and Yokoyama, Wallace and Chiou, Bor-Sen and Chen, Maoshen and Liu, Fei and Zhong, Fang
Journal: Food Bioscience (2021): 101161
Characterization of DPP-IV Inhibitory Peptides Using an In Vitro Cell Culture Model of the Intestine
Authors: Jin, Ritian and Shang, Jiaqi and Teng, Xiangyu and Zhang, Ligang and Liao, Minhe and Kang, Jiaxin and Meng, Ran and Wang, Dangfeng and Ren, Haowei and Liu, Ning
Journal: Journal of Agricultural and Food Chemistry (2021): 2711--2718
Identification of curcumin as a potential $\alpha$-glucosidase and dipeptidyl-peptidase 4 inhibitor: Molecular docking study, in vitro and in vivo biological evaluation
Authors: Cao, Wanxiu and Chen, Xin and Chin, Yaoxian and Zheng, Jinkai and Lim, Phaik Eem and Xue, Changhu and Tang, Qingjuan
Journal: Journal of Food Biochemistry (2021): e13686

References

View all 7 references: Citation Explorer
Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay
Authors: Nguyen KT, Hu X, Pei D.
Journal: Bioorg Chem (2004): 178
Activity of DPP III in human cerebrospinal fluid derived from patients with pain
Authors: Sato H, Kimura K, Yamamoto Y, Hazato T.
Journal: Masui (2003): 257
Extracellular peptidases of imaginal discs of Drosophila melanogaster
Authors: Wilson CL, Shirras AD, Isaac RE.
Journal: Peptides (2002): 2007
Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity
Authors: Tran TV, Ellis KA, Kam CM, Hudig D, Powers JC.
Journal: Arch Biochem Biophys (2002): 160
Purification and characterization of barley dipeptidyl peptidase IV
Authors: Davy A, Thomsen KK, Juliano MA, Alves LC, Svendsen I, Simpson DJ.
Journal: Plant Physiol (2000): 425
Page updated on October 28, 2024

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Physical properties

Molecular weight

443.37

Solvent

DMSO

Spectral properties

Excitation (nm)

341

Emission (nm)

441

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

CAS

115035-46-6

Platform

Fluorescence microplate reader

Excitation380 nm
Emission500 nm
Cutoff435 nm
Recommended plateSolid black
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