Gly-Pro-pNA (hydrochloride)
(Synonyms: N-甘氨酰脯氨酰-对硝基苯胺盐酸盐) 目录号 : GC18201
Gly-Pro-pNA（盐酸盐）是一种显色底物，可被循环酶二肽基肽酶 IV (DPP IV) 裂解。
Cas No.:103213-34-9
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
Mare whey protein hydrolysates |
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Preparation Method |
20 uL of sample solution and 100 uL of substrate solution (H- Gly-Pro-pNA (hydrochloride), final concentration 0.5 mM) were added. Enzyme reaction was started by adding DPP-IV. The absorbance at 405 nm was monitored after the microplate was incubated at 37°C for 0.5 h. |
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Reaction Conditions |
Final concentration 0.5 mM ，37 °C for 0.5 h. |
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Applications |
The degradation rate of the substrate Gly-Pro-pNA (hydrochloride) of DPP-IV was slowed by the addition of Mare Whey proteins. |
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References: [1]. Song JJ, Wang Q, et,al. Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates. J Dairy Sci. 2017 Sep;100(9):6885-6894. doi: 10.3168/jds.2016-11828. Epub 2017 Jul 12. PMID: 28711271. |
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Gly-Pro-pNA (hydrochloride) is a chromogenic substrate that can be cleaved by the circulating enzyme, dipeptidyl peptidase IV (DPP IV). Enzyme activity can be quantified by colorimetric detection of free p-nitroanilide at 405 nm. This substrate can be used to screen for DPP IV inhibitors.[5].
Mare whey proteins show good DPP-IV inhibitory activity in vitro，have the able to inhibit DPP-IV from degrading Gly-Pro-pNA (hydrochloride) Gly-Pro-pNA (hydrochloride) [1]. The in vitro DPP-IV inhibitory activities of quinoa protein hydrolysates can determined using Gly-Pro-pNA (hydrochloride) as a substrate[2]. Gly-Pro-pNA (hydrochloride) can also be used to evaluate the activity of DPP-IV in human serum[3]. Demonstrating that DPP IV in diabetic patients is not very different from that in normal conditions[4]. Gly-pro p-nitroanilide hydrochloride was used as a substrate to detect DPPIV level in patients with Polycystic syndrome (PCOS) ，a deregulation of DPP4 serum levels could be an additional characteristic of the metabolic imbalances associated with PCOS[5]
Using Gly-Pro-pNA (hydrochloride) as substrate can assess the enzymatic activity and biochemical status of dipeptidyl peptidase IV (DPP IV)in patients with rheumatoid arthritis[6]
References:
[1]. Song JJ, Wang Q, et,al. Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates. J Dairy Sci. 2017 Sep;100(9):6885-6894. doi: 10.3168/jds.2016-11828. Epub 2017 Jul 12. PMID: 28711271.
[2]. You H, Wu T, et,al. Preparation and identification of dipeptidyl peptidase IV inhibitory peptides from quinoa protein. Food Res Int. 2022 Jun;156:111176. doi: 10.1016/j.foodres.2022.111176. Epub 2022 Mar 19. PMID: 35651037.
[3]. Yeganeh F, Mousavi SMJ, et,al. Association of CD26/dipeptidyl peptidase IV mRNA level in peripheral blood mononuclear cells with disease activity and bone erosion in rheumatoid arthritis. Clin Rheumatol. 2018 Dec;37(12):3183-3190. doi: 10.1007/s10067-018-4268-y. Epub 2018 Aug 22. PMID: 30136129.
[4]. Sun AL, Deng JT, et,al. Dipeptidyl peptidase-IV is a potential molecular biomarker in diabetic kidney disease. Diab Vasc Dis Res. 2012 Oct;9(4):301-8. doi: 10.1177/1479164111434318. Epub 2012 Mar 2. PMID: 22388283.
[5]. Blauschmidt S, Greither T, et,al. Dipeptidyl peptidase 4 serum activity and concentration are increased in women with polycystic ovary syndrome. Clin Endocrinol (Oxf). 2017 Dec;87(6):741-747. doi: 10.1111/cen.13444. Epub 2017 Sep 13. PMID: 28799235.
[6]. Mavropoulos JC, Cuchacovich M, et,al. Anti-tumor necrosis factor-alpha therapy augments dipeptidyl peptidase IV activity and decreases autoantibodies to GRP78/BIP and phosphoglucose isomerase in patients with rheumatoid arthritis. J Rheumatol. 2005 Nov;32(11):2116-24. PMID: 16265688.
Gly-Pro-pNA（盐酸盐）是一种显色底物,可被循环酶二肽基肽酶 IV (DPP IV) 裂解。酶活性可以通过在 405 nm 处游离对硝基苯胺的比色检测来量化。该底物可用于筛选 DPP IV 抑制剂。[5]。
马乳清蛋白在体外表现出良好的DPP-IV抑制活性,能够抑制DPP-IV降解Gly-Pro-pNA (hydrochloride) Gly-Pro-pNA (hydrochloride) [1]。以 Gly-Pro-pNA（盐酸盐）为底物,可以测定藜麦蛋白水解物的体外 DPP-IV 抑制活性[2]。 Gly-Pro-pNA（盐酸盐）也可用于评价 DPP-IV 在人血清中的活性[3]。证明糖尿病患者的 DPP IV 与正常情况下的差异不大[4]。以甘氨酰对硝基苯胺盐酸盐为底物检测多囊综合征（PCOS）患者的DPPIV水平,DPP4血清水平失调可能是PCOS相关代谢失衡的另一个特征[5]
以Gly-Pro-pNA（盐酸盐）为底物可评估类风湿性关节炎患者二肽基肽酶IV（DPP IV）的酶活性和生化状态[6]
| Cas No. | 103213-34-9 | SDF | |
| 别名 | N-甘氨酰脯氨酰-对硝基苯胺盐酸盐 | ||
| 化学名 | glycyl-N-(4-nitrophenyl)-L-prolinamide, monohydrochloride | ||
| Canonical SMILES | O=C(CN)N1CCC[C@H]1C(NC2=CC=C([N+]([O-])=O)C=C2)=O.Cl | ||
| 分子式 | C13H16N4O4.HCl | 分子量 | 328.8 |
| 溶解度 | 30mg/mL in DMSO, 30mg/mL in DMF, 30mg/mL in Ethanol | 储存条件 | Store at -20°C,stored under nitrogen |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 3.0414 mL | 15.2068 mL | 30.4136 mL |
| 5 mM | 0.6083 mL | 3.0414 mL | 6.0827 mL |
| 10 mM | 0.3041 mL | 1.5207 mL | 3.0414 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)
Arch Biochem Biophys2006 Jan 1;445(1):9-18.PMID: 16364232DOI: 10.1016/j.abb.2005.11.010
Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.
Rapid detection of DPP-IV activity in porcine serum: A fluorospectrometric assay
Anal Biochem2020 Mar 1;592:113557.PMID: 31866290DOI: 10.1016/j.ab.2019.113557
Dipeptidyl peptidase IV (DPP-IV) is an aminopeptidase that cleaves the N-terminal dipeptide from peptides bearing proline or alanine residues. Currently, DPP-IV activity is quantified by spectrophotometric or fluorometric methods, which employ Gly-Pro-pNA and Gly-Pro-AMC respectively, as substrate. However, these methods require high enzyme and substrate concentrations. In this study, we adapted the DPP-IV fluorospectrometric assay using NanoDrop 3300, which requires only nanogram levels of the enzyme (30 ng crude DPP-IV) and considerably low substrate concentrations (100 μM). Fluorescence measurement required a reaction mixture of only 2 μL, thus eliminating the need for microtiter plates or cuvettes.We employed this assay to demonstrate DPP-IV activity in porcine serum for the first time. The enzymatic activity peaked at pH 8.0 in porcine (84 nM/min), human (87 nM/min) and bovine (89.1 nM/min) sera, with the optimum temperature of 37 °C. The enzyme showed maximum activity upon incubation for 40 min at 37 °C. In contrast, activity in the porcine serum was the highest after incubation for 30 min at the same optimized parameters. The IC50 values of diprotin A against DPP-IV from human, porcine, and bovine sera were 7.83, 8.62, 9.17 μM, respectively. The present assay procedure is a convenient, sensitive, accurate and high-throughput method suitable for primary screening of DPP-IV inhibitors.
An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides
Infect Immun1994 Nov;62(11):4938-47.PMID: 7523301DOI: 10.1128/iai.62.11.4938-4947.1994
An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.
Murine thymocytes possess specific cell surface-associated exoaminopeptidase activities: preferential expression by immature CD4-CD8- subpopulation
Eur J Immunol1990 Mar;20(3):459-68.PMID: 2108042DOI: 10.1002/eji.1830200302
Murine thymocytes are shown to possess at least three well-defined exo-N-aminopeptidase activities on their surface. One of them cleaves the prolyl bond in the synthetic dipeptide nitroanilide Gly-Pro-pNA (Km 0.95 mM and Vmax 8 nmol/h at pH 7.4 and 37 degrees C) and is specifically inhibited by phenylmethane sulfonyl fluoride, diprotin A, Gly-Pro-Ala and Gly-Pro-Gly-Gly. These data further support identification of this enzyme with a serine exopeptidase dipeptidyl peptidase IV (DPP IV), previously reported to be specific for collagen. The two other forms of N-exopeptidase activities are detected when Ala-pNA and Leu-pNA are used as substrates. Leu-aminopeptidase activity (Km 1.4 mM, Vmax 15 nmol/h) and Ala-aminopeptidase activity (Km 4.0 mM, Vmax 20 nmol/h) are inhibited by inhibitors for thiol- and trypsin-like proteinases, i.e. tosyl lysyl chloromethyl ketone, leupeptin and N-ethylmaleimide. Addition inhibition of Leu-aminopeptidase activity by peptstatin, a known inhibitor of carboxyl proteases, suggests that aminopeptidase activity detected with Leu-pNA is different in part from Ala-aminopeptidase activity. Among the various lymphoid cell populations tested, the three aminopeptidase activities are increased by three- to fourfold in the immature CD4-CD8- thymocyte subset as well as in the thymoma BW5147. In contrast, cortisone-resistant thymocytes, lymph node and spleen cells exhibit levels of activities almost similar to that of unfractionated thymocytes. During ontogeny, the levels of these activities are increased four- to sevenfold on fetal thymocytes (from days 14 to 16). Finally, when thymocytes or spleen cells are cultured with a mitogenic concentration of concanavalin A, their proliferative responses are correlated with an enhancement of the aminopeptidase activities (1.3- to 5-fold). From these results, a correlation between the presence of these peptidases on the cell surface of immature and mature lymphoid cells and biological responsiveness is suggested.
Identification of two isoforms of Pop in the domestic silkworm, Bombyx mori: Cloning, characterization and expression analysis
Gene2018 Aug 15;667:101-111.PMID: 29753046DOI: 10.1016/j.gene.2018.05.021
Two isoforms, Bmpop-a and Bmpop-b, were cloned and characterized, which were found to encode prolyl oligopeptidase (Pop) of the domestic silkworm Bombyx mori. The full lengths of Bmpop-a and Bmpop-b were 2497 and 2508 bp, deducing 707 and 740 amino acids, respectively. Both of them, possessing the typical characteristics of the Pop family of serine proteinase, were detected to be expressed among different tissues and development stages at the transcription and translation levels. Soluble recombinant BmPop-a (rBmPop-a) had oligopeptidase activity toward the substrates, Z-Gly-Pro-pNA, Z-Gly-Pro-AMC and angiotensin I. An inhibition assay showed that the activity of rBmPop-a was significantly inhibited by KYP-2047 and S17092 in vitro. BmPop-b was identified in the molting fluids at three different stages by Western blotting analysis, showing a predominant expression in the integument. Two isoforms of Bmpop gene and other three genes in the renin-angiotensin system (RAS) in the integument were down-regulated by starvation treatments but up-regulated by refeeding. These results suggested that BmPops may play an important role in balancing the molting fluid pressure to guarantee ecdysis normally. This study provides clues for further elucidating the function and regulation mechanisms of two isoforms of Bmpop gene.