Suc-AAPF-pNA
(Synonyms: N-琥珀酰-丙酰氨-丙酰氨-脯酰氨-苯丙氨酸对硝基酰苯胺,Suc-Ala-Ala-Pro-Phe-pNA) 目录号 : GC44960
A cathepsin substrate
Cas No.:70967-97-4
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Suc-AAPF-pNA is a chromogenic substrate that can be cleaved by cathepsin G (Km = 1.7 mM), subtilisins, chymotrypsin (Km = 60 µM), chymase (Km = 4 mM), and cyclophilin, but not neutrophil elastase. Release of p-nitroanilide is monitored at 405-410 nm. This substrate can be used for inhibitor screening and kinetic analysis.
Suc-AAPF-pNA 是一种显色底物，可被组织蛋白酶 G (Km = 1.7 mM)、枯草杆菌蛋白酶、胰凝乳蛋白酶 (Km = 60 µM)、糜蛋白酶 (Km = 4 mM) 和亲环蛋白裂解，但不能被中性粒细胞裂解弹性蛋白酶。在 405-410 nm 监测对硝基苯胺的释放。该底物可用于抑制剂筛选和动力学分析。
| Cas No. | 70967-97-4 | SDF | |
| 别名 | N-琥珀酰-丙酰氨-丙酰氨-脯酰氨-苯丙氨酸对硝基酰苯胺,Suc-Ala-Ala-Pro-Phe-pNA | ||
| Canonical SMILES | OC(CCC(N[C@@H](C)C(N[C@@H](C)C(N1CCC[C@H]1C(N[C@@H](CC2=CC=CC=C2)C(NC3=CC=C([N+]([O-])=O)C=C3)=O)=O)=O)=O)=O)=O | ||
| 分子式 | C30H36N6O9 | 分子量 | 624.7 |
| 溶解度 | DMF: 5 mg/ml,DMSO: 5 mg/ml,DMSO:PBS (pH 7.2) (1:1): 0.5 mg/ml | 储存条件 | Store at -20°C |
| 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 | 1.6008 mL | 8.0038 mL | 16.0077 mL |
| 5 mM | 0.3202 mL | 1.6008 mL | 3.2015 mL |
| 10 mM | 0.1601 mL | 0.8004 mL | 1.6008 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。
Toward tailoring the specificity of the S1 pocket of subtilisin B. lentus: chemical modification of mutant enzymes as a strategy for removing specificity limitations
Biochemistry 1999 Oct 5;38(40):13391-7.PMID:10529215DOI:10.1021/bi990861o.
In both protein chemistry studies and organic synthesis applications, it is desirable to have available a toolbox of inexpensive proteases with high selectivity and diverse substrate preferences. Toward this goal, we have generated a series of chemically modified mutant enzymes (CMMs) of subtilisin B. lentus (SBL) possessing expanded S(1) pocket specificity. Wild-type SBL shows a marked preference for substrates with large hydrophobic P(1) residues, such as the large Phe P(1) residue of the standard Suc-AAPF-pNA substrate. To confer more universal P(1) specificity on S(1), a strategy of chemical modification in combination with site-directed mutagenesis was applied. For example, WT-SBL does not readily accept small uncharged P(1) residues such as the -CH(3) side chain of alanine. Accordingly, with a view to creating a S(1) pocket that would be of reduced volume providing a better fit for small P(1) side chains, a large cyclohexyl group was introduced by the CMM approach at position S166C with the aim of partially filling up the S(1) pocket. The S166C-S-CH(2)-c-C(6)H(11) CMM thus created showed a 2-fold improvement in k(cat)/K(M) with the suc-AAPA-pNA substrate and a 51-fold improvement in suc-AAPA-pNA/Suc-AAPF-pNA selectivity relative to WT-SBL. Furthermore, WT-SBL does not readily accept positively or negatively charged P(1) residues. Therefore, to improve SBL's specificity toward positively and negatively charged P(1) residues, we applied the CMM methodology to introduce complementary negatively and positively charged groups, respectively, at position S166C in S(1). A series of mono-, di-, and trinegatively charged CMMs were generated and all showed improved k(cat)/K(M)s with the positively charged P(1) residue containing substrate, suc-AAPR-pNA. Furthermore, virtually arithmetic improvements in k(cat)/K(M) were exhibited with increasing number of negative charges on the S166C-R side chain. These increases culminated in a 9-fold improvement in k(cat)/K(M) for the suc-AAPR-pNA substrate and a 61-fold improvement in suc-AAPR-pNA/Suc-AAPF-pNA selectivity compared to WT-SBL for the trinegatively charged S166C-S-CH(2)CH(2)C(COO(-))(3) CMM. Conversely, the positively charged S166C-S-CH(2)CH(2)NH(3)(+) CMM generated showed a 19-fold improvement in k(cat)/K(M) for the suc-AAPE-pNA substrate and a 54-fold improvement in suc-AAPE-pNA/Suc-AAPF-pNA selectivity relative to WT-SBL.
Functional analysis of the two cyclophilin isoforms of Sinorhizobium meliloti
World J Microbiol Biotechnol 2017 Feb;33(2):28.PMID:28058638DOI:10.1007/s11274-016-2201-6.
The nitrogen fixing Sinorhizobium meliloti possesses two genes, ppiA and ppiB, encoding two cyclophilin isoforms which belong to the superfamily of peptidyl prolyl cis/trans isomerases (PPIase, EC: 5.2.1.8). Here, we functionally characterize the two proteins and we demonstrate that both recombinant cyclophilins are able to isomerise the Suc-AAPF-pNA synthetic peptide but neither of them displays chaperone function in the citrate synthase thermal aggregation assay. Furthermore, we observe that the expression of both enzymes increases the viability of E. coli BL21 in the presence of abiotic stress conditions such as increased heat and salt concentration. Our results support and strengthen previous high-throughput studies implicating S. meliloti cyclophilins in various stress conditions.
Secretory production of recombinant human chymase as an active form in Pichia pastoris
Yeast 2000 Mar 15;16(4):315-23.PMID:10669869DOI:10.1002/1097-0061(20000315)16:4<315::AID-YEA527>3.0.CO;2-4.
We succeeded in expressing in a Pichia pastoris (P. pastoris) host a cDNA encoding a mature human chymase (h-chymase) which was secreted directly into the culture medium. Recombinant human heart chymase (rh-chymase) was purified from the culture medium via a single one-step heparin-agarose column chromatography tracing, using succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Suc-AAPF-pNA) hydrolysing activity. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the rh-chymase showed a diffused protein band with molecular weight of 32-37 kDa. After deglycosylation, however, rh-chymase changed to a sharp protein band with molecular weight 28 kDa, which is equal in size to deglycosylated h-chymase. The rh-chymase had an activity to convert one of the natural substrates, angiotensin I, to angiotensin II. Double reciprocal plot analysis revealed that the K(m) value ofrh-chymase against Suc-AAPF-pNA was approximately 5.1 mM, which is close to that of purified h-chymase.
Production and characterization of keratinase of a feather-degrading Bacillus licheniformis PWD-1
Biosci Biotechnol Biochem 1995 Dec;59(12):2239-43.PMID:8611746DOI:10.1271/bbb.59.2239.
The keratinase produced by Bacillus licheniformis PWD-1 was induced by feather powder. Maximal enzyme production could be achieved by culturing in a medium containing 1% hammer-milled feather powder (100 mesh) at 45 degrees C for 30 h. Maximal growth of PWD-1 was achieved at 50 degrees C, and maximal enzyme induction was at 45 degrees C. The molecular mass and isoelectric point of this enzyme were 31.4 kDa and 8.5, respectively. This enzyme was stable from pH 5 to 12. The optimal reaction pHs for feather powder and casein were 8.5 and 10.5 to 11.5, respectively. The optimal reaction temperature was 50 degrees C to 55 degrees C. The relative activity of this enzyme toward casein, feather powder, keratin, elastin, and collagen was 100:52:41:18:7, and 100:56:32:3 for Suc-AAPL-pNA, Suc-AAPF-pNA, Suc-AAPM-pNA, and Suc-AAVA-pNA (Suc, succinyl; pNA, p-nitrophenylanilide).
Redirecting catalysis from proteolysis to perhydrolysis in subtilisin Carlsberg
J Biotechnol 2013 Sep 10;167(3):279-86.PMID:23835157DOI:10.1016/j.jbiotec.2013.06.017.
Enzyme promiscuity describes the ability of biocatalysts to catalyze conversions beyond their natural reactions. Enzyme engineering to promote side reactions is attractive for synthetic and industrial applications. For instance, a subtilisin Carlsberg protease variant (T58A/L216W) catalyzes in addition to its proteolytic activity the generation of peroxycarboxylic acids from corresponding esters in the presence of hydrogen peroxide. In the current study we used a semi-rational design approach to shift the specificity of subtilisin Carlsberg towards production of peroxycarboxylic acid. Among other identified amino acid substitutions, position Gly165 in the S1 binding pocket provided insights in subtilisin Carlsberg's promiscuity by promoting ester perhydrolysis. Catalytic constants of subtilisin Carlsberg for perhydrolysis of methyl-propionate, methyl-butyrate and methyl-pentanoate were increased up to 3.5-, 5.4- and 5.5-fold, respectively, while proteolysis was decreased up to 100-fold for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate (Suc-AAPF-pNA).