Ac-VDVAD-pNA

Caspase-2

Ac-DVAD-pNA、、Ac-VDVAD-pNA和Ac-LDVAD-pNA三种底物在不改变P2或P3的情况下，最明显地表现出疏水性P5残基的加入效果。这些底物被caspase-7和caspase-3水解。Caspase-3对Ac-LDVAD-pNA的催化效率最高，Km最低，kcat/Km约为Ac-DVAD-pNA的140%。同样，Ac-VDVAD-pNA的kcat/Km是Ac-DVAD-pNA值的120%。疏水性P5残基降低了caspase-7的水解效率，kcat/Km值为Ac-DVAD-pNA > Ac-VDVAD-pNA > Ac-LDVAD-pNA。在caspase-7实验中，ac - ldad - pna的kcat/Km约为Ac-DVAD-pNA的80%。这些结果表明疏水性P5残基有利于caspase-3对底物的识别和水解，而不利于caspase-7对底物的水解。这一信息是有价值的，因为它将有助于为每种半胱天冬酶设计特异性抑制剂，这在历史上一直是非常具有挑战性的。[1]

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所有3种中国仓鼠半胱天冬酶都能有效地切割相应的人类同源物。然而，在其他底物上的活性也被观察到，特别是caspase-8（图2B）。中国仓鼠caspase-8对人caspase-9的底物Ac-LEHD-pNA活性最高。它切割Ac-LEHD-pNA的速度是为人类caspase-8设计的底物Ac-IETD-pNA的1.4倍。此外，中国仓鼠caspase-8对caspase-3和-7 （Ac-DEVD-pNA）、caspase-2 （Ac-VDVAD-pNA）和caspase-6 （Ac-VEID-pNA）设计的底物也显示出相当大的酶活性。中国仓鼠caspase-2是最特异性的，因为它最有效地切割caspase-2 VDVAD五肽底物，并且对Ac-DEVD-pNA和Ac-LEHD-pNA仅显示剩余的反应性。人类caspase-9的指定底物Ac-LEHD-pNA也是中国仓鼠caspase-9的最佳底物。然而，中国仓鼠caspase-9对Ac-WEHD-pNA、Ac-VEID-pNA和Ac-IETD-pNA也表现出相当大的活性。它切割Ac-WEHD-pNA的效率（49%）几乎是切割Ac-LEHD-pNA的一半。 [2]

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每种中国仓鼠caspase样品首先与不同浓度的肽fmk抑制剂混合，包括Z-LEHD-fmk、Z-IETD-fmk、Z-VDVAD-fmk或Z-DEVD-fmk。在加入相应的pNA底物之前，将混合物在室温下孵育30分钟。由于caspase切割pNA底物，OD450的变化率被记录为图3A、3C和3E所示的动力学图。动力学曲线的初始斜率与初始酶促反应速度成正比，代表了该特定测定中酶的活性。抑制效果量化为孵育后残留酶活性与原始酶活性的比值。该比率与图3B、3D和3F中使用的不同抑制剂浓度相对应。在图3A和3B中，除阴性对照外，中国仓鼠caspase-8仅用1 μM的抑制剂孵育后就灭活了。Z-LEHD-fmk和Z-IETD-fmk对中国仓鼠caspase-9有较强的抑制作用，Z-VDVAD-fmk次之。在30 μM下，即使是Z-DEVD-fmk也能将caspase-9的活性降低到几乎为零。即使抑制剂浓度为5 μM， Z-LEHD-fmk和Z-IETD-fmk也能更有效地抑制中国仓鼠caspase-8，而抑制剂Z-VDVAD-fmk和Z-DEVD-fmk需要更高的剂量才能达到相同的效果。［2］

酶动力学测定[1]
用比色法测定caspase-3底物Ac- devd -pNA，其中Ac为乙酰基，pNA为对硝基苯胺。Caspase-3在反应缓冲液(50 mM Hepes (pH 7.5), 100 mM NaCl, 0.1% （v/v) Chaps, 10% （v/v）甘油，1 mM EDTA， 10 mM二硫苏糖醇）中室温孵育5分钟，然后加入不同浓度的底物。酶裂解释放的对硝基苯胺在405nm波长处用Polarstar Optima酶标仪测定。31 .使用SigmaPlot 9.0通过拟合反应速度得到Km和Vmax值caspase-3底物Ac-DEVD-pNA、Ac-DMQD-pNA、Ac-DVAD-pNA、Ac-VDVAD-pNA和Ac-LDVAD-pNA的催化常数kcat通过公式kcat = Vmax/[E]测定，其中[E]值在Ki测定过程中通过活性位点滴定法测定，如下所述。caspase-7也采用了同样的方法。[１]

Caspase活性测定[2]
将表达重组中国鼠半胱天冬酶的冷冻大肠杆菌细胞颗粒解冻，用1.5 ml裂解缓冲液（5 mM DTT, 10 mM HEPES pH 7.5, 2 mM EDTA, 0.1% CHAPS/NP40）裂解10分钟，并在冰上孵育。裂解物超声进一步破坏细胞和DNA等大分子。然后将细胞裂解液以13000 rpm离心5分钟。收集上清液中的细胞质提取物。通过测定595 nm处的吸光度，用Pierce's Coomasie Plus-The Better Bradford Assay Reagent测定这些裂解物的蛋白质浓度。在96孔板上，将50 μl大肠杆菌裂解液与Chemicon比色测定试剂盒提供的缓冲液和ddH2O混合至95 μl。然后加入5 μl对硝基苯胺（pNA）标记的caspase底物（Ac-DEVD-pNA、Ac-IETD-pNA、Ac-LEHD-pNA、Ac-VDVAD-pNA、Ac-WEHD-pNA和Ac-VEID-pNA）。反应混合物在37℃下孵育2小时。通过在Tecan GENios微孔板读取器上跟踪405 nm （OD405）吸光度变化，监测caspase裂解从底物释放游离pNA发色团。每个样品的Caspase活性是根据OD405的初始增加率来确定的。背景对照使用不表达重组半胱天冬酶的大肠杆菌细胞裂解物。［2］

[1]. Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition. J Mol Biol. 2006 Jul 14;360(3):654-66.
[2]. Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures. Metab Eng. 2007 Sep-Nov;9(5-6):406-18.
[3]. Talanian RV, et, al. Substrate specificities of caspase family proteases. J Biol Chem. 1997 Apr 11;272(15):9677-82.

The molecular basis for the substrate specificity of human caspase-3 has been investigated using peptide analog inhibitors and substrates that vary at the P2, P3, and P5 positions. Crystal structures were determined of caspase-3 complexes with the substrate analogs at resolutions of 1.7 Å to 2.3 Å. Differences in the interactions of caspase-3 with the analogs are consistent with the Ki values of 1.3 nM, 6.5 nM, and 12.4 nM for Ac-DEVD-Cho, Ac-VDVAD-Cho and Ac-DMQD-Cho, respectively, and relative kcat/Km values of 100%, 37% and 17% for the corresponding peptide substrates. The bound peptide analogs show very similar interactions for the main-chain atoms and the conserved P1 Asp and P4 Asp, while interactions vary for P2 and P3. P2 lies in a hydrophobic S2 groove, consistent with the weaker inhibition of Ac-DMQD-Cho with polar P2 Gln. S3 is a surface hydrophilic site with favorable polar interactions with P3 Glu in Ac-DEVD-Cho. Ac-DMQD-Cho and Ac-VDVAD-Cho have hydrophobic P3 residues that are not optimal in the polar S3 site, consistent with their weaker inhibition. A hydrophobic S5 site was identified for caspase-3, where the side-chains of Phe250 and Phe252 interact with P5 Val of Ac-VDVAD-Cho, and enclose the substrate-binding site by conformational change. The kinetic importance of hydrophobic P5 residues was confirmed by more efficient hydrolysis of caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA compared with Ac-DVAD-pNA. In contrast, caspase-7 showed less efficient hydrolysis of the substrates with P5 Val or Leu compared with Ac-DVAD-pNA. Caspase-3 and caspase-2 share similar hydrophobic S5 sites, while caspases 1, 7, 8 and 9 do not have structurally equivalent hydrophobic residues; these caspases are likely to differ in their selectivity for the P5 position of substrates. The distinct selectivity for P5 will help define the particular substrates and signaling pathways associated with each caspase. [1]

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In an attempt to investigate the molecular mechanism that leads to apoptotic death in Chinese hamster ovary (CHO) cells in batch and fed-batch cultures, we cloned caspase-2, -8 and -9 from a CHO cDNA library. Recombinant Chinese hamster caspase-2 and -9 expressed in Escherichia coli show highest activities towards commercial peptide substrates Ac-VDVAD-pNA and Ac-LEHD-pNA, the designated commercial substrates for human caspase-2 and -9, respectively. However, Chinese hamster caspase-8 shows a broad specificity profile and it cleaves the caspase-9 substrate more efficiently than it cleaves the caspase-8 substrate. The commercially available fluoromethyl ketone type of caspase inhibitors, such as Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk and Z-DEVD-fmk, were shown to completely lack specificity in inhibiting these caspases. The reversible aldehyde form of inhibitors for human caspase-8 and -9, Ac-LEHD-CHO and Ac-IETD-CHO, are equally efficient in inhibiting Chinese hamster caspase-8. Therefore, the wildly used method of utilizing the "caspase-specific" inhibitors to track the role of individual caspases in dying cells can be inaccurate and thus misleading. As an alternative, we stably expressed dominant negative (DN) mutants of Chinese hamster caspase-2, -8 and -9 to specifically inhibit these enzymes in CHO cells. Our results showed that inhibition of either endogenous caspase-8 or caspase-9 enhanced the viability of the CHO cells in both batch and fed-batch suspension cultures, but the inhibition of caspase-2 had minimal effects. These results suggest that caspase-8 and -9 are possibly involved in the apoptotic cell death in batch and fed-batch cultures of CHO cells, whereas caspase-2 is not. These findings can be valuable in the development of strategies for genetically engineering CHO cells to counter apoptotic death in batch and fed-batch cultures. [2]

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The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1beta converting enzyme (ICE) family proteases, we have examined in detail the specificities beyond the P1 position of caspase-1, -2, -3, -4, -6, and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors. [3]

C29H41N7O12

679.68

679.281

189684-53-5

25108784

Ac-Val-Asp-Val-Ala-Asp-pNA

Ac-VDVAD-pNA; VDVAD

Typically exists as solid at room temperature

1.4±0.1 g/cm3

1160.8±65.0 °C at 760 mmHg

655.8±34.3 °C

0.0±0.3 mmHg at 25°C

1.572

2.23

295.02

8

12

17

48

1230

5

CC(C(NC(C)=O)C(NC(C(NC(C(NC(C(NC(C(NC1=CC=C([N+]([O-])=O)C=C1)=O)CC(O)=O)=O)C)=O)C(C)C)=O)CC(O)=O)=O)C

SIOKOOKURWWOID-PZQVQNRFSA-N

InChI=1S/C29H41N7O12/c1-13(2)23(31-16(6)37)29(46)34-20(12-22(40)41)27(44)35-24(14(3)4)28(45)30-15(5)25(42)33-19(11-21(38)39)26(43)32-17-7-9-18(10-8-17)36(47)48/h7-10,13-15,19-20,23-24H,11-12H2,1-6H3,(H,30,45)(H,31,37)(H,32,43)(H,33,42)(H,34,46)(H,35,44)(H,38,39)(H,40,41)/t15-,19-,20-,23-,24-/m0/s1

(3S)-3-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-methylbutanoyl]amino]-3-carboxypropanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-4-(4-nitroanilino)-4-oxobutanoic acid

Ac-VDVAD-PNA; (3S)-3-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-methylbutanoyl]amino]-3-carboxypropanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-4-(4-nitroanilino)-4-oxobutanoic acid; Ac-Val-Asp-Val-Ala-Asp-PNA; Ac-VDVAD-pNA (trifluoroacetate salt); Caspase-2 substrate; SCHEMBL7885355;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。