Caspase-2

The three substrates Ac-DVAD-pNA, Ac-VDVAD-pNA and Ac-LDVAD-pNA most clearly showed the effect of adding the hydrophobic P5 residue without varying P2 or P3. These substrates were assayed for hydrolysis by caspase-7 as well as by caspase-3. Caspase-3 showed the highest catalytic efficiency and the lowest Km for Ac-LDVAD-pNA, with kcat/Km about 140% of the value for Ac-DVAD-pNA. Similarly, the kcat/Km for Ac-VDVAD-pNA was 120% of the value for Ac-DVAD-pNA. In contrast, the hydrophobic P5 residue decreased the catalytic efficiency for hydrolysis by caspase-7, and the kcat/Km values were in the order Ac-DVAD-pNA > Ac-VDVAD-pNA > Ac-LDVAD-pNA. The kcat/Km for Ac-LDVAD-pNA was about 80% of that for Ac-DVAD-pNA in the caspase-7 assay. These results demonstrated that the hydrophobic P5 residue has a favorable contribution to the recognition and hydrolysis of substrates by caspase-3 but not by caspase-7. This information is valuable because it will help to design specific inhibitors for each caspase, which has been historically very challenging. [1]

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All 3 Chinese hamster caspases were found to efficiently cleave the substrates that are designated for their corresponding human homologues. However, activities on other substrates that are not designed for their detection were also observed, especially for caspase-8 (Fig. 2B). Chinese hamster caspase-8 showed the highest activity towards Ac-LEHD-pNA which is the substrate designed for human caspase-9. It cleaves Ac-LEHD-pNA 1.4 times faster than it cleaves Ac-IETD-pNA which is the substrate designed for human caspase-8. In addition, Chinese hamster caspase-8 also showed considerable enzymatic activity against substrates designed for caspase-3 and -7 (Ac-DEVD-pNA), caspase-2 (Ac-VDVAD-pNA) and caspase-6 (Ac-VEID-pNA). Chinese hamster caspase-2 is the most specific, as it most efficiently cleaves the caspase-2 VDVAD pentapeptide substrate and showed only residual amount of reactivity against Ac-DEVD-pNA and Ac-LEHD-pNA. The designated substrate for human caspase-9, Ac-LEHD-pNA, is also the best substrate for Chinese hamster caspase-9. However, Chinese hamster caspase-9 also showed considerable activity towards Ac-WEHD-pNA, Ac-VEID-pNA and Ac-IETD-pNA. It cleaves Ac-WEHD-pNA with almost half of the efficiency (49%) as it cleaves Ac-LEHD-pNA. [2]

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Each Chinese hamster caspase sample was first mixed with different concentrations of peptide fmk inhibitors, including Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk or Z-DEVD-fmk. The mixtures were incubated at room temperature for 30 min before the addition of the respective pNA substrate. The rate of change of OD450 due to the caspase cleavage of the pNA substrate was recorded as a kinetic plot shown in Fig. 3A, 3C and 3E. The initial slope of the kinetic curve is proportional to the initial enzymatic reaction velocity and represents the activity of the enzyme in that particular assay. The inhibitory effect was quantified as the ratio of the residual enzyme activity after incubation against the original enzyme activity. This ratio was plotted against different inhibitor concentration used in Fig. 3B, 3D and 3F. In Fig. 3A and 3B, Chinese hamster caspase-8 was inactivated after incubation with just 1 μM of all of the inhibitors except the negative control. Chinese hamster caspase-9 was strongly inhibited by Z-LEHD-fmk and Z-IETD-fmk followed by Z-VDVAD-fmk. At 30 μM, even Z-DEVD-fmk was able to reduce the caspase-9 activity to nearly zero. Chinese hamster caspase-8 was more effectively inhibited by Z-LEHD-fmk and Z-IETD-fmk even at low inhibitor concentration of 5 μM, while much higher dosage was required for inhibitor Z-VDVAD-fmk and Z-DEVD-fmk to achieve the same effect. [2]

Enzyme kinetic assays [1]
CZaspase-3 activity was determined using the colorimetric caspase-3 substrate Ac-DEVD-pNA, where Ac is the acetyl group and pNA is p-nitroanilide. Caspase-3 was incubated in reaction buffer (50 mM Hepes (pH 7.5), 100 mM NaCl, 0.1% (v/v) Chaps, 10% (v/v) glycerol, 1 mM EDTA, 10 mM dithiothreitol,) at room temperature for 5 min before the addition of substrate at various concentrations. The p-nitroanilide released by enzyme cleavage was measured at a wavelength of 405 nm using a Polarstar Optima microplate reader. SigmaPlot 9.0 was used to obtain the Km and Vmax values by fitting reaction velocities as described.31 The catalytic constants kcat of caspase-3 substrates: Ac-DEVD-pNA, Ac-DMQD-pNA, Ac-DVAD-pNA, Ac-VDVAD-pNA and Ac-LDVAD-pNA were determined by using the equation kcat = Vmax/[E], where [E] values were measured by active site titration during Ki determination as described below. The same methods were used for caspase-7. [1]

Caspase activity assay [2]
Pellets of frozen E. coli cells that expressed recombinant Chinese hamster caspases were thawed and lysed with 1.5 ml of lysis buffer (5 mM DTT, 10 mM HEPES pH 7.5, 2 mM EDTA, 0.1% CHAPS/NP40) and incubated on ice for 10 min. The lysates were sonicated to further break the cells and macromolecules such as DNA. The cell lysates were then centrifuged at 13000 rpm for 5 min. The cytosolic extract in the supernatant was collected. The protein concentration of these lysates was determined by Pierce's Coomasie Plus—The Better Bradford Assay Reagent against BSA protein standards by determining absorbance at 595 nm. In a 96-well plate, 50 μl of E. coli lysates were mixed with the respective buffers provided by Chemicon's colorimetric assay kits and ddH2O to a total volume of 95 μl according to Chemicon's insturction. Then 5 μl para-nitroaniline (pNA) labeled caspase substrates (Ac-DEVD-pNA, Ac-IETD-pNA, Ac-LEHD-pNA, Ac-VDVAD-pNA, Ac-WEHD-pNA and Ac-VEID-pNA) were added. The reaction mixtures were incubated at 37 °C for up to 2 h. Release of free pNA chromophores from substrates by caspase cleavage was monitored by tracking changes in absorbance at 405 nm, OD405, on a Tecan GENios microplate reader. Caspase activity of each sample was determined from the initial rate of increase in OD405. Background control was carried out using the lysates made from E. coli cells that did not express the recombinant caspases. [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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)