Caspase-3/7

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]

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.

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]

C26H34N6O13

638.58056

638.218

C, 48.90; H, 5.37; N, 13.16; O, 32.57

189950-66-1

11527474

Ac-Asp-Glu-Val-Asp-pNA; N-Acetyl-Asp-Glu-Val-Asp-p-Nitroanilide

Ac-DEVD-p-Nitroanilide; DEVD

White to off-white solid powder

1.4±0.1 g/cm3

1187.0±65.0 °C at 760 mmHg

671.6±34.3 °C

0.0±0.3 mmHg at 25°C

1.593

1.09

8

13

17

45

1150

4

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

GGXRLUDNGFFUKI-ORGXJRBJSA-N

InChI=1S/C26H34N6O13/c1-12(2)22(26(43)30-18(11-21(38)39)24(41)28-14-4-6-15(7-5-14)32(44)45)31-23(40)16(8-9-19(34)35)29-25(42)17(10-20(36)37)27-13(3)33/h4-7,12,16-18,22H,8-11H2,1-3H3,(H,27,33)(H,28,41)(H,29,42)(H,30,43)(H,31,40)(H,34,35)(H,36,37)(H,38,39)/t16-,17-,18-,22-/m0/s1

(4S)-4-[[(2S)-2-acetamido-3-carboxypropanoyl]amino]-5-[[(2S)-1-[[(2S)-3-carboxy-1-(4-nitroanilino)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxopentanoic acid

Ac-DEVD-pNA; Ac-Asp-Glu-Val-Asp-PNA; N-Acetyl-Asp-Glu-Val-Asp p-nitroanilide; MFCD00792707; (4S)-4-[[(2S)-2-acetamido-3-carboxypropanoyl]amino]-5-[[(2S)-1-[[(2S)-3-carboxy-1-(4-nitroanilino)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxopentanoic acid; Z-VDVAD-pNA?; N-Acetyl-Asp-Glu-Val-Asp-pna; SCHEMBL2028887;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~110 mg/mL (~172.26 mM)
H2O : ~0.67 mg/mL (~1.05 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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