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.

This study explored the molecular basis of human caspase-3 substrate specificity using peptide analog inhibitors and substrates with differences at P2, P3, and P5 sites. We resolved the crystal structures of caspase-3 complexes with substrate analogs at resolutions ranging from 1.7 Å to 2.3 Å. The differences in caspase-3 interactions with these 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 the relative kcat/Km values of the corresponding peptide substrates, which are 100%, 37%, and 17%, respectively. The bound peptide analogs exhibited very similar interactions at the main chain atoms and the conserved P1 Asp and P4 Asp sites, while the interactions at P2 and P3 sites differed. P2 is located in a hydrophobic S2 groove, consistent with the weaker, polar inhibitory effect of the Gln P2 site in Ac-DMQD-Cho. S3 is a hydrophilic surface site that exhibits a favorable polar interaction with the P3 site Glu in Ac-DEVD-Cho. The P3 site residues in Ac-DMQD-Cho and Ac-VDVAD-Cho are both hydrophobic and not optimally positioned at the polar S3 site, consistent with their weaker inhibitory effects. A hydrophobic S5 site was found in caspase-3, where the side chains of Phe250 and Phe252 interact with the P5 site Val of Ac-VDVAD-Cho and surround the substrate binding site through conformational changes. The kinetic importance of the hydrophobic P5 site residues was confirmed by comparing the hydrolytic efficiency of Ac-DVAD-pNA by the caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA. In contrast, caspase-7 was less efficient at hydrolyzing substrates with a P5 site of Val or Leu than Ac-DVAD-pNA. Caspase-3 and caspase-2 have similar hydrophobic S5 sites, while the hydrophobic residues of caspase-1, 7, 8 and 9 are structurally different; these caspases may have different selectivity for substrate P5 sites. Differences in P5 site selectivity will help identify specific substrates and associated signaling pathways for each caspase. [1]

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To investigate the molecular mechanisms leading to apoptosis 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 caspases-2 and-9 expressed in E. coli exhibited the highest activity against the commercial peptide substrates Ac-VDVAD-pNA and Ac-LEHD-pNA (the designated commercial substrates for human caspases-2 and-9, respectively). However, Chinese hamster caspase-8 exhibited broad substrate specificity, cleaving caspase-9 substrates more efficiently than caspase-8 substrates. Commercially available fluoromethyl ketone caspase inhibitors, such as Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk, and Z-DEVD-fmk, were shown to completely lack specificity for inhibiting these caspases. The reversible aldehyde inhibitors of human caspases-8 and-9, Ac-LEHD-CHO and Ac-IETD-CHO, showed comparable inhibitory efficiency against Chinese hamster caspase-8. Therefore, the currently widely used approach of using "caspase-specific" inhibitors to track the role of individual caspases in cell death may be inaccurate and even misleading. As an alternative approach, we stably expressed dominant-negative (DN) mutants of Chinese hamster caspase-2, -8, and -9 to specifically inhibit these enzymes in CHO cells. The results showed that inhibition of endogenous caspase-8 or caspase-9 improved the survival rate of CHO cells in batch and fed-batch suspension cultures, while inhibition of caspase-2 had little effect. These results suggest that caspase-8 and -9 may be involved in the apoptosis process of CHO cells in batch and fed-batch cultures, while caspase-2 is not involved. These findings are of great value for developing strategies to genetically engineer CHO cells to combat apoptosis 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.)