TMPD dihydrochloride is a membrane tethering compound used to study cytochrome C for its effect on yeast cell death. TMPD dihydrochloride is also a substrate for COMPLEX IV. TMPD (N,N,N′,N′-tetramethyl-para-phenylene-diamine) is used in cell culture and microbiology to differentiate organisms that exhibit cytochrome c oxidase activity and to distinguish between Gram-negative and Gram-positive pathogenic and non-pathogenic bacteria. The concept of TMPD oxidation by microorganisms is widely used and is known as a successful colorimetric indicator for bacterial oxidases, as the radical cation TMPD+˙, formed by oxidation, shows a characteristic deep blue colour. The use of N,N,N cent,N cent-tetramethyl-p-phenylenediamine (TMPD) in high throughput microplate assays of COX activity could become the approach of choice in the screening of potential therapeutics that inhibit COX activity in vivo.
| Cell Experiment | |
|---|---|
| Cell lines | Escherichia coli & Bacillus subtilis |
| Preparation method | Bacteria culture and oxidase test Escherichia coli Bacteria were cultured in 2 × TY liquid microbial growth medium (broth), containing 16 g/L tryptone, 10 g/L yeast extract and 5.0 g/L NaCl. Growth medium was inoculated with bacteria from frozen stocks and incubated in glass culture flasks for 18 h at 37 °C in an incubator shaker. An E. coli suspension of 50 μL was transferred into a new culture flask, containing fresh growth medium. Following incubation for 3 to 4 h at 37 °C, the number of bacteria in solution was determined by optical density (OD) at a wavelength of 600 nm (OD600 of 1.0 = 8 × 108 cells/mL). When an OD600 between 0.4 and 1.8 was reached, bacteria were harvested by centrifugation for 15 min at 3000 rcf and re-suspended in pre-warmed (37 °C) PBS. Bacillus subtilis (strain PY79) Low salt growth medium (broth), containing 10 g/L tryptone, 5.0 g/L yeast extract and 5.0 g/L sodium chloride, was inoculated with cultures from frozen stocks and incubated at 30 °C for 36 h in an incubator shaker. An OD600 was determined to calculate number of bacteria in solution (OD600 of 1.0 = 5 × 108 cells/mL). Bacteria were harvested by centrifugation for 10 min at 300 rcf and re-suspended in pre-warmed (30 °C) PBS. Cultures grown on agar, containing 10 g/L tryptone, 5.0 g/L yeast extract 5.0 g/L sodium chloride and 20 g/L agar, were inoculated onto agar plates and grown at 30 °C for 36 h in a static incubator. For the oxidase test, 200 μL of a 1% (wt) N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD-2HCl) solution was added to 1 mL of cell suspension (1 × 107 cells/μL). Images were taken within 30 s of solution mixture. |
| Concentrations | 200 μL of a 1% (wt) N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD-2HCl) solution |
| Incubation time | - |
| Animal Experiment | |
|---|---|
| Animal models | |
| Formulation | |
| Dosages | |
| Administration | |
| Molecular Weight | 237.17 |
| Formula | C10H18Cl2N2 |
| CAS Number | 637-01-4 |
| Solubility (25°C) | Water ≥ 60 mg/mL DMSO ≥ 10 mg/mL |
| Storage | 4°C, protect from light, dry, sealed |
| Species | Mouse | Rat | Rabbit | Guinea pig | Hamster | Dog |
| Weight (kg) | 0.02 | 0.15 | 1.8 | 0.4 | 0.08 | 10 |
| Body Surface Area (m2) | 0.007 | 0.025 | 0.15 | 0.05 | 0.02 | 0.5 |
| Km factor | 3 | 6 | 12 | 8 | 5 | 20 |
| Animal A (mg/kg) = Animal B (mg/kg) multiplied by | Animal B Km |
| Animal A Km |
For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.
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