Biological buffer

Study of coxsackievirus B3 strain 28 (CVB3/28) stability using MOPS to improve buffering in the experimental medium revealed that MOPS (3-morpholinopropane-1-sulfonic acid) increased CVB3 stability and the effect was concentration dependent. Over the pH range 7.0-7.5, virus stability was affected by both pH and MOPS concentration. Computer-simulated molecular docking showed that MOPS can occupy the hydrophobic pocket in capsid protein VP1 where the sulfonic acid head group can form ionic and hydrogen bonds with Arg95 and Asn211 near the pocket opening. The effects of MOPS and hydrogen ion concentrations on the rate of virus decay were modeled by including corresponding parameters in a recent kinetic model. These results indicate that MOPS can directly associate with CVB3 and stabilize the virus, possibly by altering capsid conformational dynamics.[1]

The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 m Tris, while the rate of TfCut2 was 2.1-fold lower at this buffer concentration. At a Tris concentration of 1 m, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 m MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 m, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the K i values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.[2]

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Hydrolysis of PET films by LCC and TfCut2[2]
Polyethylene terephthalate films of 9 cm2 (about 150 mg) were added to reaction vials containing 0.1–2.8 μg·cm−2 of purified LCC or TfCut2 and 0.1–1 m Tris, sodium phosphate or MOPS buffer (pH 8.0) in a total volume of 1.8 mL. The pH of the buffers was adjusted at 60 °C using HCl for Tris and NaOH for MOPS buffer. The reaction vials were incubated at 60 °C on a thermo shaker (1000 rpm) for 1 h. Released hydrolysis products were quantified by RP‐HPLC 39. The sum of the released soluble products—TPA, mono‐(2‐hydroxyethyl) terephthalate (MHET), and bis‐(2‐hydroxyethyl) terephthalate (BHET) was used to determine the initial hydrolysis rate. All initial rates were determined at least in triplicate.[2]

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Inhibition of LCC and TfCut2 by Tris and MOPS[2]
Polyethylene terephthalate films of 1–9 cm2 (about 20–150 mg) were added to reaction vials containing purified LCC (1 μg) or TfCut2 (5 μg) and 0.2 m sodium phosphate buffer (pH 8.0) in a total volume of 1.8 mL. Tris (0.2–0.4 m, pH 8.0) and MOPS (0.05–0.3 m, pH 8.0) were added to the reaction mixture. The vials were incubated at 60 °C on a thermo shaker (1000 rpm) for 1 h. Released hydrolysis products were quantified by RP‐HPLC[2].

RDt3 cells (RD cells expressing truncated CAR (Cunningham et al., 2003)) and lab strain HeLa cells (Carson and Pirruccello, 2013) were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum in a 37°C incubator with 6% CO2. Five milliliters of 200 nM glutamine, 10 mL penicillin/streptomycin (10000 U/mL and 10 mg/mL, respectively) and 1.5 mL gentamicin (50 mg/mL) were added to each 1.1 liter of DMEM-10% serum (the complete medium is referred to as DMEM-10). MOPS was added to the DMEM-10 to the final experimental concentration, and pH was adjusted to target values using 4M NaOH. NaCl was added (50–200nM) to separate samples of DMEM-10 used as controls for salt effects. Separate 4mL aliquots of medium were placed in the incubator and used to determine the incubation pH at the end of each decay time course. Except at pH 7.4 (the pH of DMEM-10 in 6% CO2) the experimental pH was different than the initial adjusted pH of the DMEM-10 with MOPS.[1]

[1]. Steven D Carson, et al. MOPS and coxsackievirus B3 stability. Virology. 2017 Jan 15;501:183-187.
[2]. Juliane Schmidt, et al. Effect of Tris, MOPS, and phosphate buffers on the hydrolysis of polyethylene terephthalate films by polyester hydrolases. FEBS Open Bio. 2016 Jul 20;6(9):919-27.

3-(N-morpholinyl)propanesulfonic acid is a Goodyear buffer with a pKa of 7.2 at 20 °C. It belongs to the morpholino family, MOPS family, and organic sulfonic acid family. It is the conjugate acid of 3-(N-morpholinyl)propanesulfonate and also a tautomer of 3-(N-morpholinonyl)propanesulfonate. 3-[N-morpholinyl]propanesulfonic acid has been reported to be found in citrus fruits (Citrus reticulata) and deliciosa. As shown in this paper, MOPS plays a stabilizing role in the decay kinetics of CVB3. MOPS and MES can bind to a variety of proteins (e.g., Fitzgerald et al., 1998; Knochel et al., 1996; Long and Yang, 2009; Sigurdardottir et al., 2015), and the weak interaction between MES and liver fatty acid-binding proteins is sufficient to alter protein dynamics (Long and Yang, 2009). Molecular modeling shows that MOPS fits well into the VP1 pocket, thus it is reasonable to infer that MOPS binds to CVB3 and alters capsid dynamics through a mechanism similar to that of other pocket-binding molecules (Reisdorph et al., 2003; Tsang et al., 2000). Based on simulated docking, MES and HEPES can also fit into this pocket (not shown). The model fit data is an extension of the dynamic virus model, where the open conformation is an intermediate of the A particle (Carson, 2014). The revised kinetic equation, equation (8), incorporates a term for the competitive inhibitor (MOPS), which is mechanistically a heteroallosteric inhibitor (i.e., it inhibits the transition to the open intermediate conformation by stabilizing the closed conformation). The pH effect is empirically modeled by a factor that reaches half its maximum value at K. This is a necessary approach before gaining deeper knowledge, as pH variations affect the virus, MOPS (the proportion of unprotonated MOPS is pH-determined), and other components in the experimental environment, including any naturally occurring pocket factors. Furthermore, variations of the model indicate that the data fit is best when the unprotonated MOPS form is considered the stable form of the virus. In this interpretation, MOPS and pH have antagonistic effects on viral stability: increasing pH decreases viral stability but increases the concentration of the MOPS stabilizer. [1]
Because the pH component in the model is empirical, the data covers only a narrow pH range, and the calculated Kd value exceeds the maximum MOPS concentration tested, therefore, although K and Kd values are physiologically reasonable, they do not accurately represent the true dissociation equilibrium constant. Nevertheless, the model should still have reasonable predictive value for the relationship between viral decay rate and pH and MOPS concentration, at least within the range studied (which is typical for such experiments). The results clearly show that both MOPS and pH affect the stability of CVB3, therefore MOPS (and other Good buffers) should be avoided in studies that may be affected by increased CVB3 stability. [1]

C7H14NNAO4S

231.2451

231.054

C, 36.36; H, 6.10; N, 6.06; Na, 9.94; O, 27.67; S, 13.86

71119-22-7

71119-22-7 (sodium salt); 1132-61-2 (free acid)

3859613

Typically exists as solid at room temperature

277-282°C

0.272

0

5

4

14

233

0

S(C([H])([H])C([H])([H])C([H])([H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])(=O)(=O)[O-].[Na+]

MWEMXEWFLIDTSJ-UHFFFAOYSA-M

InChI=1S/C7H15NO4S.Na/c9-13(10,11)7-1-2-8-3-5-12-6-4-8;/h1-7H2,(H,9,10,11);/q;+1/p-1

sodium;3-morpholin-4-ylpropane-1-sulfonate

MOPS sodium salt; Sodium 3-Morpholinopropanesulfonate; MOPS-Na; 4-Morpholinepropanesulfonic acid, sodium salt; MFCD00064350; 4-Morpholinepropanesulfonic acid sodium salt; sodium 3-morpholinopropane-1-sulfonate;

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)

H2O : ~100 mg/mL (~432.43 mM)
DMSO : ~50 mg/mL (~216.22 mM)

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.)