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-morpholino)propanesulfonic acid is a Good's buffer substance, pKa = 7.2 at 20 ℃. It is a member of morpholines, a MOPS and an organosulfonic acid. It is a conjugate acid of a 3-(N-morpholino)propanesulfonate. It is a tautomer of a 3-(N-morpholiniumyl)propanesulfonate.
3[N-Morpholino]propane sulfonic acid has been reported in Citrus reticulata and Citrus deliciosa.

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MOPS has a stabilizing effect on CVB3 decay kinetics, as shown here. MOPS and MES can bind to various 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 protein is sufficient to alter protein dynamics (Long and Yang, 2009). Molecular modelling shows that MOPS is well-accommodated in the VP1 pocket, so it is reasonable to conclude that MOPS binds CVB3 and alters the capsid dynamics with a mechanism similar to that described for other pocket-binding molecules (Reisdorph et al., 2003; Tsang et al., 2000). Based on simulated docking, MES and HEPES are also accommodated in the pocket (not shown). The model fit to the data is an extension of a model for dynamic virus where the open conformation is an intermediate to the A-particle (Carson, 2014). The modified kinetic equation, equation (8), includes terms for a competitive inhibitor (MOPS) that is mechanistically a heterotropic allosteric inhibitor (i.e., it inhibits the transition to the open intermediate conformation by stabilizing the closed conformation). The pH effect was empirically modelled with a factor where the hydrogen ion effect is half-maximal when it equals K. This is a necessary approach, pending greater knowledge, because the changes in pH affect virus, MOPS (the proportion of unprotonated MOPS is determined by pH), and other components in the experimental milieu, including any naturally occurring pocket factor(s) that might be present. Moreover, variations of the model suggested that the data were fit best when the unprotonated form of MOPS was considered to be the virus-stabilizing form. In this interpretation, MOPS and pH have antagonistic effects on virus stability: increasing pH destabilizes the virus but increases the concentration of the stabilizing form of MOPS.[1]
Since the pH component of the model is empirical, the data covers a narrow range of pH values, and the calculated Kd exceeds the maximum concentration of MOPS tested, the K and Kd values, though physiologically reasonable, cannot accurately represent the true dissociation equilibrium constants. Nevertheless, the model should have reasonable predictive value for virus decay rate as a function of pH and MOPS concentration at least within the ranges studied, which are typical for such experiments. The results clearly show that MOPS and pH both affect CVB3 stability, and that MOPS (and probably other Good’s buffers) should be avoided in studies that would 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.)