The use of the highly specific, sensitive and very stable horseradish peroxidase with a chromogenic donor has proven very useful for assay systems producing hydrogen peroxide; for example, in the determination of glucose or galactose by their respective oxidases and in the determination of certain L-amino acids in conjunction with L-amino acid oxidase (Malmnstadt and Hadjiioannou 1963). A more generalized application of the principle is as follows:
To a solution of 0.05 M sodium acetate, pH 5.0, add 10 mg o-dianisidine hydrochloride, 18.6 mg EDTA, 5.0 ml of a filtered 0.1% peroxidase solution (equivalent to Worthington Code: HPOD), and 1.0 ml 10% Triton X-100. Dilute to 50 ml with sodium acetate. Protect solution from light. Add 2.0 ml of reagent to 2.0 ml of unknown. After 15 minutes at room temperature, read absorbance at 460 nm. Standard solutions containing from 1 to 3 micrograms of hydrogen peroxide per test are run simultaneously.
Do you know what is the role of TRITON X-100?
Thank You
Yours,
Héctor
The final concentration of Triton X-100 is 0.2%, which is above the critical micellar concentration. Presumably, its purpose is to keep something in solution which otherwise would not be soluble. None of the substances you mentioned is insoluble at the concentrations you gave, so there must be something else needing solubilization that you did not mention.
Thank you Addam, you always helping people.
I just copy the protocol for peroxidase. I think like you. That's why my question.
Also for disaccharidase activity ( Arne Dahlqvist, 1970, 1974) include the use of the TGO reagent. The TGO reagent is quite similar and also includes Triton-X100:
Dissolve 2 mg. glucose oxidase in 100 ml. Tris buffer (III), add 1 ml. o,-dianisidine solution (VI), 1 ml wetting agent (solution V, 20% TRITON x-100) and 0.5 ml. peroxidase solution (IV) and mix.
Same question...
Yours,
H.
Some people add Triton-100 or other non ionic detergent to avoid non specific interactions between proteins, thus avoiding aggregation, even though the protein is soluble. However, the added quantity depends on different protocols, and whether that is necessary or not, is still unclear i think.
Thank you dear Jorgat,
Additionaly, i think the role of Triton x-100 is to facilitate the solubility of o-dinisidine and oxidized o-dinisidine (dye), to read absorbance.
What do you think?
Yours
H.
Hector Nolasco I am not sure but maybe. Normally o-dinisidine is soluble at these concentrations and I have came across protocols that do not use Triton-X100 at all in a glucose assay using GO
could you send us a copy of that articles with the protocols that do not use Triton-X100 at all in a glucose assay using GO ?
H.
It's interesting that Triton X-100 is referred to as a wetting agent, rather than a detergent. This suggests that there is a need in this assay to wet a surface evenly, rather than dissolve something. The Triton X-100 helps with this by reducing surface tension.
Hector Nolasco https://aem.asm.org/content/80/2/788#sec-2 , i think it was this one
Thank you very much Adam and Jorgaq,
If we consider the solubility of o-dianisidine, it is very low in the water. The reactive generally is prepared in ethanol. The solubility is increased in acidified water, but for example TGO reagent is close to neutral pH.
According to the concentration of reactive or even la the final concentration, for example in TGO reagent, it is not possible to be prepared in water, because concentration is higher than what is soluble in water.
In TGO Reagent: 4.093e0-4 M
https://pubchem.ncbi.nlm.nih.gov/compound/o-DIANISIDINE
3.2.6 Solubility
less than 0.1 mg/mL at 68° F (NTP, 1992)
Water Solubility
2.46e-04 M YALKOWSKY,SH & DANNENFELSER,RM (1992)
In water, 60 mg/l @ 25 °C
Yalkowsky SH, Dannenfelser RM; The AQUASOL dATAbASE of Aqueous Solubility. Ver 5. Tucson, AZ: Univ AZ, College of Pharmacy (1992)
Solubility in water, g/100ml at 25 °C: 0.006.
Because of that, I consider that Triton x-100 was included to clarify the reaction, solubilizing the dye, oxidized o-dianisidine, to measure the absorbance properly,,
Yours,
H.
https://www.sigmaaldrich.com/catalog/product/sigma/d9154?lang=fr®ion=FR
Maybe, but the endproduct obtained normally is water-soluble according to Sigma
dear Jorgaq
D9154 Sigma-Aldrich
o-Dianisidine dihydrochloride
This formula contains 2 mols of HCl, thats why it is most soluble in water, as indicated before.
The TGO reagent is close to neutral.
Yours,
H.
Different effects of Triton X-100, deoxycholate, and fatty acids on the kinetics of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase.
Maiorino M, Roveri A, Gregolin C, Ursini F.
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