Ferroxidase converts FE2 to FE3. I would use kits that have ferosene s. It coordinates to fe 2 and produces a color. You can use this to monitor the disappearance of fe2.

It is ferene S

Hi Marcia,

I did use this FerenS, but I am looking for something more sensitive. How much change in absorbance at 600 nm do you see if your enzyme is really active (Sample -control). I was optimizing with commercial apoferritin and I get 0.144 unit absorbance change (sample-conrol) at 40 uM FeSo4 concentration. Is this significant ? I haven't applied this method to my expressed enzyme yet

Have you ever tried continuous assay (spectrophotmeter) ? I appreciate your response. Thank you.

Also for continuous assay I looked for abs change at 340 nm but absorbance is not very linear so I am planning to use apotransferrin with apoferritin as mentioned in some papers.

For Ferene S assay I was doing was doing in Acetate Buffer pH 5.8 incubation at 37 oC for 30 min.

I have not used ferene s myself, but for one kit with 10 nmole of iron, the absorbance is 0.8 at 593nm.

Can you just do the assay with ferroxidase, and then quantify FE2+ with the kit and not reduce the fe3 back to fe2 and not use any apoferritin. I would think all you would need to do is run the reaction, and then add the ferene s reagent.

I am not sure what the apoferritin is for? Is it for absorbing the FE3+? I thought the ferene s only reacts with ferrous ion. Check out bio visions web site.

And for the kit, I would not use all the components, just the reagent for the final fe2 determination. I would think to call the manufacturers for technical advice and then maybe you don't have to buy the kits, and just buy the ferene s reagent.

Hi Marcia, I purchased Ferene S from sigma it works fine with iron and standard curve looks great. I am concerned about the enzymatic conversion of Fe2+ to Fe3+ and how efficient my enzyme. Continuous is for Fe2+ to Fe 3+ that Fe 3+ absorbs at 340/360 nm. Only thing I am not sure is sensitivity. Thank you

Iron adsorbs best at around 500 nM I think. But here is an article that has the molar extinction coefficient of feIII in water. I don't know if this is the best reference. If you can find the extinction coefficient, you can estimate the sensitivity. I just googled 340 nM, molar extinction coefficient

Iron (III) Molar Extinction Coefficients in Light and Heavy Water Solutions

J. W. Boyle and H. A. Mahlman

Radiation Research

Vol. 16, No. 4 (Apr., 1962), pp. 416-421

There are other references.

Thank you Marcia, this will help.