Ferrozine (disodium 3-(2-pyridyl)-5,6-bis(4-phenyl sulphonate)-1,2,4-triazine) is a water-soluble Fe(II) chelator. When chelated to iron, the complex absorbs light at 562 nm (molar absorption coefficient = 27,900). Copper can also be bound by ferrozine, however copper binds more strongly to another chelating agent, neocuproine.
Iron can be released from protein through oxidation with acid-permanganate (1 hours at 60oC). Excess permanganate and released iron can then be reduced with ascorbate (RT for 30 min). Released iron must be kept at a pH of 4.5 – 5.0 for it to remain soluble. Acid-permanganate is an equal mixture of 1.2 M HCl and 0.285 M KMnO4
Given that the Mr of haemoglobin is 68000 and that for serum albumin is 66000, calculate the number of molecules of iron per molecule of protein. Use the known textbook value for the amount of iron in haemoglobin to calculate the purity of your sample.
Hint: a sensible strategy might be to start by constructing a calibration curve for the reaction of ferrozine with iron. Assays will need to use a control protein (non-Fe containing) for reference.
Another hint: A ratio of 1:5:10 would be appropriate for reagent B: acid-permanganate: sample.
Materials provided:
Haemoglobin (1.5 mg/ml)
Bovine serum albumin (5 mg/ml)
Ferrous ammonium sulphate (anhydrous?)
1.2 M HCl
4.5 % (w/v) (0.285 M) KMnO4
Reagent B containing: 6.5 mM Ferrozine
13.1 mM neocuproine
2 M ascorbic acid
5 M ammonium acetate.
Try your second method. But for estimation purpose you can club two methods. The Hb S level is ex-pressed as a percentage of total Hb by scanning the
cellulose acetate membrane with a densitometer after Hb electrophoresis at
pH 8.6 Hb S level of 30%(conventional simple transfusion) and, after 3 or more
years without recurrent stroke, an Hb S level of 50% (modified simple
transfusion). NO-modified hemoglobin formation is inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. It clears that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.
Hi, I suppose your protocol will lead to reliable results but you can try some biochemical kits which may be presence in your location.
Cheers, A.
I think iron in Hb is proportional to the absorption in electronic spectra. So take apo-Hb and measure spectra and the build calibration curve with different iron concentrations. Use this calibration curve for future estimations.
Thank you guys but the thing is I need a protocol l to do the experiment, I don't have a protocol yet, I need someone to upload or send me a link for it please, thanks.
Main thing you have to remember is to have a 0.1mM concentration of FAS and that it is most likely not anhydrous,
Basically, for your calibration curve you use the FAS as it is an iron containing protein. In order to do this you might want to mix reagent b with acid pomaganate and FAS in a 1:5:10 ratio respectively
however you must dilute FAS with water each time in order to get the curve. These must be kept in a 60 degree water bath for 60 minutes before the absorption can be measured. you can also measure it against a control containing bovine instead of FAS. I haven't worked out the rest as of yet but hopefully that will get you started.
