Hi everyone,
I’m using hemoglobin in the lab and I need it to be in the reduced state (so it is able to bind with CO2). The hemoglobin I have is a lyophilized powder from Sigma Aldrich (H7379). From information I found on their website, they have a ”procedure for reduction of Oxidized Hemoglobin from the Oxyhemoglobin derivative”, where they:
- 1st they equilibrated a Sephadex G-25 column with 0.2M phosphate buffer and 1mM EDTA
- then dissolved 0.2 g of sodium hydrosulfite (sodium dithionite) in 2ml buffer, added that to column, then added 1ml buffer to column
- then added 10 ml of hemoglobin. Then eluted it with phosphate buffer. This eluted sample is now supposedly reduced.
Here is a link for information I skipped: https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/clinical-testing-and-diagnostics-manufacturing/hematology/hemoglobin-heme-products
Has anyone done this reduction using sodium hydrosulfite (sodium dithionite) without the column? Meaning just in a tube, then maybe dialyzed the sample to remove any remaining sodium hydrosulfite?
If so, could you please share the protocol? How long should we incubate the hemoglobin with the sodium hydrosulfite for? And what should be the ratio of the concentration of the hemoglobin to the sodium hydrosulfite?
Otherwise, and even better, does anyone have a protocol not involving sodium hydrosulfite? Maybe ascorbic acid or glutathion? According to the internet they can also reduce hemoglobin but I fail to find a proper protocol. I’d appreciate the help.
Thanks for your time.
Amoon
To reduce oxidized hemoglobin (Fe3+) to its reduced form (Fe2+), you can use a reducing agent. One commonly used reducing agent for this purpose is sodium dithionite (Na2S2O4). Here's a protocol to reduce Fe3+ hemoglobin to Fe2+:
Materials :
Protocol:
Sodium metabisulphite solution (20 g/L D.W.), FRESHLY made is has nearly neutral pH and is isotonic. You have to titrate and look for the absorption peaks, till you get expected result. Oxy-hemoglobin has peaks at 576 and 540 nm, while reduced hemoglobin has a single peak at 550 nm. Met-hemoglobin has a peak at 632 nm. Here is attached diagram of absorption spectra of various hemoglobins
Another diagram of absorption spectra of hemoglobin derivatives is attached
Préparez une solution réductrice telle que la dithionite de sodium (Na2S2O4) dans de l'eau déionisée.Ajoutez l'échantillon contenant l'hémoglobine oxydée à la solution réductrice.Mélangez doucement et assurez-vous que la réduction se produit en maintenant la solution à une température constante.Contrôlez le processus en vérifiant visuellement le changement de couleur de l'échantillon, le passage de la couleur rouge (féerique) à une couleur plus claire (ferreux).Une fois la réduction terminée, vous pouvez purifier l'échantillon si nécessaire, par exemple, par filtration ou centrifugation.
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