I prepared deoxyhemoglobin by mixing a few drops of Stokes' reagent into a certain concentration of oxyhemoglobin solution. The deoxyHb was confirmed by UV-vis spectroscopy.
I also tried to use easier way to prepare deoxyHb by nitrogen bubbling of oxyHb solution for 1 day. However, the characteristic peaks of deoxyHb were not observed in UV analysis.
Can I prepare deoxyHb by only gas bubbling of oxyHb solution? If not, how can I get deoxyHb without usage of chemicals such as Stokes' reagent.
Well, it seems that you have shown it does not work very well. The problem of course is that if you have a gastight container, oxygen cannot escape, and if not, the oxygen partial pressure will only get lower, but not sufficiently low. You could play with pH to make Hb more likely to dissociate oxygen when using nitrogen bubbling. I.e. do the experiment e.g. at pH 6. Another option that does involve using two chemicals but that is commonly used for oxygen depletion is that you add glucose into you buffer, then add glucose oxidase and seal the gastight container, and the glucose oxidation by glucose oxidase will consume the oxygen.
Dear Jay Kim,
I have attached research articles on preparation of deoxyhemoglobin.
01. Article Hemoglobin. Isolation and chemical properties
02. https://lib.ugent.be/fulltxt/RUG01/002/063/879/RUG01-002063879_2013_0001_AC.pdf
I hope I have answered your question.
With Best Wishes,
Samir G. Pandya
Samir, the first paper does not use any kind of bubbling. The latter uses argon bubbling. That works better than nitrogen, I presume this is because it is heavier than oxygen and has better water solubility.
Juha-Matti Alakoskela, Argon has approximately the same solubility in water as oxygen and is 2.5 times more soluble in water than nitrogen.
Samir G. Pandya, I already read the references before. First one uses the Stokes’ reagent as I mentioned, whereas second one just uses argon bubbling to dissipate the oxygen gas. I guessed nitrogen also can do this role so that I used it instead of argon gas.
I just used commercially availavle hemoglobin. Am I do something to prepare oxyhemoglobin then?
Juha-Matti Alakoskela, I just filled nitrogen into a rubber baloon to bubble the oxyhemoglobin solution. Isn’t it enough to dissipate oxygen from oxyHb?
Thx for ur help.
I synthesized the deoxyHb sample by quenching the oxygen in the sample using sodium metabisulphite and continuous flowing of nitrogen gas.
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