I am using gel filtration chromatography to analyze Hemoglobin at varying pHs. The results of the GFC at a pH of 7.4 showed that hemoglobin was in its dimer form (I was expecting to see Tetramer). Is it possible that there are other interactions affecting the elution of the hemoglobin in the column?
Hi there,
pH variationon both sides of physiological value (7.4) affects dramatically the equilibrium between tetramer dimer and monomer. For more info see the following
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842943/
Increased ionic strength is also favoring tetramer dissociation.
Hi again,
Hb behavior during SEC is highly dependent on the sample concentration. As tetramer dimer is a fast equilibrium the retention time of the unique peak observed is variable depending on sample concentration (if it's far above Kd, retention time tends to the one of tetramer; if it's around Kd, retention time increases and get close to the one of dimer). I think the reading of the following should help:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143381/pdf/8845768.pdf
I recall answering your earlier question about Hb trimers. So you saw 0% tetramer monitoring at 215 nm or assaying for Fe? The Hb was a fresh sample of tetramer not previously treated at any other pH, right? It's unlikely the Sephacryl would interact with the tetramer as it would likely do so with the dimer as well. Superose and Superdex have also been used for Hb SEC experiments long ago and their epichlorohydrine cross linking has a slight degree of hydrophobic character as would the Sephacryl (allyl dextran cross linked with bisacrylamide) you're using.
Dissociation also depends on oxygenation state (Edelstein et al, JBC 245, 43724381, 1970). They give a dissociation constants at pH 7 of 2 uM for oxyhemoglobin, vs 0.1 uM for deoxy. So you might preserve the tetramers by pre-equilibrating the column with dithionite-treated buffer.
Hi here I like to mention several factors those are responsible for tetramer-dimer equilibrium shifting towards dimer....
low concentration Hb favors dissociation of tetramers
Hemoglobins without ligands in distal pocket are more prone to get dissociate so sixth ligand either oxygen or CO or CO2 can be used. preferential CO2 a natural ligand with high affinity.
temperature should be below 20 degree C. 4 degree C preferred. avoid temperature variation.
avoid frequent freeze-thaw
for gel filtration use superdex 75
buffer used for this CO2 saturated
20mM HEPES pH7 with 0.5mM EDTA and 1mM DTT (optional) will work
even in extreme you can use some allosteric effectors like 2,3-BPG
All the best.
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