As you all know, that widely Drabkin's method is used to estimate Hemoglobin in blood. Also, Hemoglobin exist major in tetrameric form. My question is whether the estimation done by Drabkin's is of monomeric hemoglobin or tetrameric hemoglobin?
Drabkin's method fundamentally measures the molar concentration of the heme prosthetic group in hemoglobin. Because hemoglobin contains 4 heme groups in the heme group we can reliably use this to convert molar heme concentration (monomeric) to the concentration of hemoglobin (tetrameric) in your sample. Therefore, depending on how you set up your calculation Drabkin's can give you results on either a monomeric or tetrameric basis. Unfortunately, 4 hemoglobin monomers and 1 hemoglobin tetramer both contain 4 heme groups so they will give equivalent results. This assay cannot be used to determine if you have non-tetrameric hemoglobins)
Hi, I strongly believe the Drabkin's method measures tetrameric form of haemoglobin.
The Drabkin’s method measures the concentration of heme in Hb, therefore does not recognize monomeric, dimeric or tetrameric Hb. In this method potassium ferricyanide converts Hb (ferrous heme Fe 2+) to met-Hb (ferric heme Fe 3+); then potassium cyanide converts met-Hb to cyanmethemoglobin (HiCN). The absorbance of HiCN is measured at a 540nm wavelength. The more sensitive method for determination of Hb in minute amounts is the benzidine-hydrogen peroxide method, which also does not recognize Hb monomers, dimers or tetramers. To estimate the concentration of Hb monomers, dimers or tetramers you should use the size exclusion HPLC with ie, Protein-Pak SEC column 300A, 10 um, 7.5mm x 300mm, and exclusion limit 10K-500K.
Thank you very much for your valuable inputs. However, as @@Jan Simoni Drabkin method does estimate concentration of hemoglobin on the basis that Fe ion is present. Since each Fe of monomer contribute in estimation of Hemoglobin, I think estimation is of monomer Hb. Even Benzidine-H2O2 is based on Pseudo peroxidase method, Fe ion is involved..
The Drabkin’s is unable to identify the molecular structure of Hb. Hb is a globular protein with quaternary structure. Hb tetramers (~64.5 kDa) can dissociate (under specific condition) to dimers (~32 kDa) and even to monomers (~16 kDa). Therefore the size exclusion HPLC in the proper method.
Of course that the Drabkin’s method will measure the concentration of Hb monomers; but also dimers and tetramers. I think that the question was about identification of Hb monomers using the Drabkin’s method.
Thanks Simoni for answer.... My question was which Hb type is estimated by Drabkin's method. Thank you once again..
Let me increase the existing confusion by adding my ignorance ! In step no. 1, Hb is oxidised to Hi. Normally also we do get minor amounts of Hi formed, which is reduced to native Hb form by two Hi-reductase enzymes; NADH dependant in EM pathway and NADPH dependant (which is a reserve enzyme for excessive oxidation) in PP shunt. It means that Hi has to be tetrameric. This oxidation in vitro by ferricyanide is faster at physiological pH, but slow at alkaline pH. In the second step cyanide ion is added to form the final pigment. There is no mention in any test that CN can split Hb in monomers; and I tend to believe that same may be true for Hi. For globin chain analysis, to split the molecule, different reagents are used and electrophoresis is conducted, in which heme (red pigment) migrates rapidly towards one pole, separated from globins. Now whether you calculate millimolar extinction at 540 nm of 11 for monomer or 44 for tetramer is one and same. In short, I believe it is tetramer. The existance of monomer without heme in stable form is not mentioned to the best of my knowledge. Was I successful ?
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