Application of anaerobic ion-exchange chromatography to the separation of hemoglobins in R from T conformational states.
As an allosteric protein, hemoglobin can assume at least two different quatenary structures, a deoxy or T for tense conformation and an oxy or R for relaxed conformation (2,3) depending upon its state of ligation. Because the pK values of oxy and deoxy- hemoglobins are different, molecules in the R conformation should behave differently than molecules in the T conformation on ion exchange chromatography. Kilmartin et al. (4) designed an anaerobic cation exchange chromatographic procedure with which they separated the deoxy hemoglobin from oxy hemoglobin and other R conformation hemoglobin components like CO-hemoglobin and sulfohemoglobin. In this report we describe the further development of Kilmartin's procedure to separate two electrophoretically silent hemoglobin mutants, Hb Potomac [beta 101 Glu leads to Asp] (5) a high oxygen affinity variant, and Hb M-Milwaukee [beta 67 Val leads to Glu] (6,8) a low oxygen affinity mutant from Hb A. The modifications we have made include (a) applying the hemolysate at a partial pressure of oxygen determined from an examination of the oxygen equilibrium curve to produce a favorable ratio of T to R conformations of the two hemoglobins to be separated, (b) controlling the partial pressure of oxygen tension of the column effluent during the development of the chromatogram.
D000693:Anaerobiosis; D002852:Chromatography, Ion Exchange / Q000379:methods; D006441:Hemoglobin A; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D006801:Humans; D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D011487:Protein Conformation
D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D006441:Hemoglobin A