Alkali cation effect on carbonyl-hemoglobin's and -myoglobin's conformer populations when exposed to freeze-concentration of their phosphate-buffered aqueous solutions.
Freeze-concentrated aqueous phosphate-buffered (pH 6.8) solutions of carbonyl-hemoglobin (HbCO) and -myoglobin (MbCO) were investigated by Fourier-transform infrared spectroscopy for the effect of alkali cation on the population of conformers. When using sodium phosphates as buffer components, HbCO was transformed from conformer III (at approximately 1951 cm-1) which is the dominant form at ambient temperatures, into conformer IV (at buffer concentration at a given temperature. The conformational changes started slightly below the temperature where ice began to crystallize and the remaining solution became freeze-concentrated, and they were reversible for HbCO. For MbCO in 0.5 M sodium phosphate buffer solution, however, they were irreversible and MbCO denatured completely. When potassium phosphate salts were used for preparing the buffer at the same pH of 6.8, little or no transformation of conformer III into conformer IV was observed. The conformational changes induced by sodium salts are attributed to a decrease in pH and it is shown by infrared spectroscopy that during freeze concentration drastic changes in composition of the two buffer components H2PO4-/HPO(2)4- occur, the acid component increasing strongly relative to the base component. Supersaturation is also important because change from conformer III to IV requires a minimum concentration of sodium salts: whereas 0.1 M sodium phosphate buffer concentration shows a strong effect, 0.03 M concentration does not and therefore behaves like a potassium phosphate buffer.
Astl
G
G
Institut für Anorganische und Analytische Chemie, Universität Innsbruck, Austria.
Mayer
E
E
D000818:Animals; D002021:Buffers; D002263:Carboxyhemoglobin / Q000737:chemistry*; D003460:Crystallization; D005583:Fourier Analysis; D005615:Freezing; D006736:Horses; D006801:Humans; D009211:Myoglobin / Q000737:chemistry*; D010710:Phosphates; D011487:Protein Conformation; D012996:Solutions; D013055:Spectrophotometry, Infrared / Q000379:methods; D013816:Thermodynamics
D002021:Buffers; D009211:Myoglobin; D010710:Phosphates; D012996:Solutions; C018013:carboxymyoglobin; D002263:Carboxyhemoglobin