Separation of the valence intermediates of human haemoglobin by high-performance chromatofocusing.
Investigations into the properties of haemoglobin often require the isolation of the valence intermediates (alpha o2 beta+)2 and (alpha+ beta o2)2. Chromatofocusing with an anion-exchange gel (Mono PTM; Pharmacia, particle size 10 micron) in an HR5/20 column at various temperatures (10-25 degrees C) provides an excellent method for this task. A linearly decreasing pH gradient (8 to 7, generated by Polybuffer 96, Pharmacia) eluted sequentially the species methaemoglobin, (alpha o2 beta+)2, (alpha+ beta o2)2 and oxygenated haemoglobin. Calibration graphs help in quantitative analyses. This method is simpler and less time consuming and provides a similar or even better resolution than the traditional ion-exchange or isoelectric focusing methods.
Bolzacchini
E
E
Istituto Scientifico San Raffaele, Milan, Italy.
Fermo
I
I
Rovida
E
E
Colombo
R
R
Samaja
M
M
D002852:Chromatography, Ion Exchange; D006454:Hemoglobins / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008706:Methemoglobin / Q000302:isolation & purification; D013696:Temperature
D006454:Hemoglobins; D008706:Methemoglobin