A straightforward method for purification of myo-inositol 1,3,4,5,6-pentakisphosphate from avian erythrocytes for subsequent application in studies on hemoglobin allosteric effect.
myo-Inositol 1,3,4,5,6-pentakisphosphate (Ins-P5) is the main allosteric effector of hemoglobin in avian erythrocytes. Molecular studies on the functioning of hemoglobin have frequently been performed with the hexaphosphate form of inositol, since it is difficult to obtain Ins-P5 of appropriate purity and in sufficient quantity for oxygen-affinity studies. This paper provides a detailed description of a method for adequate isolation of Ins-P5 from avian erythrocytes. The method is based on fractionation of erythrocyte organic phosphates by chromatography. The fractions corresponding to the peak of Ins-P5 were retained for further purification and concentration. This process includes gel filtration, cation-exchange chromatography, and lyophilization. At the end of the process about 15 mg of Ins-P5, free from other organic phosphates or salts, was obtained from 45 ml of pigeon blood. Trials performed on avian stripped hemoglobin solutions with Ins-P5 obtained in this way confirm that this phosphate is the main factor responsible for the decrease in oxygen affinity in intact erythrocytes, and that it is preferable to Ins-P6 for hemoglobin oxygen-affinity studies.
Prats
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Departament de Bioquimica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Spain.
Riera
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D000495:Allosteric Site; D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D010856:Columbidae; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism*; D005612:Freeze Drying / Q000379:methods; D006454:Hemoglobins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000097:blood* / Q000302:isolation & purification*; D007700:Kinetics; D010108:Oxyhemoglobins / Q000378:metabolism
D006454:Hemoglobins; D007295:Inositol Phosphates; D010108:Oxyhemoglobins; C013571:inositol pentaphosphate