Lactoferrin from human breast milk and from neutrophil granulocytes. Comparative studies of isolation, quantitation, characterization and iron binding properties.
The isolation and properties of lactoferrin from human breast milk and from neutrophilic granulocytes were investigated. Human breast milk lactoferrin was purified by means of heparin-sepharose or Cibacron Blue affinity chromatography. Quantitative recovery using these two methods was comparable but Cibacron Blue affinity chromatography allowed for isolation of a more homogenous protein. Lactoferrin could only be isolated from human neutrophilic granulocytes by sequential use of antibody affinity followed by non-specific affinity chromatography. Both breast milk lactoferrin and granulocyte lactoferrin were separated into apo and iron-rich species by SDS polyacrylamide gel chromatography. Iron binding is accompanied by a conformational change in tertiary structure associated with more rapid electrophoretic migration. The isoelectric point of both human breast milk lactoferrin and human granulocyte lactoferrin is 5.5-6.2. Both types of lactoferrin have similar iron binding properties with release of iron from the one binding site occurring at pH 5.2-6.0 while the other binding site holds on to iron down to pH 3.6-3.2. Despite the high affinity for iron the percentage saturation of native lactoferrin is low, that for breast milk lactoferrin averaging 12-25% and that for granulocyte lactoferrin less than 10%.
Department of Medicine, Haematology/Oncology, University of the Witwatersand Medical School, Johannesburg, South Africa.
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Bezwoda
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Mansoor
D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007501:Iron / Q000378:metabolism; D007525:Isoelectric Focusing; D007781:Lactoferrin / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D007782:Lactoglobulins / Q000302:isolation & purification*; D008895:Milk, Human / Q000032:analysis*; D008970:Molecular Weight; D009504:Neutrophils / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding
D007782:Lactoglobulins; D007501:Iron; D007781:Lactoferrin