Thermostable Superoxide-Producing Complex Between NADPH-Containing Lipoprotein (NCL) And Fe (III) From Boiled Cow Milk: Activation Of Immune Cells NADPH Oxidase By NCL In Vitro

Abstract

О2−-producing complexes or associates have recently been isolated from blood serum, goat milk, erythrocyte, leukocyte membranes, and medicinal plant membranes. Fe(III) ions in these complexes act as bridges for electron transfer from NADPH-containing lipoprotein (NCL) to molecular oxygen, reducing it up to О2−. On the other hand, NADPH oxidase (Nox) of erythrocytes and leukocytosis are activated by NCL in vitro. In fact, both serum and milk contain NCL. The main systems for the production of О2− in mammalian milk and blood, as previously stated, are polymorphonuclear leukocytes, as well as О2−-producing complex between NCL and Fe (III). At the same time, Fe(III) ions can be found in milk, and anemia can result from a Fe(III) deficiency in milk. The superoxide (О2−) - producing thermostable complex between NCL and Fe(III): NCL-Fe(III) was isolated and purified from raw and boiled cow milk, for the first time. The specific О2−-producing activity of NCL-Fe(III) complex from milk, before and after boiling, practically does not change as a result of milk boiling (this activity decreases by only 7-8%). After incubating an aqueous mixture of NCL (5 mg/ml) with an aqueous solution of Nox1 + Nox2 (erythrocytes or leukocytes membranes) isoforms, 5 mg/ml, at 37°C for 40 minutes, an ion exchange chromatography was performed on the column of DE cellulose, equilibrated by water at pH9.5. The hNCL-Nox associate eluates from this column with water at pH9.5 The specific О2−-producing activity of hNCL-Nox isoforms of EM and LM basically does not decrease when compared to the NCL-Fe(III) complex. This is a promising finding, suggesting that milk NCL has stimulating the Nox from erythrocytes or leukocyte membranes in vitro does not decrease even after boiling. In contrast to these О2−-producing associates or complexes, NCL isolated from them, suppresses the oxidation of adrenaline to adrenochrome, exerting a reducing-antioxidant effect due to NADPH electrons in its composition (the Cu, Zn-SOD does not inhibit this process). The NCL isolated from this complex, at the expense of NADPH, has a reductive (antioxidant) influence and forms a hybrid О2−-producing associate with isoforms of the NADPH oxidase from erythrocytes and leukocytes membranes. Thus the properties of the thermostable complex NCL-Fe(III): О2−-producing activity, as well as the reductive (antioxidant) activity of NCL, are practically preserved after milk boiling during 10-12 min.