Identification and Purification of a New Non-Heme,Non-Ferritin Iron Protein

Abstract

In a previous paper Najean et al. have proposed a “mamillary” model to account for iron metabolism in man and animals under normal conditions (1). The model is based on the concept that the biexponential fall-off curve of plasma radioactivity after in vivo labeling of transferrin with ⁵⁹Fe is due to a small non-heme, noncirculating iron pool (pool Q2 in the model, ref. 1) which can exchange iron with plasma transferrin. According to this model, the Q2 pool must be distributed throughout the whole body. If the model is to be validated, the characteristics of this small iron pool must be in good agreement with those calculated for Q2, from in vivo studies, using the mamillary model.

Materials and Methods. Adult Wistar rats fed with a standardized iron diet were injected intravenously with a tracer dose of ⁵⁹Fe (40 μCi/100 g, below U.I.B.C.) diluted in buffered saline. Blood samples were then withdrawn in order to calculate the variations in specific activity of pool Q2 according to the model (1). The animals were sacrificed at appropriate intervals following infusion of the tracer. Lung, liver, spleen, kidney and small intestine were then removed and washed or perfused in cold buffered saline. The tissues were homogenized in cold 0.006 M phosphate buffer, pH 7.2 and the homogenates were centrifuged at 80,000g at 4° for 1 hr. The clear supernatants were counted for ⁵⁹Fe radioactivity prior to a 5 hr dialysis in an ultrathimble 1 against the homogenizing buffer. The dialyzed and concentrated supernatants were counted once more for ⁵⁹Fe activity, in order to estimate the possible loss of iron due to dialysis. The dialysates were then subjected to ion-exchange chromatography on DEAE-cellulose, using the following stepwise elution process: (a) 0.006 phosphate buffer pH 7.