Microspectrophotometric Determination of Nonheme Iron in Maturing Erythroblasts and its Relationship to the Endocellular Hemoglobin Formation

The strong near-UV absorption of inorganic iron makes possible the microspectrophotometric determination of nonheme intracellular iron as distinct from hemoglobin-bound iron. Erythroid cells of Salamandra Maculosa were therefore studied microspectrophotometrically in the near ultraviolet and visible region. Absorption curves from cytoplasmic areas in living and dried cells were obtained from 310 to 700 mµ., and two component analyses were made to determine the presence and amount of hemoglobin and nonhemoglobin absorption. Under the experimental conditions, intracellular hemoglobin was present largely as the carbon monoxide derivative, a stable form which provided a well defined standard to correct for nonspecific absorption and other spectral distortion due to the optical properties of single cells.

Absorption curves from the youngest cells measured were found to differ from those of mature cells, the difference curve agreeing most closely with that of inorganic iron or ferritin in solution. In the youngest cells, nonheme iron appeared to exceed 6 per cent by weight of hemoglobin, or over 20 times molar hemoglobin concentration, dropping and remaining at about twice the hemoglobin molarity from the time hemoglobin synthesis was one-third complete until maturity. Allowing for the increase of total cell mass with growth, the data suggest that although young erythroblasts may already have enough Fe for subsequent Hb synthesis, they continue to incorporate excess Fe throughout maturation and may thus contain a reserve pool of nonheme iron.