PROPERTIES OF LEGHAEMOGLOBIN IN VIVO, AND ITS ISOLATION AS FERROUS OXYLEGHAEMOGLOBIN

1. 1. Leghaemoglobin (Lb) appears to function as an O2-transporting rather than N2-transporting or N2-activating pigment during symbiotic N2 fixation. 2. 2. Direct spectrophotometry of soybean root nodules under near-physiological conditions establishes that Lb exists in vivo in its ferrous form (Lb2+), which has a myoglobin (Mb)-like spectrum with no haemochrome characteristics. This Lb2+ does not form a spectroscopically-distinguished N2 complex, nor undergo a natural cycle of oxidation and reduction. Equilibration with air or O2 causes the reversible, in vivo, formation of ferrous oxyleghaemoglobin (Lb2+O2). At an external O2 tension of 142 mm Hg, the Lb2+ of a 2.5-mm diameter nodule was 20-25% oxygenated. 3. 3. The natural pH of soybean nodules is close to 6.4 and extracts made in 0.01 M phosphate (pH 6.4) contain much Lb2+O2. At pH 6.4 this stable, purified Lb2+O2 has an α absorption band at 574 nm (εmm = 15.1). β band at 541 nm (εmm = 15.0) and γ band at 411 nm (εmm = 139); it shows Mb-like rather than haemochrome spectra when deoxygenated to Lb2+ with Na2S2O4 or oxidized to ferrileghaemoglobin (Lb3+) with K3Fe(CN)6. © 1969.