BACTERIAL EXPRESSION AND SPECTROSCOPIC CHARACTERIZATION OF SOYBEAN LEGHEMOGLOBIN A

A gene encoding leghaemoglobin a from soybean has been constructed and the soluble recombinant protein expressed in E. coli. The integrity o f the recombinant protein has been assessed by a range of spectroscopi c techniques. Electrospray mass spectrometry of the protein indicates that the molecular mass of the protein corresponds to the predicted am ino acid sequence. Circular dichroism spectra of the ferric derivative and UV-visible spectra of various ferric and ferrous derivatives (pH 6.99, mu = 0.10 M, 25.0 degrees C) are consistent with published data for the wild-type protein. For the ferric derivative, UV-visible (298 and 77 K) and EPR (10 K) spectra indicate the existence of a thermal e quilibrium between high-and low-spin forms. Titration of the protein ( 0.10 M NaCl, mu = 0.10 M, 25.0 degrees C) between pHs 6.68 and 10.35 i ndicate formation (pK(a) = 8.3 +/- 0.03) of a 6-coordinate, hydroxide- bound form of the protein at high pH. All of the above data are consis tent with the behaviour of the wild-type protein.