Structure of the extracellular hemoglobin of Biomphalaria glabrata

The hemoglobin of Biomphalaria glabrata was purified to homogeneity by gel filtration column followed by anion exchange chromatography. The dissociation products were analyzed by a 5–15% gradient polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) giving a band of 270 kDa and a band of 180 kDa after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5% agarose gel electrophoresis containing SDS (SDS-AGE) but showed additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers, since they showed a good correlation in a plot of R_f versus log M_r. After partial reduction in a two-dimensional SDS-AGE, the proposed monomers and dimers produced two and four bands, respectively, likely indicating one to four chains crosslinked by disulfide bridges. Digestion with four different proteases yielded several equivalent fragments with molecular weights multiples of its minimum molecular weight (17.7 kDa). The circular dichroism spectrum of the protein showed a characteristic high α-helix content (70%). It was proposed that this hemoglobin is a pentamer with a molecular weight of aproximately 1.8×10³ kDa, assembled by five 360-kDa subunits, each formed by two 180-kDa chains linked in pairs by disulfide bridges and each of these chains, in turn, comprised by ten heme binding domains linked in tandem. These data are compared to the published information for other planorbid extracellular hemoglobins.