STRUCTURE OF THE EXTRACELLULAR HEMOGLOBIN OF BIOPHALARIA-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) givi ng a band of 270 kDa and a band of 180 kDa after reduction with P-merc aptoethanol. The same profile was obtained in a 3.5% agarose gel elect rophoresis containing SDS (SDS-AGE) but showed additional bands of hig her molecular weight. These bands were proposed to be monomers, dimers and trimers, since they showed a good correlation in a plot of R-f ve rsus log M-r. After partial reduction in a two-dimensional SDS-AGE the proposed monomers and dimers produced two and four bands, respectivel y, likely indicating one to four chains crosslinked by disulfide bridg es. Digestion with four different proteases yielded several equivalent fragments with molecular weights multiples of its minimum molecular w eight (17.7 kDa). The circular dichroism spectrum of the protein showe d a characteristic high a-helix content (similar to 70%). It was propo sed that this hemoglobin is a pentamer with a molecular weight of apro ximately 1.8 x 10(3) kDa, assembled by five 360-kDa subunits, each for med by two 180-kDa chains linked in pairs by disulfide bridges and eac h of these chains, in turn, comprised by ten heme binding domains link ed in tandem. These data are compared to the published information for other planorbid extracellular hemoglobins. (C) 1998 Elsevier Science Inc. All rights reserved.